Patent Application
20070134261
The present invention relates generally to peptides and specifically to peptides effective as therapeutics and for drug discovery related to pathologies resulting from microbial infections and for modulating innate immunity or inflammation.
Infectious diseases are the leading cause of death worldwide. According to a 1999 World Health Organization study, over 13 million people die from infectious diseases each year. Infectious diseases are the third leading cause of death in North America, accounting for 20% of deaths annually and increasing by 50% since 1980. The success of many medical and surgical treatments also hinges on the control of infectious diseases. The discovery and use of antibiotics has been one of the great achievements of modem medicine. Without antibiotics, physicians would be unable to perform complex surgery, chemotherapy or most medical interventions such as catheterization.
Current sales of antibiotics are US$26 billion worldwide. However, the overuse and sometimes unwarranted use of antibiotics have resulted in the evolution of new antibiotic-resistant strains of bacteria. Antibiotic resistance has become part of the medical landscape. Bacteria such as vancomycin-resistant Enterococcus (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) strains cannot be treated with antibiotics and often, patients suffering from infections with such bacteria die. Antibiotic discovery has proven to he one of the most difficult areas for new drug development and many large pharmaceutical companies have cut back or completely halted their antibiotic development programs. However, with the dramatic rise of antibiotic resistance, including the emergence of untreatable infections, there is a clear unmet medical need for novel types of anti-microbial therapies, and agents that impact on innate immunity would be one such class of agents.
The innate immune system is a highly effective and evolved general defense system. Elements of innate immunity are always present at low levels and are activated very rapidly when stimulated. Stimulation can include interaction of bacterial signaling molecules with pattern recognition receptors on the surface of the body's cells or other mechanisms of disease. Every day, humans are exposed to tens of thousands of potential pathogenic microorganisms through the food and water we ingest, the air we breathe and the surfaces, pets and people that we touch. The innate immune system acts to prevent these pathogens from causing disease. The innate immune system differs from so-called adaptive immunity (which includes antibodies and antigen-specific B- and T-lymphocytes) because it is always present, effective immediately, and relatively non-specific for any given pathogen. The adaptive immune system requires amplification of specific recognition elements and thus takes days to weeks to respond. Even when adaptive immunity is pre-stimulated by vaccination, it may take three days or more to respond to a pathogen whereas innate immunity is immediately or rapidly (hours) available. Innate immunity involves a variety of effector functions including phagocytic cells, complement, etc, but is generally incompletely understood. Generally speaking many known innate immune responses are “triggered” by the binding of microbial signaling molecules with pattern recognition receptors such as Toll-like receptors (TLR) on the surface of host cells. We now know that Toll/Interleukin-1 Receptor (TIR) domain-containing proteins play a pivotal role in initiating aspects of the inflammatory responses. Many of these effector functions are grouped together in the inflammatory response. However, too severe an inflammatory response can result in responses that are harmful to the body, and, in an extreme case, sepsis and potentially death can occur. Thus, a therapeutic intervention to boost innate immunity, which is based on stimulation of TLR signaling (for example using a TLR agonist), has the potential disadvantage that it could stimulate a potentially harmful inflammatory response and/or exacerbate the natural inflammatory response to infection.
Early responses to infection, collectively termed innate immunity and/or acute inflammation, are substantially orchestrated by various mechanisms, for example, the interaction of bacterial molecules with TLR. It has been shown that a breakdown in the appropriate regulation of the TLR pathway can cause common chronic inflammatory diseases including inflammatory bowel disease (IBD), cardiovascular disease, arthritis, and chronic interstitial nephritis. Further, TLR engagement by conserved microbial molecules results in the translocation of the pivotal transcription factor NFκB and the transcription of ‘early-response’ genes encoding, for example, cytokines, chemokines, selected antimicrobial/host defense peptides, acute phase proteins, cell adhesion molecules, co-stimulatory molecules and proteins required for negative feedback to suppress these responses. Alternatively, an exaggerated response to bacterial stimuli underlies a clinical condition called Systemic Inflammatory Response Syndrome, or sepsis, in which high levels of cytokines and inflammatory mediators become destructive, causing organ failure, cardiovascular shock and/or death.
Sepsis occurs in approximately 780,000 patients in North America annually. Sepsis may develop as a result of infections acquired in the community such as pneumonia, or it may be a complication of the treatment of trauma, cancer or major surgery. Severe sepsis occurs when the body is overwhelmed by the inflammatory response and body organs begin to fail. Up to 120,000 deaths occur annually in the United Stated due to sepsis. Sepsis may also involve pathogenic microorganisms or toxins in the blood (e.g., septicemia), which is a leading cause of death among humans. Gram-negative bacteria are the organisms most commonly associated with such diseases. However, gram-positive bacteria are an increasing cause of infections. Gram-negative and Gram-positive bacteria and their components can all cause sepsis.
The presence of microbial components induces the release of pro-inflammatory cytokines of which tumor necrosis factor-α (TNF-α) is of extreme importance. TNF-α and other pro-inflammatory cytokines can then cause the release of other pro-inflammatory mediators and lead to an inflammatory cascade. Gram-negative sepsis is usually caused by the release of the bacterial outer membrane component, lipopolysaccharide (LPS; also referred to as endotoxin). Endotoxin in the blood, called endotoxemia comes primarily from a bacterial infection, and may be released during treatment with antibiotics. Gram-positive sepsis can be caused by the release of bacterial cell wall components such as lipoteichoic acid (LTA), peptidoglycan (PG)i rhamnose-glucose polymers made by Streptococci, or capsular polysaccharides made by Staphylococci. Bacterial or other non-mammalian DNA that, unlike mammalian DNA, frequently contains unmethylated cytosine-guanosine dimers (CpG DNA) has also been shown to induce septic conditions including the production of TNF-α. Mammalian DNA contains CpG dinucleotides at a much lower frequency, often in a methylated form. In addition to their natural release during bacterial infections, antibiotic treatment can also cause release of the bacterial cell wall components LPS and LTA and probably also bacterial DNA. This can then hinder recovery from infection or even cause sepsis.
In humans, inhalation of the Gram-negative bacterial component lipopolysaccharide (LPS), a TLR4 ligand, results in increased cytokine and chemokine (TNFα, IL1β, IL6, IL8) mRNA and protein expression within 4-6 hr of inhalation. In mutant mice lacking responsiveness to LPS animals do not develop septic shock, demonstrating that the response to endotoxin is sufficient to promote sepsis. Other TLRs exist in humans and can be engaged by other pathogen molecules to drive septic responses. For example, TLR2 is engaged by the signature cell wall-associated molecule lipoteichoic acid (LTA) from Gram positive bacteria, while DNA containing the signature dinucleotide pair unmethylated CpG engages TLR9 and can also stimulate proinflammatory Cytokine production. The nature, duration and intensity of inflammatory/septic responses are considered to involve the interplay between TLR and other receptors, different adaptor molecules such as MyD88, TIRAP/Mal and TRIF, and different signaling pathways. An ideal therapeutic regulator of the inflammatory response would be antagonistic to potentially lethal conditions such as septic shock by interacting with inflammatory signaling pathways but maintain innate immune defenses against bacterial infections, thus sustaining a balance between the protective and destructive components of inflammation.
Cationic host defense peptides (also known as antimicrobial peptides) are crucial molecules in host defense against pathogenic microbe challenge. These peptides have been demonstrated to have a wide range of functions ranging from direct antimicrobial activity to a broad range of immunomodulatory functions. They are widely distributed in nature, existing in organisms from insects to plants to mammals. The family includes defensins, cathelicidins, and histatins. Cathelicidins are small (12 to around 50 amino acids) cationic peptides and are amphipathic in nature with ˜50% hydrophobic residues. Mammalian cathelicidins are synthesized in a precursor pro-form that requires (generally-extracellular) proteolytic processing to generate the mature peptide. The only endogenous cathelicidin in humans is hCAP-18 (SEQ ID NO: 1) which is found at high concentrations in its unprocessed form (hCAP-18) in the granules of neutrophils and is processed upon degranulation and release. It is also produced by epithelial cells and keratinocytes, etc., as the hCAP-18 precursor form, and is found as the processed 37-amino acid peptide SEQ ID NO: 1 in a number of tissues and bodily fluids including gastric juices, saliva, semen, sweat, plasma, airway surface liquid and breast milk.
Cationic peptides are being increasingly recognized as a form of defense against infection, and although the major effects recognized in the scientific and patent literature were the antimicrobial effects (Hancock, R. E. W., and R. Lehrer. 1998. Cationic peptides: a new source of antibiotics. Trends in Biotechnology 16: 82-88.), it is now becoming increasingly clear that they are effectors in other aspects of innate immunity (Hancock, R. E. W. and G. Diamond. 2000. The role of cationic peptides in innate host defenses. Trends in Microbiology 8:402-410.; Hancock, R. E. W. 2001. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infectious Diseases 1 :156-164).
Some cationic peptides have an affinity for binding bacterial products such as LPS and LTA. Such cationic peptides can suppress cytokine production in response to LPS, and to varying extents can prevent lethal shock. However it has not been proven as to whether such effects are due to binding of the peptides to LPS and LTA, or due to a direct interaction of the peptides with host cells. Cationic peptides are induced, in response to challenge by microbes or microbial signaling molecules like LPS, by a regulatory pathway similar to that used by the mammalian immune system (involving Toll receptors and the transcription factor; NFκB). Cationic peptides therefore appear to have a key role in innate immunity. Mutations that affect the induction of antibacterial peptides can reduce survival in response to bacterial challenge. As well, mutations of the Toll pathway of Drosophila that lead to decreased antiftingal peptide expression result in increased susceptibility to lethal fungal infections. In humans, patients with specific granule deficiency syndrome, completely lacking in α-defensins, suffer from frequent and severe bacterial infections. Other evidence includes the inducibility of some peptides by infectious agents, and the very high concentrations of such peptides that have been recorded at sites of inflammation. Cationic peptides may also regulate cell migration, to promote the ability of leukocytes to combat bacterial infections. For example, two human α-defensin peptides, HNP-1 and HNP-2, have been indicated to have direct chemotactic activity for murine and human T cells and monocytes, and human β-defensins appear to act as chemoattractants for immature dendritic cells and memory T cells through interaction with CCR6. Similarly, the porcine cationic peptide PR-39 was found to be chemotactic for neutrophils. It is unclear however as to whether peptides of different structures and compositions share these properties.
The single known cathelicidin from humans, SEQ ID NO: 1, is produced by myeloid precursors, testis, and human keratinocytes during inflammatory disorders and airway epithelium. The characteristic feature of cathelicidin peptides is a high level of sequence identity at the N-terminus prepro regions termed the cathelin domain. Cathelicidin peptides are stored as inactive propeptide precursors that, upon stimulation, are processed into active peptides.
The present invention is based on the seminal discovery that based on patterns of polynucleotide expression regulated by endotoxic lipopolysaccharide, lipoteichoic acid, CpG DNA, or other cellular components (e.g., microbe or their cellular components), and affected by cationic peptides, one can screen for novel compounds that block or reduce sepsis and/or inflammation in a subject. Further, based on the use of cationic peptides as a tool, one can identify selective enhancers of innate immunity that do not trigger the sepsis reaction and that can block/dampen inflammatory and/or septic responses.
Thus, in one embodiment, a method of identifying a polynucleotide or pattern of polynucleotides regulated by one or more sepsis or inflammatory inducing agents and inhibited by a cationic peptide, is provided. The method of the invention includes contacting cells containing polynucleotide or polynucleotides with one or more sepsis or inflammatory inducing agents and contacting the cells containing polynucleotide or polynucleotides with a cationic peptide either simultaneously or immediately thereafter. Differences in expression are detected in the presence and absence of the cationic peptide, and a change in expression, either up- or down-regulation, is indicative of a polynucleotide or pattern of polynucleotides that is regulated by a sepsis or inflammatory inducing agent and inhibited by a cationic peptide. In another aspect the invention provides a polynucleotide or polynucleotides identified by the above method. Examples of sepsis or inflammatory regulatory agents include LPS, LTA or CpG DNA or microbial components (or any combination thereof), or related agents.
In another embodiment, the invention provides a method of identi fying an agent that blocks sepsis or inflammation including combining a polynucleotide identified by the method set forth above with an agent wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression in the absence of the agent and wherein the modulation in expression affects an inflammatory or septic response.
In another embodiment, the invention provides a method of identifying a pattern of polynucleotide expression for inhibition of an inflammatory or septic response by 1) contacting cells with LPS, LTA and/or CpG DNA in the presence or absence of a cationic peptide and 2) detecting a pattern of polynucleotide expression for the cells in the presence and absence of the peptide. The pattern obtained in the presence of the peptide represents inhibition of an inflammatory or septic response. In another aspect the pattern obtained in the presence of the peptide is compared to the pattern of a test compound to identify a compound that provides a. similar pattern. In another aspect the invention provides a compound identified by the foregoing method.
In another embodiment, the invention provides a method of identifying an agent that selectively enhances innate immunity by contacting cells containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein expression of the polynucleotide in the presence of the agent is modulated as compared with expression of the polynucleotide in the absence of the agent and wherein the modulated expression results in enhancement of innate immunity. Preferably, the agent does not stimulate a sepsis reaction in a subject. In one aspect, the agent increases the expression of an anti-inflammatory polynucleotide. Exemplary, but non-limiting anti-inflammatory polynucleotides encode proteins such as IL-1 R antagonist homolog 1 (AI167887), IL-10 R beta (AA486393), IL-10 R alpha (U00672) TNF Receptor member 1B (AA150416), TNF receptor member 5 (H98636), TNF receptor member 11b (AA194983), IK cytokine down-regulator of HLA II (R39227), TGF-B inducible early growth response 2 (AI473938), CD2 (AA927710), IL-19 (NM—013371) or IL-10 (M57627). In one aspect, the agent decreases the expression of polynucleotides encoding proteasome subunits involved in NF-κB activation such as proteasome subunit 26S (D78151). In one aspect, the agent may act as an antagonist of protein kinases. In one aspect, the agent is a peptide selected from SEQ ID NO:4-54.
In another embodiment, the invention provides a method of identifying an agent that selectively suppresses the proinflammatory response of cells containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity. The method includes contacting the cells with microbes, or the TLR ligands and agonists derived from those microbes, and further contacting the cells with an agent of interest, wherein the agent decreases the expression of a proinflammatory gene encoding the polynucleotide as compared with expression of the proinflammatory gene in the absence of the agent. In one aspect, the modulated expression results in suppression of proinflammatory and septic responses. Preferably, the agent does not stimulate a sepsis reaction in a subject. Exemplary, but non-limiting proinflammatory genes include TNFα, TNFAIP2, IL-1β. IL-6, NFKB1 and RELA.
In another embodiment, the invention provides a method of identifying an agent that enhances innate immunity by contacting cells containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein the agent suppresses inflammation and sepsis while increasing the expression of an anti-inflammatory gene encoding the polynucleotide as compared with expression of the anti-inflammatory gene in the absence of the agent and wherein the modulated expression results in enhancement of innate immunity. In one aspect, the agent inhibits the expression of proinflammatory molecules such as TNFα, IL1-β, IL-6, TNFα, TNFAIP2, or the p50 or p65 subunits of transcription factor NFκB. In another aspect, inflammation is induced by a microbe or a microbial ligand acting on a Toll-like receptor such as Toll-like receptor-2, Toll-like receptor-4, or Toll-like receptor-9. Microbial ligands include, but are not limited to a bacterial endotoxin, lipopolysaccharide, lipoteichoic acid or CpG DNA. Exemplary, but non-limiting anti-inflammatory genes include ZNF83, NFKBIA, Q9P188, INVS, DIAPHI, IER3, Q9H640, GBP2, NANS, Q86XN7, Q9H9M1, TNFAIP3, Q96MJ8, Q9BSE2, Q9H753, NTNG1, INHBE, BCL6, CXCL1, EHD1, RELB, HRK, CCL4, SESN2, NAB1, EBI3, DDX21, XBP1, SLURP1, ARS, HDAC10, MEP1A, RAP2C, GYS1, RARRES3, PPY, NFKB1, MTL4_HUMAN, Q9H040, and Q9NUP6.
In another embodiment, the invention provides a method of identifying an agent that is capable of selectively enhancing innate immunity by contacting cells containing one or more genes that encode a polypeptide involved in innate immunity and protection against an infection, with an agent of interest, wherein expression of the one or more genes in the presence of the agent is modulated as compared with expression of the one or more genes in the absence of the agent, and wherein the modulated expression results in enhancement of innate immunity. In one aspect, the invention includes agents identified by the methods. In another aspect, the agent does not stimulate a septic reaction, but does stimulate expression of the one or more genes. Exemplary, but non-limiting genes include any of the genes listed in Table 69. In one embodiment, the one or more genes encode G-coupled protein receptors that initiate signaling from extracellular ligands. Exemplary, but non-limiting genes encoding G-coupled protein receptors that initiate signaling from extracellular ligands include GPR55, GPR6, GPR30, GPCR42, CASR, and EDG2. In another embodiment, the one or more genes encode chemokines or interleukins that attract imrnune cells. Exemplary, but non-limiting genes encoding chemokines or interleukins that attract immune cells include MCP-1, MCP-3, IL-8, CXCL-1, IL-17C, and IL-19. In another embodiment, the one or more genes encode receptors for chemokines. An exemplary, but non-limiting gene encoding a receptor for chemokines includes CCR7. In another embodiment, the one or more genes encode transcription factors that mediate selective gene expression. Exemplary, but non-limiting genes encoding transcription factors that mediate selective gene expression include JAK1, STAT1, ELF1, Q9Y4C1, ETV4, POU1F1, ZNF254, ZNF292, ZNF78L1, HOXD3, and DLX5. In another embodiment, the one or more genes encode tyrosine-protein kinase or tyrosine-protein kinase receptors. Exemplary, but non-limiting genes encoding tyrosine-protein kinase or tyrosine-protein kinase receptors include MAP2K6, NTRK3, PLCG1, EFNA2, and NCK1. In another embodiment, the one or more genes encode adhesion molecules that mediate cell attachment and interaction. Exemplary, but non-limiting adhesion molecules that mediate cell attachment and interaction include the ICAM, NCAM families, and PTPRF. Exemplary, but non-limiting genes encoding adhesion molecules that mediate cell attachment and interaction include ICAM3, NCAM2, and PTPRF. In another embodiment, the one or more genes are involved in actin polymerization or cytoskeletal remodeling. Exemplary, but non-limiting genes involved in actin polymerization or cytoskeletal remodeling include Integrin-α, EPHA4, ARHGAP6, and DST. In another embodiment, the one or more genes encode regulators of transcription factors. Exemplary, but non-limiting genes encoding regulators of transcription factors include TRIP4, GMEB2, GSK3B, ARNT, BACH, ARID3A, HIPK2, POLR2D, TGIF, SSBP3, and FYB. In another embodiment, the one or more genes encode transmembrane receptors and adapters of signaling pathways. Exemplary, but non-limiting genes encoding transmembrane receptors and adapters of signaling pathways include WNT5B, FZD10, TIRAP, and REPS1. In another embodiment, the one or more genes encode proteins involved in antiviral activity. Exemplary, but non-limiting genes encoding proteins involved in antiviral activity include IFNA2, STAT1, MNDA, and IFNA2. In another embodiment, the agent stimulates the JAK-STAT pathway. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of JAK2, STAT1, STAT3, SOCS1, and. IL-19. In another embodiment, the agent stimulates the P13K pathway. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of BACH2/PIK3CB, Akt, CREB, IL-6, and MCP-3. In another embodiment, the agent stimulates the ERK1/2 mitogen activated kinase pathway. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of MAP3K1 and PP2A. In another embodiment, the agent stimulates the p38 mitogen activated kinase pathway. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of MINK1/MAP4K6, MAP2K6, and MAP2K4. In another embodiment, the agent transiently stimulates the NFκB pathway. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of TIRAP, NFκB2 (p52), DUSP14, ICAM3, TRIP4, MMP17, ITGB4, ZNF36, ZNF251, BNIP1, CD226, NRXN1, and TNC. In another embodiment, the agent stimulates the AP-1, JNK or Wnt pathways. In another embodiment, the agent stimulates expression of one or more genes selected from the group consisting of TRIP4, TIRAP, HIPK2, GSK3B, and FZD10.
In another embodiment, the invention provides a method of identifying a pattern of gene expression for identification of an agent that selectively enhances innate immunity by contacting a cell containing one or more genes that encode a polypeptide involved in innate immunity and defense against infections, with an agent of interest, wherein expression of the one or more genes in the presence of the agent is modulated as compared with expression of the one or more genes in the absence of the agent, and wherein the modulated expression results in enhancement of innate immunity. In one embodiment, the modulated expression is a marker of enhancement of innate immunity. In another embodiment, the method further includes determining the efficacy of compounds that enhance innate immunity. In another embodiment, the one or more genes are any gene shown in Table 69. In another embodiment, the one or more genes express IL-8, IL-6, IL-19, CXCL-1, MCP-3, or MCP-1. In another embodiment, the modulated expression occurs in the presence of a bacterial signature molecule. The bacterial signature molecule may be a Toll-like receptor agonist such as bacterial lipopolysaccharide, lipoteichoic acid, and CpG bacterial signature DNA. In another embodiment, the one or more genes are any gene shown in Table 71.
In another embodiment, the invention provides a method of identifying an agent that is capable of selectively enhancing innate immunity in the presence of an infection or bacterial signature molecule by contacting a cell containing one or more genes that encode a polypeptide involved in innate immunity, with an agent of interest in the presence of a bacterial signature molecule, wherein expression of the one or more genes in the presence of the agent and bacterial signature molecule is modulated as compared with expression of the one or more genes in the absence of the agent and bacterial signature molecule, and wherein the modulated expression results in enhancement of innate immunity. In one aspect, the invention includes agents identified by the methods. In another aspect, the bacterial signature molecule is a Toll-like receptor agonist such as bacterial lipopolysaccharide, lipoteichoic acid, and CpG bacterial signature DNA. In another embodiment, the one or genes are any gene shown in Table 71. In another embodiment, the agent does not stimulate a septic reaction. In another embodiment, the agent has anti-endotoxic activity. In another embodiment, the one ore more genes are selected from the group consisting of GPD1, Q8NI35, FEZ2, NRXN1, PLCG1, Q7RTU0, ALDOB, Q9H5P1, SYT11, UBXD2, PROZ, PLAC8, Q96PN6, ASTN2, O60290, FTCD, NFKB2, CTLA4, PSMA1, CCL2, HNF4A, MAFF, FBXO32, TNFα, NPAS2, ICAM3, Q8NC30, Q81UC6, O94940, CGI-117, KDELR1, IFITM1 and COL7A1. In another embodiment, the agent stimulates transient IκBα degradation or transient NFκB subunit p50 translocation. In another embodiment, the method further includes contacting the cell with IL-1β. In another embodiment, the one or more genes encode chemokines. Exemplary, but non-limiting genes that encode chemokines include CCL20, CCL23, IL-6, and MCP-3. In another embodiment, the one or more genes encode cytokine receptors. Exemplary, but non-limiting genes that encode chemokines include EBI3 and IL7R. In another embodiment, the one or more genes encode factors involved in lymphocyte activation. Exemplary, but non-limiting genes that encode factors involved in lymphocyte activation include SLAMF1, CD58, and IL32. In another embodiment, the one or more genes encode regulators of signal transduction. Exemplary, but non-limiting genes that encode regulators of signal transduction include MAP2K2, DUSP5, MAPK8IP3, RIN2, RANBP9, IP3 3-kinase A, BATF, IRAK3, NM1, SP3, RAP2C, PNRC1, NEK1, CHC1, ZNF219, ZNF593, WIF1, PIM2, CD79A, and LATS2. In another embodiment, the one or more genes encode substrate transporters. Exemplary, but non-limiting genes that encode substrate transporters include SLC23A3 and SLC17A5. In another embodiment, the one or more genes encode apoptosis regulators. Exemplary, but non-limiting genes that encode apoptosis regulators include BOK, BIRC3, TNFRSF6, and CASP9. In another embodiment, the one or more genes encode genes associated with plasma membrane. Exemplary, but non-limiting genes that encode genes associated with plasma membrane include STIM1, BPAG1, PTPN4, TRIM36, SDK1, and FNDC5. In another embodiment, the one or more genes encode genes involved in selective ion transport and in mediating selective ion-channels. Exemplary, but non-limiting genes that encode genes involved in selective ion transport and in mediating selective ion-channels include VGCNL1, TRPC5, CACNA1B, KCNA6, KCNJ2, KCNA10, and AQP9. In another embodiment, the one or more genes encode growth modulating genes or genes involved in wound healing. xemplary, but non-limiting genes that encode growth modulating genes or genes involved in wound healing include FGF10 and AREG. In another embodiment, the one or more genes encode inflammatory mediators. Exemplary, but non-limiting genes that encode inflammatory mediators include PTGS2, SOD2, TNFAIP8, and TNIP3. In another embodiment, the method further includes contacting the cell with IL-1β, wherein the agent stimulates the PI3 kinase pathway. In another embodiment, the agent stimulates transient IκBα phosphorylation and p50 nuclear translocation. In another embodiment, the one or more genes encodes a G-protein coupled receptor or a purinergic receptor. An exemplary, but non-limiting purinergic receptor is P2X7. In another embodiment, the agent fturther stimulates phosphorylation of Akt, which stimulates activation of CREB.
In another embodiment, the invention provides a method of identifying an agent that selectively reduces inflammation by contacting a cell containing one or more genes that encode a polypeptide involved in sepsis, with an agent of interest, wherein the agent reduces expression of the one or more genes compared with expression of the one or more genes in the absence of the agent. In another embodiment, the one or more genes are selected from the group consisting of GPD1, Q8NI35, FEZ2, NRXN, PLCG1, Q7RTU0, ALDOB, Q9H5P1, SYT11, UBXD2, PROZ, PLAC8, Q96PN6, ASTN2, O60290, FTCD, NFKB2, CTLA4, PSMA1, CCL2, HNF4A, MAFF, FBXO32, TNF, NPAS2, ICAM3, Q8NC30, Q81UC6, O94940, CGI-117, KDELR1, IFITM1, and COL7A1.
In another embodiment, the invention provides a method of identifying an agent that selectively suppresses sepsis by contacting cells containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein the agent suppresses expression of a proinflammatory gene while maintaining expression of an anti-inflammatory gene encoding the polynucleotide as compared with expression of the anti-inflammatory gene in the absence of the agent. In one aspect, the agent inhibits the expression of proinflammatory molecules such as TNFα, IL1-β, IL-6, TNFα, TNFAIP2, or the p50 or p65 subunits of transcription factor NFκB. In another aspect, inflammation is induced by a microbe or a microbial ligand acting on a Toll-like receptor such as Toll-like receptor-2, Toll-like receptor-4, or Toll-like receptor-9. Microbial ligands include, but are not limited to a bacterial endotoxin, lipopolysaccharide, lipoteichoic acid or CpG DNA. Exemplary, but non-limiting anti-inflammatory genes include ZNF83, NFKBIA, Q9P188, INVS, DIAPH1, IER3, Q9H640, GBP2, NANS, Q86XN7, Q9H9M1, TNFAIP3, Q96MJ8, Q9BSE2, Q9H753, NTNG1, INHBE, BCL6, CXCL1, EHD1, RELB, HRK, CCL4, SESN2, NAB1, EBI3, DDX21, XBP1, SLURP1, ARS, HDAC10, MEP1A, RAP2C, GYS1, RARRES3, PPY, NFKB1, MTL4_HUMAN, Q9H040, and Q9NUP6. Exemplary, but non-limiting proinflammatory genes include LC2A6, SLC4A5, MCL1, Q86XN7, Q9H9M1, Q86UU3, Q8NAA1, C15orf2, TNFRSF5, FACL6, Q8IW99, Q96AU7, PRB4, Q9NWP0, Q8NF24, Q8TEE5, PDE4DIP, NUDT4, DUSP2, LMAN2, RELB, SNF1LK, TNFα, GHRHR, TNFSF6, ENSG00000181873, IRAK2, CKB, CASR, KRTAP4-10, ARHGEF3, CYP3A4, CYP3A7, GPR27, PAX8, GAP43, Q96M75, Q9H568, AGTRL1, C1orf22, EHD1, ADRA1B, SSTR2, SYNE1, ENSG00000139977, PTPRK, O15059, Q9NZ16, N4BP3, KIAA0341, Q8IVT2, Q9NV39, HIP1R, HIP12, KIAA0655, IL-6, TNFAIP2, RCV1, FBLN2, TWIST2, PARD6B, DCK, TULP4, LK10, SPAP1, IBRDC2, JAM2, NRG2, CBARA1, DLG2, PRKCBP1, MGLL, Q9BYE1, MARCKS, Q96N98, Q8NBY1, Q96AF2, Q9BS16, PPP2CA, RAB38, VCAM1, TTTY8, HTR2A, SERPINB10, O75121, Q9BVE1, ZCCHC2, CXCL2, GADD45B, KARS, SCG2, SLC17A2, FLT4, Q9NXT0, Q96L19, BICD1, HCK, Q8N9T8, Q9H978, PPP1R1A, PAX7, EBI3, THRA, SLC16A10, INPP5E, Q9H967, NFKB1, MKL1, SS18L2, TNFRSF9, TNFAIP6, Q9Y2K2, ING5, IL1A, TMH, HDAC4, KPTN, SEC61G, Q9Y484, FRAS1, IER5, Q8N137, Q8NCB8, Q96HQ0, Q9H5P0, TXNRD1, CAV2, SCARB1, MAP3K5, PDHX, TCEB3, C21orf55, MPHOSPH10, PDE8A, TFR2, FARP1, SERPINA1, MYO15A, RABGGTA, KCNMB4, Q9BR02, APOB, MYC, FARP2, TFAP2BL1, Q86U90, Q9H5F8, USH1C, IL-8, SOX2, Q9NVC3, NEIL2, TNIP1, ADRA1D, PCDHB9, Q12987, TNFRSF6, C20orf72, DNAJA3, MAB21 L1, BIRC2, MYST1, CNN3, CXCL3, CD80, CSRP2, RAD51L1, ADARB1, TNFSF8, Q8IW74, UXS1, ENSG00000182364, TNFRSF7, MYBL2, RAB33A, ATIC, CAMK1, CCNT1, KCNE4, BOK, NF2, PDP2, and KIAA1348.
In another embodiment, the invention provides a method of identifying an agent that selectively suppresses sepsis by contacting cells containing a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity, with an agent of interest, wherein the agent induces signaling of the JAK-STAT pathway and suppresses expression of a proinflammatory gene while maintaining expression of an anti-inflammatory gene encoding the polynucleotide as compared with expression of the anti-inflammatory gene in the absence of the agent. In one aspect, the agent inhibits the expression of proinflammatory molecules such as TNFα, NFκB2, IL1-β, IL-6, IL-8, CXCL-1, TNFAIP2, or the p50 or p65 subunits of transcription factor NFκB. In another aspect, inflammation is induced by a microbe or a microbial ligand acting on a Toll-like receptor such as Toll-like receptor-2, Toll-like receptor-4, or Toll-like receptor-9. Microbial ligands include, but are not limited to a bacterial endotoxin, lipopolysaccharide, lipoteichoic acid or CpG DNA. Exemplary, but non-limiting anti-inflammatory genes include one or more genes listed in Table 69. Exemplary, but non-limiting proinflammatory genes include one or more genes listed in Table 72.
In another embodiment, the invention provides a method of identifying a pattern of polynucleotide expression for identification of a compound that selectively enhances innate immunity. The invention includes detecting a pattern of polynucleotide expression for cells contacted in the presence and absence of a cationic peptide, wherein the pattern in the presence of the peptide represents stimulation of innate immunity; detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide, is indicative of a compound that enhances innate immunity.
In another embodiment, the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by an increase in polynucleotide expression of at least 2 polynucleotides in Table 50, 51 and or 52, as compared to a non-infected subject. Also included is a polynucleotide expression pattern obtained by any of the methods described above.
In another aspect a cationic peptide that is an antagonist of CXCR-4 is provided. In still another aspect, a method of identifying a cationic peptide that is an antagonist of CXCR-4 by contacting T cells with SDF-1 in the presence of absence of a test peptide and measuring chemotaxis is provided. A decrease in chemotaxis in the presence of the test peptide is indicative of a peptide that is an antagonist of CXCR-4. Cationic peptide also acts to reduce the expression of the SDF-1 receptor polynucleotide (NM—012428).
In all of the above described methods, the compounds or agents of the invention include but are not limited to peptides, cationic peptides, peptidomimetics, chemical compounds, polypeptides, nucleic acid molecules and the like.
In still another aspect the invention provides an isolated cationic peptide. An isolated cationic peptide of the invention is represented by one of the following general formulas and the single letter amino acid code:
Additionally, in another aspect the invention provides isolated cationic peptides
Also provided are nucleic acid sequences encoding the cationic peptides of the invention, vectors including such polynucleotides and host cells containing the vectors.
In another embodiment, the invention provides methods for stimulating or enhancing innate immunity in a subject comprising administering to the subject a peptide of the invention, for example, peptides set forth in SEQ ID NO:1-4, 11, 18, 25, 32, 39, 46, 53 or 54. As shown in the Examples herein, innate immunity can be evidenced by monocyte activation, proliferation, differentiation, or MAP kinase pathway activation just by way of example. In one aspect, the method includes further administering a serum factor such as GM-CSF to the subject. The subject is preferably any mammal and more particularly a human subject.
In another embodiment, the invention provides a method of stimulating innate immunity in a subject having or at risk of having an infection including administering to the subject a sub-optimal concentration of an antibiotic in combination with a peptide of the invention. In one aspect, the peptide is SEQ ID NO:1 or SEQ ID NO:7.
In all of the above described embodiments, the methods may be performed ex vivo.
inhibition (⊥), or suppression (→). Other abbreviations used are phosphorylation (P) and ubiquitination (
).
The present invention provides novel cationic peptides, characterized by a group of generic formulas (SEQ ID NO: 4, 11, 18, 25, 32, 39, 46), which have ability to modulate (e.g., up- and/or down regulate) polynucleotide expression, thereby regulating sepsis and inflammatory responses and/or innate immunity.
“Innate immunity” as used herein refers to the natural ability of an organism to defend itself against invasions by pathogens. Pathogens or microbes as used herein, may include, but are not limited to bacteria, fungi, parasite, and viruses. Innate immunity is contrasted with acquired/adaptive immunity in which the organism develops a defensive mechanism based substantially on antibodies and/or immune lymphocytes that is characterized by specificity, amplifiability and self vs. non-self dsicrimination. With innate immunity, broad, nonspecific immunity is provided and there is no immunologic memory of prior exposure. The hallmarks of innate immunity are effectiveness against a broad variety of potential pathogens, independence of prior exposure to a pathogen, and immediate effectiveness (in contrast to the specific immune response which takes days to weeks to be elicited). In addition, innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like.
As used herein, the term “cationic peptide” refers to a sequence of amino acids from about 5 to about 50 amino acids in length. In one aspect, the cationic peptide of the invention is from about 10 to about 35 amino acids in length. A peptide is “cationic” if it possesses sufficient positively charged amino acids to have a pI greater than about 9.0, where pl (isoelectric point)=pH when the net charge of the peptide is neutral. Typically, at least two of the amino acid residues of the cationic peptide will be positively charged, for example, lysine or arginine. “Positively charged” refers to the side chains of the amino acid residues which have a net positive charge at pH 7.0. Examples of naturally occurring cationic antimicrobial peptides which can be recombinantly produced according to the invention include defensins, cathelicidins, magainins, melittin, and cecropins, bactenecins, indolicidins, polyphemusins, tachyplesins, and analogs thereof. A variety of organisms make cationic peptides, molecules used as part of a non-specific defense mechanism against microorganisms. When isolated, these peptides are toxic to a wide variety of microorganisms, including bacteria, fuingi, and certain enveloped viruses. While cationic peptides act against many.pathogens, notable exceptions and varying degrees of toxicity exist. However this patent reveals additional cationic peptides with no toxicity towards microorganisms but an ability to protect against infections through stimulation of innate immunity, and this invention is not limited to cationic peptides with antimicrobial activity. In fact, many peptides useful in the present invention do not have antimicrobial activity.
Cationic peptides known in the art include for example, the human cathelicidin LL-37, and the bovine neutrophil peptide indolicidin and the bovine variant of bactenecin, Bac2A.
Although SEQ ID NO: 1 is often defined as an antimicrobial (direct killing) peptide it has been suggested that at physiological salt conditions, this peptide is not antimicrobial at the concentrations (1-5 μg/ml) normally found in adults at mucosal surfaces (Bowdish, D. M. E., D. J. Davidson, Y. E. Lau, K. Lee, M. G. Scott, and R. E. W. Hancock. 2005. Impact of LL-37 on anti-infective immunity. J. Leukocyte Biol. 77:451-459). Moreover under these conditions and at these concentrations, SEQ ID NO: 1 exhibits a variety of immunomodulatory functions. This could help to explain why SEQ ID NO: 1 administration can protect mice against certain bacterial infections, due to its ability to modulate immunity. SEQ ID NO: 1 is also able to protect mice and rats against endotoxemialsepsis induced by pure LPS indicating that SEQ ID NO: 1 can suppress potentially harmful pro-inflammatory responses.
Accordingly, the present invention provides evidence that human host defense peptide SEQ ID NO: 1 has potent anti-endotoxin properties, at very low (≦1 μg/ml) concentrations and physiological salt conditions reflecting those found in vivo. It is further demonstrated here that SEQ ID NO: 1 had a general anti-inflammatory effect on TLR stimulation, inhibiting pro-inflammatory cytokine release from human monocytic cells stimulated with TLR2, TLR4 and TLR9 agonists. The suppression of inflammatory responses by SEQ ID NO: 1 in LPS-stimulated cells is selective, as SEQ ID NO: 1 does not block the expression of certain (pro-inflammatory) genes required for cell recruitment and movement, yet abrogates pro-inflammatory cytokine responses that can potentially lead to sepsis. The anti-inflammatory activity of SEQ ID NO: 1 is apparently mediated through a diversity of mechanisms.
In innate immunity, the immune response is not dependent upon antigens. The innate immunity process may include the production of secretory molecules and cellular components as set forth above. In innate immunity, the pathogens are recognized by receptors (for example, Toll-like receptors) that have broad specificity, are capable of recognizing many pathogens, and are encoded in the germline. These Toll-like receptors have broad specificity and are capable of recognizing many pathogens. When cationic peptides are present in the immune response, they aid in the host response to pathogens. This change in the immune response induces the release of chemokines, which promote the recruitment of immune cells to the site of infection.
Chemokines, or chemoattractant cytokines, are a subgroup of immune factors that mediate chemotactic and other pro-inflammatory phenomena (See, Schall, 1991, Cytokine 3:165-183). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into two subgroups, α which have two N-terminal cysteines separated by a single amino acid (CxC) and β which have two adjacent cysteines at the N terminus (CC). RANTES, MIP-1α and MIP-1β are members of the β subgroup (reviewed by Horuk, R., 1994, Trends Pharmacol. Sci, 15:159-165; Murphy, P. M., 1994, Annu. Rev. Immunol., 12:593-633). The amino terminus of the β chemokines RANTES, MCP-1, and MCP-3 have been implicated in the mediation of cell migration and inflammation induced by these chemokines. This involvement is suggested by the observation that the deletion of the amino terminal 8 residues of MCP-1, amino terminal 9 residues of MCP-3, and amino terminal 8 residues of RANTES and the addition of a methionine to the amino terminus of RANTES, antagonize the chemotaxis, calcium mobilization and/or enzyme release stimulated by their native counterparts (Gong et al., 1996 J. Biol. Chem. 271:10521-10527; Proudfoot et al., 1996 J Biol. Chem. 271:2599-2603). Additionally, α chemokine-like chemotactic activity has been introduced into MCP-1 via a double mutation of Tyr 28 and Arg 30 to leucine and valine, respectively, indicating that internal regions of this protein also play a role in regulating chemotactic activity (Beall et al., 1992, J. Biol. Chem. 267:3455-3459).
The monomeric forms of all chemokines characterized thus far share significant structural homology, although the quaternary structures of α and β groups are distinct. While the monomeric structures of the β and a chemokines are very similar, the dimeric structures of the two groups are completely different. An additional chemokine, lymphotactin, which has only one N terminal cysteine has also been identified and may represent an additional subgroup (γ) of chemokines (Yoshida et al., 1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science 266:1395-1399).
Receptors for chemokines belong to the large family of G-protein coupled, 7 transmembrane domain receptors (GCR's) (See, reviews by Horuk, R., 1994, Trends Pharmacol. Sci. 15:159-165; and Murphy, P. M., 1994, Annu. Rev. Immunol. 12:593-633). Competition binding and cross-desensitization studies have shown that chemokine receptors exhibit considerable promiscuity in ligand binding. Examples demonstrating the promiscuity among β chemokine receptors include: CC CKR-1, which binds RANTES and MIP-1α (Neote et al., 1993, Cell 72: 415-425), CC CKR-4, which binds RANTES, MIP-1α, and MCP-1 (Power et al., 1995, J. BioL. Chem. 270:19495-19500), and CC CKR-5, which binds RANTES, MIP-1α, and MIP-1β (Alkhatib et al., 1996, Science, in press and Dragic et al., 1996, Nature 381:667-674). Erythrocytes possess a receptor (known as the Duffy antigen) which binds both α and β chemokines (Horuk et al., 1994, J. Biol. Chem. 269:17730-17733; Neote et al., 1994, Blood 84:44-52; and Neote et al., 1993, J. Biol. Chem. 268:12247-12249). Thus the sequence and structural homologies evident among chemokines and their receptors allows some overlap in receptor-ligand interactions.
In one aspect, the present invention provides the use of compounds including peptides of the invention to reduce sepsis and inflammatory responses by acting directly on host cells. In this aspect, a method of identification of a polynucleotide or polynucleotides that are regulated by one or more sepsis or inflammatory inducing agents is provided, where the regulation is altered by a cationic peptide. Such sepsis or inflammatory inducing agents include, but are not limited to endotoxic lipopolysaccharide (LPS), lipoteichoic acid (LTA) and/or CpG DNA or intact bacteria or other bacterial components. The identification is performed by contacting the polynucleotide or polynucleotides with the sepsis or inflammatory inducing agents and further contacting with a cationic peptide either simultaneously or immediately after. The expression of the polynucleotide in the presence and absence of the cationic peptide is observed and a change in expression is indicative of a polynucleotide or pattern of polynucleotides that is regulated by a sepsis or inflammatory inducing agent and inhibited by a cationic peptide. In another aspect, the invention provides a polynucleotide identified by the method.
Once identified, such polynucleotides will be useful in methods of screening for compounds that can block sepsis or inflammation by affecting the expression of the polynucleotide. Such an effect on expression may be either up regulation or down regulation of expression. By identifying compounds that do not trigger the sepsis reaction and that can block or dampen inflammatory or septic responses, the present invention also presents a method of identifying enhancers of innate immunity. Additionally, the present invention provides compounds that are used or identified in the above methods.
Candidate compounds are obtained from a wide variety of sources including libraries of synthetic. or natural compounds. For example, numerous means are available for, random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs. Candidate agents are also found among biomolecules including, but not limited to: peptides, peptidiomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives, structural analogs or combinations thereof.
Incubating components of a screening assay includes conditions which allow contact between the test compound and the polynucleotides of interest. Contacting includes in solution and in solid phase, in a cell, or on a cell surface. The test compound may optionally be a combinatorial library for screening a plurality of compounds. Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a compound.
Generally, in the methods of the invention, a cationic peptide is utilized to detect and locate a polynucleotide that is essential in the process of sepsis or inflammation. Once identified, a pattern of polynucleotide expression may be obtained by observing the expression in the presence and absence of the cationic peptide. The pattern obtained in the presence of the cationic peptide is then useful in identifying additional compounds that can inhibit expression of the polynucleotide and therefore block sepsis or inflammation. It is well known to one of skill in the art that non-peptidic chemicals and peptidomimetics can mimic the ability of peptides to bind to receptors and enzyme binding sites and thus can be used to block or stimulate biological reactions. Where an additional compound of interest provides a pattern of polynucleotide expression similar to that of the expression in the presence of a cationic peptide, that compound is also useful in the modulation of sepsis or an innate immune response. In this manner, the cationic peptides of the invention, which are known inhibitors of sepsis and inflammation and enhancers of innate immunity are useful as tools in the identification of additional compounds that inhibit sepsis and inflammation and enhance innate immunity.
As can be seen in the Examples below, peptides of the invention have a widespread ability to reduce the expression of polynucleotides regulated by LPS. High levels of endotoxin in the blood-are responsible for many of the symptoms seen during a serious infection or inflammation such as fever and an elevated white blood cell count. Endotoxin is a component of the cell wall of Gram-negative bacteria and is a potent trigger of the pathophysiology of sepsis. The basic mechanisms of inflammation and sepsis are related. In Example 1, polynucleotide arrays were utilized to determine the effect of cationic peptides on the transcriptional response of epithelial cells. Specifically, the effects on over 14,000 different specific polynucleotide probes induced by LPS were observed. The tables show the changes seen with cells treated with peptide compared to control cells. The resulting data indicated that the peptides have the ability to reduce the expression of polynucleotides induced by LPS.
Example 2, similarly, shows that peptides of the invention are capable of neutralizing the stimulation of immune cells by Gram positive and Gram negative bacterial products. Additionally, it is noted that certain pro-inflammatory polynucleotides are down-regulated by cationic peptides, as set forth in table 24 such as TLR1 (AI339155), TLR2 (T57791), TLR5 (N41021), TNF receptor-associated factor 2 (T55353), TNF receptor-associated factor 3 (AA504259), TNF receptor superfamily, member 12 (W71984), TNF receptor superfamily, member 17 (AA987627), small inducible cytokine subfamily B, member 6 (AI889554), IL-12R beta 2 (AA977194), IL-18 receptor 1 (AA482489), while anti-inflammatory polynucleotides are up-regulated by cationic peptides, as seen in table 25 such as IL-1 R antagonist homolog 1 (AI167887), IL-10 R beta (AA486393), TNF Receptor member 1B (AA150416), TNF receptor member 5 (H98636), TNF receptor member 11b (AA194983), IK cytokine down-regulator of HLA II (R39227), TGF-B inducible early growth response 2 (AI473938), or CD2 (AA927710). The relevance and application of these results are confirmed by an in vivo application to mice.
In another aspect, the invention provides a method of identifying an agent that enhances innate immunity. In-the method, a polynucleotide or polynucleotides that encode a polypeptide involved in innate immunity is contacted with an agent of interest. Expression of the polynucleotide is determined, both in the presence and absence of the agent. The expression is compared and of the specific modulation of expression was indicative of an enhancement of innate immunity. In another aspect, the agent does not stimulate a septic reaction as revealed by the lack of upregulation of the pro-inflammatory cytokine TNF-α. In still another aspect the agent reduces or blocks the inflammatory or septic response. In yet another aspect, the agent reduces the expression of TNF-αand/or interleukins including, but not limited to, IL-1β, IL-6, IL-12 p40, IL-12 p70, and IL-8.
In another aspect, the invention provides methods of direct polynucleotide regulation by cationic peptides and the use of compounds including cationic peptides to stimulate elements of innate immunity. In this aspect, the invention provides a method of identification of a pattern of polynucleotide expression for identification of a compound that enhances innate immunity. In the method of the invention, an initial detection of a pattern of polynucleotide expression for cells contacted in the presence and absence of a cationic peptide is made. The pattern resulting from polynucleotide expression in the presence of the peptide represents stimulation of innate immunity. A pattern of polynucleotide expression is then detected in the presence of a test compound, where a resulting pattern with the test compound that is similar to the pattern observed in the presence of the cationic peptide is indicative of a compound that enhances innate immunity. In another aspect, the invention provides compounds that are identified in the above methods. In another aspect, the compound of the invention stimulates chemokine or chemokine receptor expression. Chemokine or chemokine receptors may include, but are not limited to CXCR4, CXCR1, CXCR2, CCR2, CCR4, CCR5, CCR6, MIP-1 alpha, MDC, MIP-3 alpha, MCP-1, MCP-2, MCP-3, MCP4, MCP-5, anid RANTES. In still another aspect, the compound is a peptide, peptidomimetic, chemical compound, or a nucleic acid molecule.
In still another aspect the polynucleotide expression pattern includes expression of pro-inflammatory polynucleotides. Such pro-inflammatory polynucleotides may include, but are not limited to, ring finger protein 10 (D87451), serine/threonine protein kinase MASK (AB040057), KIAA0912 protein (AB020719), KIAA0239 protein (D87076), RAP1, GTPase activating protein 1 (M64788), FEM-1-like death receptor binding protein (AB007856), cathepsin S (M90696), hypothetical protein FLJ20308 (AK000315), pim-1 oncogene (M54915), proteasome subunit beta type 5 (D2901 1), KIAA0239 protein (D87076), mucin 5 subtype B tracheobronchial (AJ001403), cAMP response element-binding protein CREBPa, integrin alpha M (J03925), Rho-associated kinase 2 (NM—004850), PTD017 protein (AL050361) unknown genes (AK00143, AK034348, AL049250, AL161991, AL031983) and any combination thereof In still another aspect the polynucleotide expression pattern includes expression of cell surface receptors that may include but is not limited to retinoic acid receptor (X06614), G protein-coupled receptors (Z94155, X81892, U52219, U22491, AF015257, U66579) chemokine (C-C motif) receptor 7 (L31584), tumor necrosis factor receptor superfamily member 17 (Z29575), interferon gamma receptor 2 (U05875), cytokine receptor-like factor 1 (AF059293), class I cytokine receptor (AF053004), coagulation factor II (thrombin) receptor-like 2 (U9297 1), leukemia inhibitory factor receptor (NM—002310), interferon gamma receptor 1 (AL050337).
In Example 4 it can be seen that the cationic peptides of the invention alter polynucleotide expression in macrophage and epithelial cells. The results of this example-show that pro-inflammatory polynucleotides are down-regulated by cationic peptides (Table 24) whereas anti-inflammatory polynucleotides are up-regulated by cationic peptides (Table 25).
It is shown below, for example, in tables 1-15, that cationic peptides can neutralize the host response to the signaling molecules of infectious agents as well as modify the transcriptional responses of host cells, mainly by down-regulating the pro-inflammatory response and/or up-regulating the anti-inflammatory response. Example 5 shows that the cationic peptides can aid in the host response to pathogens by inducing the release of chemokines, which promote the recruitment of immune cells to the site of infection. The results are confirmed by an in vivo application to mice.
It is seen from the examples below that cationic peptides have a substantial influence on the host response to pathogens in that they assist in regulation of the host immune response by inducing selective pro-inflammatory responses that for example promote the recruitment of immune cells to the site of infection but not inducing potentially harmful pro-inflammatory cytokines. Sepsis appears to be caused in part by an overwhelming pro-inflammatory response to infectious agents. Peptides can aid the host in a “balanced” response to pathogens by inducing an anti-inflammatory response and suppressing certain potentially harmful pro-inflammatory responses.
In Example 7, the activation of selected MAP kinases was examined, to study the basic mechanisms behind the effects of interaction of cationic peptides with cells. Macrophages activate MEK/ERK kinases in response to bacterial infection. MEK is a MAP kinase kinase that when activated, phosphorylates the downstream kinase ERK (extracellular regulated kinase), which then dimerizes and translocates to the nucleus where it activates transcription factors such as Elk-1 to modify polynucleotide expression. MEK/ERK kinases have been shown to impair replication of Salmonella within macrophages. Signal transduction by MEK kinase and NADPH oxidase may play an important role in innate host defense against intracellular pathogens. By affecting the MAP kinases as shown below the cationic peptides have an effect on bacterial infection. The cationic peptides can directly affect kinases. Table 21 demonstrates but is not limited to MAP kinase polynucleotide expression changes in response to peptide. The kinases include MAP kinase kinase 6 (H070920), MAP kinase kinase 5 (W69649), MAP kinase 7 (H39192), MAP kinase 12 (AI936909) and MAP kinase-activated protein kinase 3 (W6828 1).
In another method, the methods of the invention may be used in combination, to identify an agent with multiple characteristics, i.e. a peptide with anti-inflammatory/anti-sepsis activity, and the ability to enhance innate immunity, in part by inducing chemokines in vivo.
In another aspect, the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by an increase in polynucleotide expression of at least 2 polynucleotides in Table 55 as compared to a non-infected subject. In another aspect the invention provides a method for inferring a state of infection in a mammalian subject from a nucleic acid sample of the subject by identifying in the nucleic acid sample a polynucleotide expression pattern exemplified by a polynucleotide expression of at least 2 polynucleotides in Table 56 or Table 57 as compared to a non-infected subject. In one aspect of the invention, the state of infection is due to infectious agents or signaling molecules derived therefrom, such as, but not limited to, Gram negative bacteria and Gram positive bacteria, viral, fungal or parasitic agents. In still another aspect the invention provides a polynucleotide expression pattern of a subject having a state of infection identified by the above method. Once identified, such polynucleotides will be useful in methods of diagnosis of a condition associated with the activity or presence of such infectious agents or signaling molecules.
Example 10 below demonstrates this aspect of the invention. Specifically, table 61 demonstrates that both MEK and the NADPH oxidase inhibitors can limit bacterial replication (infection of IFN-γ-primed macrophages by S. typhimurium triggers a MEK kinase). This is an example of how bacterial survival can be impacted by changing host cell signaling molecules.
In still another aspect of the invention, compounds are presented that inhibit stromal derived factor-1 (SDF-1) induced chemotaxis of T cells. Compounds are also presented which decrease expression of SDF-1 receptor. Such compounds also may act as an antagonist or inhibitor of CXCR-4. In one aspect the invention provides a cationic peptide that is an antagonist of CXCR-4. In another aspect the invention provides a method of identifying a cationic peptide that is an antagonist of CXCR-4. The method includes contacting T cells with SDF-1 in the presence of absence of a test peptide and measuring chemotaxis. A decrease in chemotaxis in the presence of the test peptide is then indicative of a peptide that is an antagonist of CXCR-4. Such compounds and methods are useful in therapeutic applications in HIV patients. These types of compounds and the utility thereof is demonstrated, for example, in Example 11 (see also Tables 62, 63). In that example, cationic peptides are shown to inhibit cell migration and therefore antiviral activity.
In one embodiment, the invention provides an isolated cationic peptides having an amino acid sequence of the general formula (Formula A): X1X2X3IX4PX4IPX5X2X1 (SEQ ID NO: 4), wherein X1 is one or two of R, L or K, X2 is one of C, S or A, X3 is one of R or P, X4 is one of A or V and X5 is one of V or W. Examples of-the peptides of the invention include, but are not limited to: LLCRIVPVIPWCK (SEQ ID NO: 5), LRCPIAPVIPVCKK (SEQ ID NO: 6), KSRIVPAIPVSLL (SEQ ID NO: 7), KKSPIAPAIPWSR (SEQ ID NO: 8), RRARIVPAIPVARR (SEQ ID NO: 9) and LSRIAPAIPWAKL (SEQ ID NO: 10).
In another embodiment, the invention provides an isolated linear cationic peptide having an amino acid sequence of the general formula (Formula B):
X1LX2X3KX4X2X5X3PX3X1 (SEQ ID NO: 11), wherein XI is one or two of D, E, S, T or N, X2 is one or two of P, G or D, X3 is one of G, A, V, L, I or Y, X4 is one of R, K or H and X5 is one of S, T, C, M or R. Examples of the peptides of the invention include, but are not limited to:
In another embodiment, the invention provides an isolated linear cationic peptide having an amino acid sequence of the general formula (Formula C): X1X2X3X4WX4WX4X5K (SEQ ID NO: 18) (this formula includes CP12a and CP12d), wherein X1 is one to four chosen from A, P or R, X2 is one or two aromatic amino acids (F, Y and W), X3 is one of P or K, X4 is one, two or none chosen from A, P, Y or W and X5 is one to three chosen from R or P. Examples of the peptides of the invention include, but are not limited to:
In another embodiment, the invention provides an isolated hexadecameric cationic peptide having an amino acid sequence of the general formula (Formula D):
X1X2X3X4X1VX3X4RGX4X3X4X1X3X1 (SEQ ID NO: 25) wherein X1 is one or two of R or K, X2 is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is C, S, M, D or A and X4 is F, I, V, M or R. Examples of the peptides of the invention include, but are not limited to: RRMCIKVCVRGVCRRKCRK (SEQ ID NO: 26), KRSCFKVSMRGVSRRRCK (SEQ ID NO: 27), KKDAIKKVDIRGMDMRRAR (SEQ ID NO: 28), RKMVKVDVRGIMIRKDRR (SEQ ID NO: 29), KQCVKVAMRGMALRRCK (SEQ ID NO: 30) and RREAIRRVAMRGRDMKRMRR (SEQ ID NO: 31).
In still another embodiment, the invention provides an isolated hexadecameric cationic peptide having an amino acid sequence of the general formula (Formula E): X1X2X3X4X1VX5X4RGX4X5X4X1X3X1 (SEQ ID NO: 32), wherein XI is one or two of R or K, X2 is a polar or charged amino acid (S, T, M, N, Q, D, E, K, R and H), X3 is one of C, S, M, D or A, X4 is one of F, I, V, M or R and X5 is one of A, I, S, M, D or R. Examples of the peptides of the invention include, but are not limited to: RTCVKRVAMRGIIRKRCR (SEQ ID NO: 33), KKQMMKRVDVRGISVKRKR (SEQ ID NO: 34), KESIKVIIRGMMVRMKK (SEQ ID NO: 35), RRDCRRVMVRGIDIKAK (SEQ ID NO: 36), KRTAIKKVSRRGMSVKARR (SEQ ID NO: 37) and RHCIRRVSMRGIIMRRCK (SEQ ID NO: 38).
In another embodiment, the invention provides an isolated longer cationic peptide having an amino acid sequence of the general formula (Formula F): KX1KX2FX2KMLMX2ALKKX3 (SEQ ID NO: 39), wherein X1 is a polar amino acid (C, S, T, M, N and Q); X2 is one of A, L, S or K and X3 is 1-17 amino acids chosen from G, A, V, L, I, P, F, S, T, K and H. Examples of the peptides of the invention include, but are not limited to:
In yet another embodiment, the invention provides an isolated longer cationic peptide having an amino acid sequence of the general formula (Formula G): KWKX2X1X1X2X2X1X2X2XX1X2X2IFHTALKPISS (SEQ ID NO: 46), wherein X1 is a hydrophobic amino acid and X2 is a hydrophilic amino acid. Examples of the peptides of the invention include, but are not limited to:
In still another embodiment, the invention provides an isolated cationic peptide having an amino acid sequence of the formula: KWKSFLRTFKSPVRTVFHTALKPISS (SEQ ID NO: 53) or KWKSYAHTIMSPVRLVFHTALKPISS (SEQ ID NO: 54).
The termn “isolated” as used herein refers to a peptide that is substantially free of other proteins, lipids, and nucleic acids (e.g., cellular components with which an in vivo-produced peptide would naturally be associated). Preferably, the peptide is at least 70%, 80%, or most preferably 90% pure by weight and when assessed in exclusion of counter-ion.
The invention also includes analogs, derivatives, conservative variations, and cationic peptide variants of the enumerated polypeptides, provided that the analog, derivative, conservative variation, or variant has a detectable activity in which it enhances innate immunity or has anti-inflammatory activity. It is not necessary that the analog, derivative, variation, or variant have activity identical to the activity of the peptide from which the analog, derivative, conservative variation, or variant is derived.
A cationic peptide “variant” is a peptide that is an altered form of a referenced cationic peptide. For example, the term “variant” includes a cationic peptide in which at least one amino acid of a reference peptide is substituted in an expression library. The term “reference” peptide means any of the cationic peptides of the invention (e.g. as defined in the above formulas), from which a variant, derivative, analog, or conservative variation is derived. Included within the term “derivative” is a hybrid peptide that includes at least a portion of each of two cationic peptides (e.g., 30-80% of each of two cationic peptides). Also included are peptides in which one or more amino acids are deleted from the sequence of a peptide enumerated herein, provided that the derivative has activity in which it enhances innate immunity or has anti-inflammatory activity. This can lead to the development of a smaller active molecule which would also have utility. For example, amino or carboxy terminal amino acids which may not be required for enhancing innate immunity or anti-inflammatory activity of a peptide can be removed. Likewise, additional derivatives can be produced by adding one or a few (e.g., less than 5) amino acids to a cationic peptide without completely inhibiting the activity of the peptide. In addition, C-terminal derivatives, e.g., C-terminal methyl esters, and N-terminal derivatives can be produced and are encompassed by the invention. Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein remains. Also included is the reverse sequence of a peptide encompassed by the general formulas set forth above. Additionally, an amino acid of “D” configuration may be substituted with an amino acid of “L” configuration and vice versa. Alternatively the peptide may be cyclized chemically or by the addition of two or more cysteine residues within the sequence and oxidized to form disulphide bonds.
The invention also includes peptides that are conservative variations of those peptides exemplified herein. The term “conservative variation” as used herein denotes a polypeptide in which at least one amino acid is replaced by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. The term “conservative variation” also encompasses a peptide having a substituted amino acid in place of an unsubstituted parent amino acid. Such substituted amino acids may include amino acids that have been methylated or amidated. Other substitutions will be known to those of skill in the art. In one aspect, antibodies raised to a substituted polypeptide will also specifically bind the unsubstituted polypeptide.
Peptides of the invention can be synthesized by commonly used methods such as those that include t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise synthesis in which a single amino acid is added at each step starting from the C-terminus of the peptide (See, Coligan, et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods such as those described by Merrifield, J. Am. Chem. Soc., 85:2149, 1962) and Stewart and Young, Solid Phase Peptides Synthesis, Freeman, San Francisco, 1969, pp. 27-62) using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about ¼-1 hours at 0° C. After evaporation of the reagents, the peptides are extracted from the polymer with a 1% acetic acid solution, which is then lyophilized to yield the crude material. The peptides can be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column eluate yield homogeneous peptide, which can then be characterized by standard techniques such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, or measuring solubility. If desired, the peptides can be quantitated by the solid phase Edman degradation.
The invention also includes isolated nucleic acids (e.g., DNA, cDNA, or RNA) encoding the peptides of the invention. Included are nucleic acids that encode analogs, mutants, conservative variations, and variants of the peptides described herein. The term “isolated” as used herein refers to a nucleic acid that is substantially free of proteins, lipids, and other nucleic acids with which an in vivo-produced nucleic acids naturally associated. Preferably, the nucleic acid is at least 70%, 80%, or preferably 90% pure by weight, and conventional methods for synthesizing nucleic acids in vitro can be used in lieu of in vivo methods. As used herein, “nucleic acid” refers to a polymer of deoxyribo-nucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger genetic construct (e.g., by operably linking a promoter to a nucleic acid encoding a peptide of the invention). Numerous genetic constructs (e.g., plasmids and other expression vectors) are known in the art and can be used to produce the peptides of the invention in cell-free systems or prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian) cells. By taking into account the degeneracy of the genetic code, one of ordinary skill in the art can readily synthesize nucleic acids encoding the polypeptides of the invention. The nucleic acids of the invention can readily be used in conventional molecular biology methods to produce the peptides of the invention.
DNA encoding the cationic peptides of the invention can be inserted into an “expression vector.” The term “expression vector” refers to a genetic construct such as a plasmid, virus or other vehicle known in the art that can be engineered to contain a nucleic acid encoding a polypeptide of the invention. Such expression vectors are preferably plasmids that contain a promoter sequence that facilitates transcription of the inserted genetic sequence in a host cell. The expression vector typically contains an origin of replication, and a promoter, as well as polynucleotides that allow phenotypic selection of the transformed cells (e.g., an antibiotic resistance polynucleotide). Various promoters, including inducible and constitutive promoters, can be utilized in the invention. Typically, the expression vector contains a replicon site and control sequences that are derived from a species compatible with the host cell.
Transformation or transfection of a recipient with a nucleic acid of the invention can be carried out using conventional techniques well known to those skilled in the art. For example, where the host cell is E. coli, competent cells that are capable of DNA uptake can be prepared using the CaCI2, MgCl2 or RbCl methods known in the art. Alternatively, physical means, such as electroporation or microinjection can be used. Electroporation allows transfer of a nucleic acid into a cell by high voltage electric impulse. Additionally, nucleic acids can be introduced into host cells by protoplast fusion, using methods well known in the art. Suitable methods for transforming eukaryotic cells, such as electroporation and lipofection, also are known.
“Host cells” or “Recipient cells” encompassed by of the invention are any cells in which the nucleic acids of the invention can be used to express the polypeptides of the invention. The term also includes any progeny of a recipient or host cell. Preferred recipient or host cells of the invention include E. coli, S. aureus and P. aeruginosa, although other Gram-negative and Gram-positive bacterial, fungal and mammalian cells and organ isms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.
The cationic peptide polynucleotide sequence used according to the method of the invention can be isolated from an organism or syrithesized in the laboratory. Specific DNA sequences encoding the cationic peptide of interest can be obtained by: 1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the cationic peptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually fo)rmed which is generally referred to as cDNA.
The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known. In the present invention, the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons which are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation. In addition, virtually any peptide can be synthesized, including those encoding natural cationic peptides, variants of the same, or synthetic peptides.
When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the cationic peptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay, et al., Nuc. Acid Res., 11:2325, 1983).
The peptide of the invention can be administered parenterally by injection or by gradual infusion over time. Preferably the peptide is administered in a therapeutically effective amount to enhance or to stimulate an innate immune response. Innate immunity has been described herein, however examples of indicators of stimulation of innate immunity include but are not limited to monocyte activation, proliferation, differentiation or MAP kinase pathway activation.
The peptide can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. Preferred methods for delivery of the peptide include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation. Other methods of administration will be known to those skilled in the art.
Preparations for parenteral administration of a peptide of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
In one embodiment, the invention provides a method for synergistic therapy. For example, peptides as described herein can be used in synergistic combination with sub-inhibitory concentrations of antibiotics. Examples of particular classes of antibiotics useful for synergistic therapy with the peptides of the invention include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., ciprofloxacin), carbapenems (e.g., imipenem), tetracyclines and macrolides (e.g., erythromycin and clarithromycin). Further to the antibiotics listed above, typical antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, tobramycin, s-treptomycin, azithromycin, clarithromycin, erythromycin, erythromycin estolate/ethyl-succinate/gluceptate/lactobionate/stearate), beta-lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin and piperacillin), or cephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefiuroxime, cefonicid, cefmnetazole, cefotetan, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, and cefsulodin). Other classes of antibiotics include carbapenems (e.g., imipenem), monobactams (e.g.,aztreonam), quinolones (e.g., fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin), tetracyclines (e.g., doxycycline, minocycline, tetracycline), and glycopeptides (e.g., vancomycin, teicoplanin), for example. Other antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin and the cationic peptides.
The efficacy of peptides was evaluated therapeutically alone and in combination with sub-optimal concentrations of antibiotics in models of infection. S. aureus is an important Gram positive pathogen and a leading cause of antibiotic resistant infections. Briefly, peptides were tested for therapeutic efficacy in the S. aureus infection model by injecting them alone and in combination with sub-optimal doses of antibiotics 6 hours after the onset of infection. This would simulate the circumstances of antibiotic resistance developing during an infection, such that the MIC of the resistant bacterium was too high to permit successful therapy (i.e the antibiotic dose applied was sub-optimal). It was demonstrated that the combination of antibiotic and peptide resulted in improved efficacy and suggests the potential for combination therapy (see Example 12).
The invention will now be described in greater detail by reference to the following non-limiting examples. While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
Polynucleotide arrays were utilized to determine the effect of cationic peptides on the transcriptional response of epithelial cells. The A549 human epithelial cell line was maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Medicorp). The A549 cells were plated in 100 mm tissue culture dishes at 2.5×106 cells/dish, cultured overnight and then incubated with 100 ng/ml E.coli O111:B4 LPS (Sigma), without (control) or with 50 μg/ml peptide or medium alone for 4 h. After stimulation, the cells were washed once with diethyl pyrocarbonate-treated phosphate buffered saline (PBS), and detached from the dish using a cell scraper. Total RNA was isolated using RNAqueous (Ambion, Austin, Tex.). The RNA pellet was resuspended in RNase-free water containing Superase-In (RNase inhibitor; Ambion). DNA contamination was removed with DNA-free kit, Ambion). The quality of the RNA was assessed by gel electrophoresis on a 1% agarose gel.
The polynucleotide arrays used were the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 10 μg of total RNA and labeled with Cy3 or Cy5 labeled dUTP. The probes were purified and hybridized to printed glass slides overnight at 42° C. and washed. After washing, the image was captured using a Perkin Elmer array scanner. The image processing software (Imapolynucleotide 5.0, Marina Del Rey, Calif.) determines the spot mean intensity, median intensities, and background intensities. A “homemade” program was used to remove background. The program calculates the bottom 10% intensity for each subgrid and subtracts this for each grid. Analysis was performed with Genespring software (Redwood City, Calif.). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in Tables 1 and 2. These tables reflect only those polynucleotides that demonstrated significant changes in expression of the 14,000 polynucleotides that were tested for altered expression. The data indicate that the peptides have a widespread ability to reduce the expression of polynucleotides that were induced by LPS.
In Table 1, the peptide, SEQ ID NO: 27 is shown to potently reduce the expression of many of the polynucleotides up-regulated by E. coli O1111:B4 LPS as studied by polynucleotide microarrays. Peptide (50 μg/ml) and LPS (0.1 μg/ml) or LPS alone was incubated with the A549 cells for 4 h and the RNA was isolated. Five μg total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 1. The “Ratio: LPS/control” column refers to the intensity of polynucleotide expression in LPS simulated cells divided by in the intensity of unstimulated cells. The “Ratio: LPS+ID 27/control” column refers to the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.
aAll Accession Numbers in Table 1 through Table 64 refer to GenBank Accession Numbers.
In Table 2, the cationic peptides at a concentration of 50 μg/ml were shown to potently reduce the expression of many of the polynucleotides up-regulated by 100 ng/ml E. coli O111:B4 LPS as studied by polynucleotide microarrays. Peptide and LPS or LPS alone was incubated with the A549 cells for 4 h and the RNA was isolated. 5 μg total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the third column of Table 2. The “Ratio: LPS/control” column refers to the intensity of polynucleotide expression in LPS-simulated cells divided by in the intensity of unstimulated cells. The other columns refer to the intensity of polynucleotide expression in cells stimulated with LPS and peptide divided by unstimulated cells.
The ability of compounds to neutralize the stimulation of immune cells by both Gram-negative and Gram-positive bacterial products was tested. Bacterial products stimulate cells of the immune system to produce inflammatory cytokines and when unchecked this can lead to sepsis. Initial experiments utilized the murine macrophage cell line RAW 264.7, which was obtained from the American Type Culture Collection, (Manassas, Va.), the human epithelial cell line, A549, and primary macrophages derived from the bone marrow of BALB/c mice (Charles River Laboratories, Wilmington, Mass.). The cells from mouse bone marrow were cultured in 150-mm plates in Dulbecco's modified Eagle medium (DMEM; Life Technologies, Burlington, ON) supplemented with 20% FBS (Sigma Chemical Co, St. Louis, Mo.) and 20% L cell-conditioned medium as a source of M-CSF. Once macrophages were 60-80% confluent, they were deprived of L cell-conditioned medium for 14-16 h to render the cells quiescent and then were subjected to treatments with 100 ng/ml LPS or 100 ng/ml LPS+20 μg/ml peptide for 24 hours. The release of cytokines into the culture supernatant was determined by ELISA (R&D Systems, Minneapolis, Minn.). The cell lines, RAW 264.7 and A549, were maintained in DMEM supplemented with 10% fetal calf serum. RAW 264.7 cells were seeded in 24 well plates at a density of 106 cells per well in DMEM and A549 cells were seeded in 24 well plates at a density of 105 cells per well in DMEM and both were incubated at 37° C. in 5% CO2 overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. In some experiments, blood from volunteer human donors was collected (according to procedures accepted by UBC Clinical Research Ethics Board, certificate C00-0537) by venipuncture into tubes (Becton Dickinson, Franklin Lakes, N.J.) containing 14.3 USP units heparin/ml blood. The blood was mixed with LPS with or without peptide in polypropylene tubes at 37° C. for 6 h. The samples were centrifuged for 5 min at 2000×g, the plasma was collected and then stored at −20° C. until being analyzed for IL-8 by ELISA (R&D Systems). In the experiments with cells, LPS or other bacterial products were incubated with the cells for 6-24 hr at 37° C. in 5% CO2. S. typhimurium LPS and E. coli 0111:B4 LPS were purchased from Sigma. Lipoteichoic acid (LTA) from S. aureus (Sigma) was resuspended in endotoxin free water (Sigma). The Limulus amoebocyte lysate assay (Sigma) was performed on LTA preparations to confirm that lots were not significantly contaminated by endotoxin. Endotoxin contamination was less than 1 ng/ml, a concentration that did not cause significant cytokine production in the RAW 264.7 cells. Non-capped lipoarabinomannan (AraLAM ) was a gift from Dr. John T. Belisle of Colorado State University. The AraLAM from Mycobacterium was filter sterilized and the endotoxin contamination was found to be 3.75 ng per 1.0 mg of LAM as determined by Limulus Amebocyte assay. At the same time as LPS addition (or later where specifically described), cationic peptides were added at a range of concentrations. The supernatants were removed and tested for cytokine production by ELISA (R&D Systems). All assays were performed at least three times with similar results. To confirm the anti-sepsis activity in vivo, sepsis was induced by intraperitoneal injection of 2 or 3 μg of E. coli O111:B4 LPS in phosphate-buffered saline (PBS; pH 7.2) into galactosamine-sensitized 8- to 10-week-old female CD-1 or BALB/c mice. In experiments involving peptides, 200 μg in 100 μl of sterile water was injected at separate intraperitoneal sites within 10 min of LPS injection. In other experiments, CD-1 mice were injected with 400 μg E. coli 011 I :B4 LPS and 10 min later peptide (200 μg) was introduced by intraperitoneal injection. Survival was monitored for 48 hours post injection.
Hyperproduction of TNF-α has been classically linked to development of sepsis. The three types of LPS, LTA or AraLAM used in this example represented products released by both Gram-negative and Gram-positive bacteria. Peptide, SEQ ID NO: 1, was able to significantly reduce TNF-A production stimulated by S. typhimurium, B. cepacia, and E. coli O111:B4 LPS, with the former being affected to a somewhat lesser extent (Table 3). At concentrations as low as 1 μg/ml of peptide (0.25 nM) substantial reduction of TNF-α production was observed in the latter two cases. A different peptide, SEQ ID NO: 3 did not reduce LPS-induced production of TNF-α in RAW macrophage cells, demonstrating that this is not a uniform and predictable property of cationic peptides. Representative peptides from each Formula were also tested for their ability to affect TNF-α production stimulated by E. coli O111:B4 LPS (Table 4). The peptides had a varied ability to reduce TNF-α production although many of them lowered TNF-α by at least 60%.
At certain concentrations peptides SEQ ID NO: 1 and SEQ ID NO: 2, could also reduce the ability of bacterial products to stimulate the production of IL-8 by an epithelial cell line. LPS is a known potent stimulus of IL-8 production by epithelial cells. Peptides, at low concentrations (1-20 μg/ml), neutralized the IL-8 induction responses of epithelial cells to LPS (Tables 5-7). Peptide SEQ ID 2 also inhibited LPS-induced production of IL-8 in whole human blood (Table 4). Conversely, high concentrations of peptide SEQ ID NO: 1 (50 to 100 μg/ml) actually resulted in increased levels of IL-8 (Table 5). This suggests that the peptides have different effects at different concentrations.
The effect of peptides on inflammatory stimuli was also demonstrated in primary murine. cells, in that peptide SEQ ID NO: 1 significantly reduced TNF-α production (>90%) by bone marrow-derived macrophages from BALB/c mice that had been stimulated with 100 ng/ml E. coli 0111 :B4 LPS (Table 8). These experiments were performed in the presence of serum, which contains LPS-binding protein (LBP), a protein that can mediate the rapid binding of LPS to CD14. Delayed addition of SEQ ID NO: 1 to the supernatants of macrophages one hour after stimulation with 100 ng/ml E. coli LPS still resulted in substantial reduction (70%) of TNF-α production (Table 9).
Consistent with the ability of SEQ ID NO: 1 to prevent LPS-induced production of TNF-α in vitro, certain peptides also protected mice against lethal shock induced by high concentrations of LPS. In some experiments, CD-1 mice were sensitized to LPS with a prior injection of galactosamine. Galactosamine-sensitized mice that were injected with 3 μg of E. coli 0111:B4 LPS were all killed within 4-6 hours. When 200 μg of SEQ ID NO: 1 was injected 15 min after the LPS, 50% of the mice survived (Table 10). In other experiments when a higher concentration of LPS was injected into BALB/c mice with no D-galactosamine, peptide protected 100% compared to the control group in which there was no survival (Table 13). Selected other peptides were also found to be protective in these models (Tables 11,12).
Cationic peptides were also able to lower the stimulation of macrophages by Gram-positive bacterial products such as Mycobacterium non-capped lipoarabinomannan (AraLAM) and S. aureus LTA. For example, SEQ ID NO: 1 inhibited induction of TNF-α in RAW 264.7 cells by the Gram-positive bacterial products, LTA (Table 14) and to a lesser extent AraLAM (Table 15). Another peptide, SEQ ID NO: 2, was also found to reduce LTA-induced TNF-α production by RAW 264.7 cells. At a concentration of 1 μg/ml SEQ ID NO: 1 was able to substantially reduce (>75%) the induction of TNF-α production by 1 μg/ml S. aureus LTA. At 20 μg/ml SEQ ID NO: 1, there was >60% inhibition of AraLAM induced TNF-α. Polymyxin B (PMB) was included as a control to demonstrate that contaminating endotoxin was not a significant factor in the inhibition by SEQ ID NO: 1 of AraLAM induced TNF-α. These results demonstrate that cationic peptides can reduce the pro-inflammatory cytokine response of the immune system to bacterial products.
RAW 264.7 mouse macrophage cells were stimulated with 100 ng/ml S. typhimurium LPS, 100 ng/ml B. cepacia LPS and 100 ng/ml E. coli 0111:B4 LPS in the presence of the indicated concentrations of SEQ ID 1 for 6 hr. The concentrations of TNF-α released into the culture supernatants were determined by ELISA. 100% represents the amount of TNF-α resulting from RAW 264.7 cells incubated with LPS alone for 6 hours (S. typhimurium LPS = 34.5 ± 3.2 ng/ml,
RAW 264.7 mouse macrophage cells were stimulated with 100 ng/ml E. coli 0111:B4 LPS in the presence of the indicated concentrations of cationic peptides for 6 h. The concentrations of TNF-α released into the culture supernatants were determined by ELISA. Background levels of TNF-α production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-α levels ranging from 0.037-0.192 ng/ml. The data is from duplicate samples and
A549 cells were stimulated with increasing concentrations of SEQ ID 1 in the presence of LPS (100 ng/ml E. coli O111:B4) for 24 hours. The concentration of IL-8 in the culture supernatants was determined by ELISA. The background levels of IL-8 from cells alone was 0.172 ± 0.029 ng/ml. The data is presented as the mean of three experiments + standard error.
Human A549 epithelial cells were stimulated with increasing concentrations of SEQ ID NO: 2 in the presence of LPS (100 ng/ml E. coli O111:B4) for 24 hours. The concentration of IL-8 in the culture supernatants was determined by ELISA. The data is presented as the mean of three experiments + standard error.
Whole human blood was stimulated with increasing concentrations of peptide and E. coli O111:B4 LPS for 4 hr. The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA. The data is presented as the average of 2 donors.
BALB/c Mouse bone marrow-derived macrophages were cultured for either 6 h or 24 h with 100 ng/ml E. coli 0111:B4 LPS in the presence or absence of 20 μg/ml of peptide. The supernatant was collected and tested for levels of TNF-α by ELISA. The data represents the amount of TNF-α resulting from duplicate wells of bone marrow-derived macrophages incubated with LPS alone for 6 h (1.1 ± 0.09 ng/ml) or 24 h (1.7 ± 0.2 ng/ml). Background levels of TNF-α were
Peptide (20 μg/ml) was added at increasing time points to wells already containing A549 human epithelial cells and 100 ng/ml E. coli 0111:B4 LPS. The supernatant was collected after 6 hours and tested for levels of TNF-α by ELISA. The data is presented as the mean of three experiments ± standard error.
E. coli
CD-1 mice (9 weeks-old) were sensitized to endotoxin by three intraperitoneal injections of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli 0111:B4 LPS (3 μg in 0.1 ml PBS). Peptide, SEQ ID NO: 1, (200 μg/mouse = 8 mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS. The mice were monitored for 48 hours and the results were recorded.
E. coli 0111:B4
CD-1 mice (9 weeks-old) were sensitized to endotoxin by intraperitoneal injection of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli 0111:B4 LPS (2 μg in 0.1 ml PBS). Peptide (200 μg/mouse = 8 mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS. The mice were monitored for 48 hours and the results were recorded.
E. coli
BALB/c mice (8 weeks-old) were sensitized to endotoxin by intraperitoneal injection of galactosamine (20 mg in 0.1 ml sterile PBS). Then endotoxic shock was induced by intraperitoneal injection of E. coli 0111:B4 LPS (2 μg in 0.1 ml PBS). Peptide (200 μg/mouse = 8 mg/kg) was injected at a separate intraperitoneal site 15 min after injection of LPS. The mice were monitored
E. coli
BALB/c mice were injected intraperitoneal with 400 μg E. coli 0111:B4 LPS. Peptide (200 μg/mouse = 8 mg/kg) was injected at a separate intraperitoneal site and the mice were monitored for 48 hours and the results were recorded.
RAW 264.7 mouse macrophage cells were stimulated with 1 μg/ml S. aureus LTA in the absence and presence of increasing concentrations of peptide. The supernatant was collected and tested for levels of TNF-α by ELISA. Background levels of TNF-α production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-α levels ranging from 0.037-0.192 ng/ml. The data is presented as the mean of three or more experiments + standard error.
RAW 264.7 mouse macrophage cells were stimulated with 1 μg/ml AraLAM in the absence and presence of 20 μg/ml peptide or Polymyxin B. The supernatant was collected and tested for levels of TNF-α by ELISA. Background levels of TNF-α production by the RAW 264.7 cells cultured with no stimuli for 6 hours resulted in TNF-α levels ranging from 0.037-0.192 ng/ml. The data is
The potential toxicity of the peptides was measured in two ways. First, the Cytotoxicity Detection Kit (Roche) (Lactate dehydrogenase-LDH) Assay was used. It is a colorimetric assay for the quantification of cell death and cell lysis, based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant. LDH is a stable cytoplasmic enzyme present in all cells and it is released into the cell culture supernatant upon damage of the plasma membrane. An increase in the amount of dead or plasma membrane-damaged cells results in an increase of the LDH enzyme activity in the culture supernatant as measured with an ELISA plate reader, OD490 nm (the amount of color formed in the assay is proportional to the number of lysed cells). In this assay, human bronchial epithelial cells (I6HBEo14, HBE) cells were incubated with 100 μg of peptide for 24 hours, the supernatant removed and tested for LDH. The other assay used to measure toxicity of the cationic peptides was the WST-1 assay (Roche). This assay is a colorimetric assay for the quantification of cell proliferation and cell viability, based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells (a non-radioactive alternative to the [3H]-thymidine incorporation assay). In this assay, HBE cells were incubated with 100 μg of peptide for 24 hours, and then 10 μl/well Cell Proliferation Reagent WST-1 was added. The cells are incubated with the reagent and the plate is then measured with an ELISA plate reader, OD490 nm.
The results shown below in Tables 16 and 17 demonstrate that most of the peptides are not toxic to the cells tested. However, four of the peptides from Formula F (SEQ ID NOS: 40, 41, 42 and 43) did induce membrane damage as measured by both assays.
Human HBE bronchial epithelial cells were incubated with 100 μg/ml peptide or Polymyxin B for 24 hours. LDH activity was assayed in the supernatant of the cell cultures. As a control for 100% LDH release, Triton X-100 was added. The data is presented as the mean ± standard deviation. Only peptides SEQ ID 40, 41, 42 and 43 showed any significant toxicity.
HBE cells were incubated with 100 μg/ml peptide or Polymyxin B for 24 hours and cell viability was tested. The data is presented as the mean ± standard deviation. As a control for 100% LDH release, Triton X-100 was added. Only peptides SEQ ID NOS: 40, 41, 42 and 43 showed any significant toxicity.
Polynucleotide arrays were utilized to determine the effect of cationic peptides by themselves on the transcriptional response of macrophages and epithelial cells. Mouse macrophage RAW 264.7, Human Bronchial cells (HBE), or A549 human epithelial cells were plated in 150 mm tissue culture dishes at 5.6×106 cells/dish, cultured overnight and then incubated with 50 μg/ml peptide or medium alone for 4 h. After stimulation, the cells were washed once with diethyl pyrocarbonate-treated PBS, and detached from the dish using a cell scraper. Total RNA was isolated using Trizol (Gibco Life Technologies). The RNA pellet was resuspended in RNase-free water containing RNase inhibitor (Ambion, Austin, Tex.). The RNA was treated with DNaseI (Clontech, Palo Alto, Calif.) for 1 h at 37° C. After adding termination mix (0.1 M EDTA [pH 8.0], 1 mg/ml glycogen), the samples were extracted once with phenol:chloroform:isoamyl alcohol (25:24:1), and once with chloroform. The RNA was then precipitated by adding 2.5 volumes of 100% ethanol and 1/10th volume sodium acetate, pH 5.2. The RNA was resuspended in RNase-free water with RNase inhibitor (Ambion) and stored at −70° C. The quality of the RNA was assessed by gel electrophoresis on a 1% agarose gel. Lack of genomic DNA contamination was assessed by using the isolated RNA as a template for PCR amplification with P-actin-specific primers (5′-GTCCCTGTATGCCTCTGGTC-3′ (SEQ ID NO: 55) and 5′-GATGTCACGCACGATTTCC-3′(SEQ ID NO: 56)). Agarose gel electrophoresis and ethidium bromide staining confirmed the absence of an amplicon after 35 cycles.
Atlas cDNA Expression Arrays (Clontech, Palo Alto, Calif.), which consist of 588 selected mouse cDNAs spotted in duplicate on positively charged membranes were used for early polynucleotide array studies (Tables 18 and 19). 32P-radiolabeled cDNA probes prepared from 5 μg total RNA were incubated with the arrays overnight at 71° C. The filters were washed extensively and then exposed to a phosphoimager screen (Molecular Dynamics, Sunnyvale, Calif.) for 3 days at 4° C. The image was captured using a Molecular Dynamics PSI phosphoimager. The hybridization signals were analyzed using Atlaslmage 1.0 Image Analysis software (Clontech) and Excel (Microsoft, Redmond, Wash.). The intensities for each spot were corrected for background levels and normalized for differences in probe labeling using the average values for 5 polynucleotides observed to vary little between the stimulation conditions: β-actin, ubiquitin, ribosomal protein S29, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Ca2+ binding protein. When the normalized hybridization intensity for a given cDNA was less than 20, it was assigned a value of 20 to calculate the ratios and relative expression.
The next polynucleotide arrays used (Tables 21-26) were the Resgen Human cDNA arrays (identification number for the genome is PRHU03-S3), which consist of 7,458 human cDNAs spotted in duplicate. Probes were prepared from 15-20 μg of total RNA and labeled with Cy3 labeled dUTP. The probes were purified and hybridized to printed glass slides overnight at 42° C. and washed. After washing, the image was captured using a Virtek slide reader. The image processing software (Imagene 4.1, Marina Del Rey, Calif.) determines the spot mean intensity, median intensities, and background intensities. Normalization and analysis was performed with Genespring software (Redwood City, Calif.). Intensity values were calculated by subtracting the mean background intensity from the mean intensity value determined by Imagene. The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in the Tables below.
The other polynucleotide arrays used (Tables 27-35) were the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 10 μg of total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated in 100 mm tissue culture dishes at 2.5×106 cells/dish. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with DNA-free kit (Ambion). The probes prepared from total RNA were purified and hybridized to printed glass slides overnight at 42° C. and washed. After washing, the image was captured using a Perkin Elmer array scanner. The image processing software (Imagene 5.0, Marina Del Rey, Calif.) determines the spot mean intensity, median intensities, and background intensities. An “in house” program was used to remove background. The program calculates the bottom 10% intensity for each subgrid and subtracts this for each grid. Analysis was performed with Genespring software (Redwood City, Calif.). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with peptide compared to control cells can be found in the Tables below.
Semi-quantitative RT-PCR was performed to confirm polynucleotide array results. 1 μg RNA samples were incubated with 1 μl oligodT (500 μg/ml) and 1 μl mixed dNTP stock at 1 mM, in a 12 μl volume with DEPC treated water at 65° C. for 5 min in a thermocycler. 4 μl 5× First Strand buffer, 2 μl 0.1M DTT, and 1 μl RNaseOUT recombinant ribonuclease inhibitor (40 units/μl) were added and incubated at 42° C. for 2 min, followed by the addition of 1 μl (200 units) of Superscript II (Invitrogen, Burlington, ON). Negative controls for each RNA source were generated using parallel reactions in the absence of Superscript II. cDNAs were amplified in the presence of 5′ and 3′ primers (1.0 μM), 0.2 mM dNTP mixture, 1.5 mM MgCl, 1 U of Taq DNA polymerase (New England Biolabs, Missisauga, ON), and 1× PCR buffer. Each PCR was performed with a thermal cycler by using 30-40 cycles consisting of 30s of denaturation at 94° C., 30s of annealing at either 52° C. or 55° C. and 40s of extension at 72° C. The number of cycles of PCR was optimized to lie in the linear phase of the reaction for each primer and set of RNA samples. A housekeeping polynucleotide 0-actin was amplified in each experiment to evaluate extraction procedure and to estimate the amount of RNA. The reaction product was visualized by electrophoresis and analyzed by densitometry, with relative starting RNA concentrations calculated with reference to β-actin amplification.
Table 18 demonstrates that SEQ ID NO: 1 treatment of RAW 264.7 cells up-regulated the expression of more than 30 different polynucleotides on small Atlas microarrays with selected known polynucleotides. The polynucleotides up-regulated by peptide, SEQ ID NO: 1, were mainly from two categories: one that includes receptors (growth, chemokine, interleukin, interferon, hormone, neurotransmitter), cell surface antigens and cell adhesion and another one that includes cell-cell communication (growth factors, cytokines, chemokines, interleukin, interferons, hormones), cytoskeleton, motility, and protein turnover. The specific polynucleotides up-regulated included those encoding chemokine MCP-3, the anti-inflammatory cytokine IL-10, macrophage colony stimulating factor, and receptors such as IL-1R-2 (a putative antagonist of productive IL-1 binding to IL-1R1), PDGF receptor B, NOTCH4, LIF receptor, LFA-1, TGFβ receptor 1, G-CSF receptor, and IFNγ receptor. The peptide also up-regulated polynucleotides encoding several metalloproteinases, and inhibitors thereof, including the bone morphogenetic proteins BMP-1, BMP-2, BMP-8a, TIMP2 and TIMP3. As well, the peptide up-regulated specific transcription factors, including JunD, and-the YY and LIM-1 transcription factors, and kinases such as Etk1 and Csk demonstrating its widespread effects. It was also discovered from the polynucleotide array studies that SEQ ID NO: 1 down-regulated at least 20 polynucleotides in RAW 264.7 macrophage cells (Table 19). The polynucleotides down-regulated by peptide included DNA repair proteins and several inflammatory mediators such as MIP-1α, oncostatin M and IL-12. A number of the effects of peptide on polynucleotide expression were confirmed by RT-PCR (Table 20). The peptides, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 19, and SEQ ID NO: 1, and representative peptides from each of the formulas also altered the transcriptional responses in a human epithelial cell line using mid-sized microarrays (7835 polynucleotides). The effect of SEQ ID NO: 1 on polynucleotide expression was compared in 2 human epithelial cell lines, A549 and HBE. Polynucleotides related to the host immune response that were up-regulated by 2 peptides or more by a ratio of 2-fold more than unstimulated cells are described in Table 21. Polynucleotides that were down-regulated by 2 peptides or more by a ratio of 2-fold more than unstimulated cells are described in Table 22. In Table 23 and Table 24, the human epithelial pro-inflammatory polynucleotides that are up- and down-regulated respectively are shown. In Table 25 and Table 26 the anti-inflammatory polynucleotides affected by cationic peptides are shown. The trend becomes clear that the cationic peptides up-regulate the anti-inflammatory response and down-regulate the pro-inflammatory response. It was very difficult to find a polynucleotide related to the anti-inflammatory response that was down-regulated (Table 26). The pro-inflammatory polynucleotides upregulated by cationic peptides were mainly polynucleotides related to migration and adhesion. Of the down-regulated pro-inflammatory polynucleotides, it should be noted that all the cationic peptides affected several toll-like receptor (TLR) polynucleotides, which are very important in signaling the host response to infectious agents. An important anti-inflammatory polynucleotide that was up-regulated by all the peptides is the IL-10 receptor. IL-10 is an important cytokine involved in regulating the pro-inflammatory cytokines. These polynucleotide expression effects were also observed using primary human macrophages as observed for peptide SEQ ID NO: 6 in Tables 27 and 28. The effect of representative. peptides from each of the formulas on human epithelial cell expression of selected polynucleotides (out of 14,000 examined) is shown in Tables 31-37 below. At least 6 peptides from each formula were tested for their ability to alter human epithelial polynucleotide expression and indeed they had a wide range of stimulatory effects. In each of the formulas there were at least 50 polynucleotides commonly up-regulated by each of the peptides in the group.
The cationic peptides at a concentration of 50 μg/ml were shown to potently induce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of unstimulated cells is shown in the third column. The “Ratio Peptide:Unstimulated” column refers to
The changes in the normalized intensities of the housekeeping polynucleotides ranged from 0.8-1.2 fold, validating the use of these polynucleotides for normalization. When the normalized hybridization intensity for a given cDNA was less than 20, it was assigned a value of 20 to calculate the ratios and relative expression. The array experiments were repeated 3 times with different RNA
The cationic peptides at a concentration of 50 μg/ml were shown to reduce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays. The intensity of unstimulated cells is shown in the third column. The “Ratio Peptide:Unstimulated” column refers to the
RAW 264.7 macrophage cells were incubated with 50 μg/ml of peptide or media only for 4 hours and total RNA isolated and subjected to semi-quantitative RT-PCR. Specific primer pairs for each polynucleotide were used for amplification of RNA. Amplification of β-actin was used as a positive control and for standardization. Densitometric analysis of RT-PCR products was used. The results refer
The cationic peptides at concentrations of 50 μg/ml were shown to increase the expression of several polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns refers to the intensity
The cationic peptides at concentrations of 50 μg/ml were shown to decrease the expression of several polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns refers to the intensity
The cationic peptides at concentrations of 50 μg/ml were shown to increase the expression of certain pro-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns
The cationic peptides at concentrations of 50 μg/ml were shown to decrease the expression of certain pro-inflammatory polynucleotides (data is a subset of Table 22). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns
The cationic peptides at concentrations of 50 μg/ml were shown to increase the expression of certain anti-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns
The cationic peptides at concentrations of 50 μg/ml were shown to increase the expression of certain anti-inflammatory polynucleotides (data is a subset of Table 21). Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human cDNA arrays ID#PRHU03-S3. The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Unstimulated” columns
The peptide SEQ ID NO: 6 at a concentration of 50 μg/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human macrophages for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio peptide
The peptide SEQ ID NO: 6 at a concentration of 50 μg/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human macrophages for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio of Peptide:Control” columns refer to the intensity of
H. sapiens CREB gene
The peptide SEQ ID NO: 1 at a concentration of 50 μg/ml was shown to increase the expression of many polynucleotides. Peptide was incubated with the human HBE epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the second column. The “Ratio Peptide:Control” columns refer to the intensity of
The peptide SEQ ID NO: 1 at a concentration of 50 μg/ml was shown to decrease the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in unstimulated cells is shown in the third column. The “Ratio Peptide:Control” columns refer to the intensity of
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively.
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively.
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides. Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04). The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively.
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04).
The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively.
The “ID#: Control” columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Homo
sapiens BNPI
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04).
The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and Cy5 respectively.
The “ID#: Control” columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Human Operon arrays (PRHU04).
The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labeling of cDNA with the dyes Cy3 and CyS respectively.
The “Ratio ID#: Control” columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
The peptides at a concentration of 50 μg/ml were shown to increase the expression of many polynucleotides.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labelled cDNA probes and hybridised to Human Operon arrays (PRHU04).
The intensity of polynucleotides in control, unstimulated cells are shown in the second and third columns for labelling of cDNA with the dyes Cy3 and Cy5 respectively.
The “Ratio ID#: Control” columns refer to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
Accession numbers and gene designations are U00115, zinc finger protein; M91036, hemoglobin gamma G; K000070, hypothetical protein; AF055899, solute carrier family 27; AK001490, hypothetical protein; X97674, nuclear receptor coactivator 2; AB022847, unknown; AJ275986, transcription factor; D10495, protein kinase C, delta; L36642, EphA7; M31166, pentaxin-related gene; AF176012,
The murine macrophage cell line RAW 264.7, THP-1 cells (human monocytes), a human epithelial cell line (A549), human bronchial epithelial cells (16HBEo14), and whole human blood were used. HBE cells were grown in MEM with Earle's. THP-1 cells were grown and maintained in RPMI 1640 medium. The RAW and A549 cell lines were maintained in DMEM supplemented with 10% fetal calf serum. The cells were seeded in 24 well plates at a density of 106 cells per well in DMEM (see above) and A549 cells were seeded in 24 well plates at a density of 105 cells per well in DMEM (see above) and both were incubated at 37° C. in 5% CO2 overnight. DMEM was aspirated from cells grown overnight and replaced with fresh medium. After incubation of the cells with peptide, the release of chemokines into the culture supernatant was determined by ELISA (R&D Systems, Minneapolis, Minn.).
Animal studies were approved by the UBC Animal Care Committee.(UBC ACC # A01-0008). BALB/c mice were purchased from Charles River Laboratories and housed in standard animal facilities. Age, sex and weight matched adult mice were anaesthetized with an intraperitoneal injection of Avertin (4.4 mM 2-2-2-tribromoethanol, 2.5% 2-methyl-2-butanol, in distilled water), using 200 ill per 10 g body weight. The instillation was performed using a non-surgical, intratracheal instillation method adapted from Ho and Furst 1973. Briefly, the anaesthetized mouse was placed with its upper teeth hooked over a wire at the top of a support frame with its jaw held open and a spring pushing the thorax forward to position the pharynx, larynx and trachea in a vertical straight line. The airway was illuminated externally and an intubation catheter was inserted into the clearly illuminated tracheal lumen. Twenty-μl of peptide suspension or sterile water was placed in a well at the proximal end of the catheter and gently instilled into the trachea with 200 μl of air. The animals were maintained in an upright position for 2 minutes after instillation to allow the fluid to drain into the respiratory tree. After 4 hours the mice were euthanaised by intraperitoneal injection of 300 mg/kg of pentobarbital. The trachea was exposed; an intravenous catheter was passed into the proximal trachea and tied in place with suture thread. Lavage was performed by introducing 0.75 ml sterile PBS into the lungs via the tracheal cannula and then after a few seconds, withdrawing the fluid. This was repeated 3 times with the same sample of PBS. The lavage fluid was placed in a tube on ice and the total recovery volume per mouse was approximately 0.5 ml. The bronchoalveolar.lavage (BAL) fluid was centrifuged at 1200 rpm for 10 min, the clear supernatant removed and tested for TNF-α and MCP-1 by ELISA.
The up-regulation of chemokines by cationic peptides was confirmed in several different systems. The murine MCP-1, a homologue of the human MCP-1, is a member of the β(C-C) chemokine family. MCP-1 has been demonstrated to recruit monocytes, NK cells and some T lymphocytes. When RAW 264.7 macrophage cells and whole human blood from 3 donors were stimulated with increasing concentrations of peptide, SEQ ID NO: 1, they produced significant levels of MCP-1 in their supernatant, as judged by ELISA (Table 36). RAW 264.7 cells stimulated with peptide concentrations ranging from 20-50 μg/ml for 24 hr produced significant levels of MCP-1(200-400 pg/ml above background). When the cells (24 h) and whole blood (4 h) were stimulated with 100 μg/ml of SEQ ID NO: 1, high levels of MCP-1 were produced.
The effect of cationic peptides on chemokine induction was also examined in a completely different cell system, A549 human epithelial cells. Interestingly, although these cells produce MCP-1 in response to LPS, and this response could be antagonized by peptide; there was no production of MCP-1 by A549 cells in direct response to peptide, SEQ ID NO: 1. Peptide SEQ ID NO: 1 at high concentrations, did however induce production of IL-8, a neutrophil specific chemokine (Table 37). Thus, SEQ ID NO: 1 can induce a different spectrum of responses from different cell types and at different concentrations. A number of peptides from each of the formula groups were tested for their ability to induce IL-8 in A549 cells (Table 38). Many of these peptides at a low concentration, 10 μg/ml induced IL-8 above background levels. At high concentrations (100 μg/ml) SEQ ID NO: 13 was also found to induce IL-8 in whole human blood (Table 39). Peptide SEQ ID NO: 2 also significantly induced IL-8 in HBE cells (Table 40) and undifferentiated THP-1 cells (Table 41).
BALB/c mice were given SEQ ID NO: 1 or endotoxin-free water by intratracheal instillation and the levels of MCP-1 and TNF-α examined in the bronchioalveolar lavage fluid after 3-4 hr. It was found that the mice treated with 50 μg/ml peptide, SEQ ID NO: 1 produced significantly increased levels of MCP-1 over mice given water or anesthetic alone (Table 42). This was not a pro-inflammatory response to peptide, SEQ ID NO: 1 since peptide did not significantly induce more TNF-α than mice given water or anesthetic alone. peptide, SEQ ID NO: 1 was also found not to significantly induce TNF-α production by RAW 264.7 cells and bone marrow-derived macrophages treated with peptide, SEQ ID NO: 1 (up to 100 μg/ml) (Table 43). Thus, peptide, SEQ ID NO: 1 selectively induces the production of chemokines without inducing the production of inflammatory mediators such as TNF-α. This illustrates the dual role of peptide, SEQ ID NO: 1 as a factor that can block bacterial product-induced inflammation while helping to recruit phagocytes that can clear infections.
RAW 264.7 mouse macrophage cells or whole human blood were stimulated with increasing concentrations of SEQ ID NO: 1 for 4 hr. The human blood samples were centrifuged and the serum was removed and tested for MCP-1 by ELISA along with the supernatants from the RAW 264.7 cells. The RAW cell data presented is the mean of three or more experiments ± standard error and the human blood data represents the mean ± standard error from three separate donors.
A549 cells or whole human blood were stimulated with increasing concentrations of peptide for 24 and 4 hr respectively. The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA along with the supernatants from the A549 cells. The A549 cell data presented is the mean of three or more experiments ± standard error and the human blood data represents the mean ± standard error from three separate donors.
A549 human epithelial cells were stimulated with 10 μg of peptide for 24 hr. The supernatant was removed and tested for IL-8 by ELISA.
Whole human blood was stimulated with increasing concentrations of peptide for 4 hr. The human blood samples were centrifuged and the serum was removed and tested for IL-8 by ELISA. The data shown is the average 2 donors.
Increasing concentrations of the peptide were incubated with HBE cells for 8 h, the supernantant removed and tested for IL-8. The data is presented as the mean of three or more experiments ± standard error.
The human monocyte THP-1 cells were incubated with indicated concentrations of peptide for 8 hr. The supernatant was removed and tested for IL-8 by ELISA.
BALB/c mice were anaesthetised with avertin and given intratracheal instillation of peptide or water or no instillation (no treatment). The mice were monitored for 4 hours, anaesthetised and the BAL fluid was isolated and analyzed for MCP-1 and TNF-α concentrations by ELISA. The data shown is the mean of 4 or 5 mice for each condition ± standard error.
RAW 264.7 macrophage cells were incubated with indicated peptides (40 μg/ml) for 6 hours. The supernatant was collected and tested for levels of TNF-α by ELISA. The data is presented as the mean of three or more experiments + standard error.
To analyze cell surface expression of IL-8RB, CXCR-4, CCR2, and LFA-1, RAW macrophage cells were stained with 10 μg/ml of the appropriate primary antibody (Santa Cruz Biotechnology) followed by FITC-conjugated goat anti-rabbit IgG [IL-8RB and CXCR-4 (Jackson ImmunoResearch Laboratories, West Grove, Pa.)] or FITC-conjugated donkey anti-goat IgG (Santa Cruz). The cells were analyzed using a FACscan, counting 10,000 events and gating on forward and side scatter to exclude cell debris.
The polynucleotide array data suggested that some peptides up-regulate the expression of the chemokine receptors IL-8RB, CXCR-4 and CCR2 by 10, 4 and 1.4 fold above unstimulated cells respectively. To confirm the polynucleotide array data, the surface expression was examined by flow cytometry of these receptors on RAW cells stimulated with peptide for 4 hr. When 50 μg/ml of peptide was incubated with RAW cells for 4 hr, IL-8RB was upregulated an average of 2.4-fold above unstimulated cells, CXCR-4 was up-regulated an average of 1.6-fold above unstimulated cells and CCR2 was up-regulated 1.8-fold above unstimulated cells (Table 46). As a control CEMA was demonstrated to cause similar up-regulation. SEQ ID NO: 3 was the only peptide to show significant up-regulation of LFA-1 (3.8 fold higher than control cells).
RAW macrophage cells were stimulated with peptide for 4 hr. The cells were washed and stained with the appropriate primary and FITC-labeled secondary antibodies. The data shown represents the average (fold change of RAW cells stimulated with peptide from media) ± standard error.
The cells were seeded at 2.5×105−5×105 cells/ml and left overnight. They were washed once in media, serum starved in the morning (serum free media—4 hrs). The media was removed and replaced with PBS, then sat at 37° C. for 15 minutes and then brought to room temp for 15 minutes. Peptide was added (concentrations 0.1 μg/ml−50 μg/ml) or H2O and incubated 10 min. The PBS was very quickly removed and replaced with ice-cold radioimmunoprecipitation (RIPA) buffer with inhibitors (NaF, B-glycerophosphate, MOL, Vanadate, PMSF, Leupeptin Aprotinin). The plates were shaken on ice for 10-15 min or until the cells were lysed and the lysates collected. The procedure for THP-1 cells was slightly different; more cells (2×106) were used. They were serum starved overnight, and to stop the reaction 1 ml of ice-cold PBS was added then they sat on ice 5-10 min, were spun down then resuspended in RIPA. Protein concentrations were determined using a protein assay (Pierce, Rockford, Ill.). Cell lysates (20 μg of protein) were separated by SDS-PAGE and transferred to nitrocellulose filters. The filters were blocked for 1 h with 10 mM Tris-HCI, pH 7.5, 150 mM NaCl (TBS)/5% skim milk powder and then incubated overnight in the cold with primary antibody in TBS/0.05% Tween 20. After washing for 30 min with TBS/0.05% Tween 20, the filters were incubated for 1 h at room temperature with 1 μg/ml secondary antibody in TBS. The filters were washed for 30 min with TBS/0.05% Tween 20 and then incubated 1 h at room temperature with horseradish peroxidase-conjugated sheep anti-mouse IgG (1:10,000 in TBS/0.05% Tween 20). After washing the filters for 30 min with TBS/0.1% Tween 20, immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection. For experiments with peripheral blood mononuclear cells: The peripheral blood (50-100 ml) was collected from all subjects. Mononuclear cells were isolated from the peripheral blood by density gradient centrifugation on Ficoll-Hypaque. Interphase cells (mononuclear cells) were recovered, washed and then resuspended in recommended primary medium for cell culture (RPMI-1640) with 10% fetal calf serum (FCS) and 1% L-glutamine. Cells were added to 6 well culture plates at 4×106 cells/well and were allowed to adhere at 37° C. in 5% CO2 atmosphere for 1 hour. The supernatant medium and non-adherent cells were washed off and the appropriate media with peptide was added. The freshly harvested cells were consistently >99% viable as assessed by their ability to exclude trypan blue. After stimulation with peptide, lysates were collected by lysing the cells in RIPA buffer in the presence of various phosphatase- and kinase-inhibitors. Protein content was analyzed and approximately 30 μg of each sample was loaded in a 12% SDS-PAGE gel. The gels were blotted onto nitrocellulose, blocked for 1 hour with 5% skim milk powder in Tris buffered saline (TBS) with 1% TritonX100. Phosphorylation was detected with phosphorylation-specific antibodies.
The results of peptide-induced phosphorylation are summarized in Table 46. SEQ ID NO: 2 was found to cause dose dependent phosphorylation of p38 and ERK1/2 in the mouse macrophage RAW cell line and the HBE cells. SEQ ID NO: 3 caused phosphorylation of MAP kinases in THP-1 human monocyte cell line and phosphorylation of ERK1/2 in the mouse RAW cell line.
BALB/c mice were given 1×105 Salmonella and cationic peptide (200 μg) by intraperitoneal injection. The mice were monitored for 24 hours at which point they were euthanized, the spleen removed, homogenized and resuspended in PBS and plated on Luria Broth agar plates with Kanamycin (50 μg/ml). The plates were incubated overnight at 37° C. and counted for viable bacteria (Table 49 and 50). CD-1 mice were given 1×108 S. aureus in 5% porcine mucin and cationic peptide (200 μg) by intraperitoneal injection (Table 51). The mice were monitored for 3 days at which point they were euthanized, blood removed and plated for viable counts. CD-1 male mice were given 5.8×106 CFU EHEC bacteria and cationic peptide (200 μg) by intraperitoneal (IP) injection and monitored for 3 days (Table 52). In each of these animal models a subset of the peptides demonstrated protection against infections. The most protective peptides in the Salmonella model demonstrated an ability to induce a common subset of genes in epithelial cells (Table 53) when comparing the protection assay results in Tables 50 and 51 to the gene expression results in Tables 31-37. This clearly indicates that there is a pattern of gene expression that is consistent with the ability of a peptide to demonstrate protection. Many of the cationic peptides were shown not to be directly antimicrobial as tested by the Minimum Inhibitory Concentration (MIC) assay (Table 54). This demonstrates that the ability of peptides to protect against infection relies on the ability of the peptide to stimulate host innate immunity rather than on direct antimicrobial activity.
The BALB/c mice were injected IP with Salmonella and Peptide, and 24 h later the animals were euthanized, the spleen removed, homogenized, diluted in PBS and plate counts were done to determine bacteria viability.
The BALB/c mice were injected intraperitoneally with Salmonella and Peptide, and 24 h later the animals were euthanized, the spleen removed, homogenized, diluted in PBS and plate counts were done to determine bacteria viability.
CD-1 mice were given 1 × 108 bacteria in 5% porcine mucin via intraperitoneal (IP) injection. Cationic peptide (200 μg) was given via a separate IP injection. The mice were monitored for 3 days at which point they were euthanized, blood removed and plated for viable counts. The following peptides were not effective in controlling S. aureus infection: SEQ ID NO: 48, SEQ ID NO: 26.
CD-1 male mice (5 weeks old) were given 5.8 × 106 CFU EHEC bacteria via intraperitoneal (IP) injection. Cationic peptide (200 μg) was given via a separate IP injection. The mice were monitored for 3 days.
The peptides SEQ ID NO: 30, SEQ ID NO: 7 and SEQ ID NO: 13 at concentrations of 50 μg/ml were each shown to increase the expression of a pattern of genes after 4 h treatment.
Peptide was incubated with the human A549 epithelial cells for 4 h and the RNA was isolated, converted into labelled cDNA probes and hybridised to Human Operon arrays (PRHU04).
The intensity of polynucleotides in control, unstimulated cells are shown in the second columns for labelling of cDNA (average of Cy3 and Cy5).
The Fold Up regulation column refers to the intensity of polynucleotide expression in peptide-simulated cells divided by the intensity of unstimulated cells.
The SEQ ID NO: 37 peptide was included as a negative control that was not active in the murine infection models.
E. coli
S. aureus
P. aerug.
S. typhim.
C. rhod.
Most cationic peptides studied here and especially the cationic peptides effective in infection models are not significantly antimicrobial. A dilution series of peptide was incubated with the indicated bacteria overnight in a 96-well plate. The lowest concentration of peptide that killed the bacteria was used as the MIC.
The symbol > indicates the MIC is too large to measure. An MIC of 4 μg/ml or less was considered clinically meaningful activity.
Abbreviations:
E. coli, Escherichia coli;
S. aureus, Staphylococcus aureus;
P. aerug, Pseudomonas aeruginosa;
S. Typhim, Salmonella enteritidis ssp. typhimurium;
C. rhod, Citobacter rhodensis;
EHEC, Enterohaemorrhagic E. coli.
S. typhimurium LPS and E. coli 0111:B4 LPS were purchased from Sigma Chemical Co. (St. Louis, Mo.). LTA (Sigma) from S. aureus, was resuspended in endotoxin free water (Sigma). The Limulus amoebocyte lysate assay (Sigma) was performed on LTA preparations to confirm that lots were not significantly contaminated by endotoxin (i.e. <1 ng/ml, a concentration that did not cause significant cytokine production in the RAW cell assay). The CpG oligodeoxynucleotides were synthesized with an Applied Biosystems Inc., Model 392 DNA/RNA Synthesizer, Mississauga, ON., then purified and resuspended in endotoxin-free water (Sigma). The following sequences were used CpG: 5′-TCATGACGTTCCTGACGTT-3′ (SEQ ID NO: 57) and nonCpG: 5′-TTCAGGACTTTCCTCAGGTT-3′ (SEQ ID NO: 58). The nonCpG oligo was tested for its ability to stimulate production of cytokines and was found to cause no significant production of TNF-α or IL-6 and therefore was considered as a negative control. RNA was isolated from RAW 264.7 cells that had been incubated for 4 h with medium alone, 100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA, or 1 μM CpG (concentrations that led to optimal induction of tumor necrosis factor (TNF-α) in RAW cells). The RNA was used to polynucleotiderate cDNA probes that were hybridized to Clontech Atlas polynucleotide array filters, as described above. The hybridization of the cDNA probes to each immobilized DNA was visualized by autoradiography and quantified using a phosphorimager. Results from at least 2 to 3 independent experiments are summarized in Tables 55-59. It was found that LPS treatment of RAW 264.7 cells resulted in increased expression of more than 60 polynucleotides including polynucleotides encoding inflammatory proteins such as IL-1β, inducible nitric oxide synthase (iNOS), MIP-1α, MIP-1β, MIP-2α, CD40, and a variety of transcription factors. When the changes in polynucleotide expression induced by LPS, LTA, and CpG DNA were compared, it was found that all three of these bacterial products increased the expression of pro-inflammatory polynucleotides such as iNOS, MIP-1α, MIP-2α, IL-1β, IL-15, TNFR1 and NF-κB to a similar extent (Table 57). Table 57 describes 19 polynucleotides that were up-regulated by the bacterial products to similar extents in that their stimulation ratios differed by less than 1.5 fold between the three bacterial products. There were also several polynucleotides that were down-regulated by LPS, LTA and CpG to a similar extent. It was also found that there were a number of polynucleotides that were differentially regulated in response to the three bacterial products (Table 58), which includes many of these polynucleotides that differed in expression levels by more than 1.5 fold between one or more bacterial products). LTA treatment differentially influenced expression of the largest subset of polynucleotides compared to LPS or CpG, including hyperstimulation of expression of Jun-D, Jun-B, Elk-1 and cyclins G2 and A1. There were only a few polynucleotides whose expression was altered more by LPS or CpG treatment. Polynucleotides that had preferentially increased expression due to LPS treatment compared to LTA or CpG treatment included the cAMP response element DNA-binding protein 1 (CRE-BPI), interferon inducible protein 1 and CACCC Box-binding protein BKLF. Polynucleotides that had preferentially increased expression after CpG treatment compared to LPS or LTA treatment included leukemia inhibitory factor (LIF) and protease nexin 1 (PN-1). These results indicate that although LPS, LTA, and CpG DNA stimulate largely overlapping polynucleotide expression responses, they also exhibit differential abilities to regulate certain subsets of polynucleotides.
The other polynucleotide arrays used are the Human Operon arrays (identification number for the genome is PRHU04-S1), which consist of about 14,000 human oligos spotted in duplicate. Probes were prepared from 5 μg of total RNA and labeled with Cy3 or Cy5 labeled dUTP. In these experiments, A549 epithelial cells were plated in 100 mm tissue culture dishes at 2.5×106 cells/dish, incubated overnight and then stimulated with 100 ng/ml E. coli O111:B4 LPS for 4 h. Total RNA was isolated using RNAqueous (Ambion). DNA contamination was removed with DNA-free kit (Ambion). The probes prepared from total RNA were purified and hybridized to printed glass slides overnight at 42° C. and washed. After washing, the image was. captured using a Perkin Elmer array scanner. The image processing software (Imapolynucleotide 5.0, Marina Del Rey, Calif.) determines the spot mean intensity, median intensities, and background intensities. An “in house” program was used to remove background. The program calculates the bottom 10% intensity for each subgrid and subtracts this for each grid. Analysis was performed with Polynucleotidespring software (Redwood City, Calif.). The intensities for each spot were normalized by taking the median spot intensity value from the population of spot values within a slide and comparing this value to the values of all slides in the experiment. The relative changes seen with cells treated with LPS compared to control cells can be found in the Tables below. A number of previously unreported changes that would be useful in diagnosing infection are described in Table 60.
To confirm and assess the functional significance of these changes, the levels of selected mRNAs and proteins were assessed and quantified by densitometry. Northern blots using a CD14, vimentin, and tristetraprolin-specific probe confirmed similar expression after stimulation with all 3 bacterial products (Table 60). Similarly measurement of the enzymatic activity of nitric oxide synthetase, iNOS, using Griess reagent to assess levels of the inflammatory mediator NO, demonstrated comparable levels of NO produced after 24 h, consistent with the similar up-regulation of iNOS expression (Table 59). Western blot analysis confirmed the preferential stimulation of leukaemia inhibitory factor (LIF, a member of the IL-6 family of cytokines) by CpG (Table 59). Other confirmatory experiments demonstrated that LPS up-regulated the expression of TNF-α and IL-6 as assessed by ELISA, and the up-regulated expression of MIP-2α, and IL-1β mRNA and down-regulation of DP-1 and cyclin D mRNA as assessed by Northern blot analysis. The analysis was expanded to a more clinically relevant ex vivo system, by examining the ability of the bacterial elements to stimulate pro-inflammatory cytokine production in whole human blood. It was found that E. coli LPS, S. typhimurium LPS, and S. aureus LTA all stimulated similar amounts of serum TNF-α, and IL-1β. CpG also stimulated production of these cytokines, albeit to much lower levels, confirming in part the cell line data.
E. coli O111:B4 LPS (100 ng/ml) increased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays. LPS was incubated with the A549 cells for 4 h and the RNA was isolated. 5 μg total RNA was used to make Cy3/Cy5 labelled cDNA probes and hybridised onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the second column of Table 55. The “Ratio: LPS/control” column refers to the intensity of polynucleotide
E. coli O111:B4 LPS (100 ng/ml) decreased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays. LPS was incubated with the A549 cells for 4 h and the RNA was isolated. 5 μg total RNA was used to make Cy3/Cy5 labeled cDNA probes and hybridized onto Human Operon arrays (PRHU04). The intensity of unstimulated cells is shown in the second column of the Table. The “Ratio: LPS/control” column refers to the intensity of polynucleotide
Bacterial products (100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA or 1 μM CpG) were shown to potently induce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays.
The intensity of control, unstimulated cells is shown in the second column.
The “Ratio LPS/LTA/CpG: Control” column refers to the intensity of polynucleotide expression in bacterial product-simulated cells divided by the intensity of unstimulated cells.
Bacterial products (100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA or 1 μM CpG) were shown to potently induce the expression of several polynucleotides. Peptide was incubated with the RAW cells for 4 h and the RNA was isolated, converted into labeled cDNA probes and hybridized to Atlas arrays.
The intensity of control, unstimulated cells is shown in the second column.
The “Ratio LPS/LTA/CpG: Control” column refers to the intensity of polynucleotide expression in bacterial product-simulated cells divided by the intensity of unstimulated cells.
aTotal RNA was isolated from unstimulated RAW macrophage cells and cells treated for 4 hr with 100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA, 1 μM CpG DNA or media alone and Northern blots were performed the membrane was probed for GAPDH, CD14, vimentin, and tristetraprolin as described previously [Scott et al]. The hybridization
bRAW 264.7 cells were stimulated with 100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA, 1 μM CpG DNA or media alone for 24 hours. Protein lysates were prepared, run on SDS PAGE gels and western blots were performed to detect LIF (R&D Systems). These experiments were repeated at least three times and the data shown is the relative levels of LIF
cSupernatant was collected from RAW macrophage cells treated with 100 ng/ml S. typhimurium LPS, 1 μg/ml S. aureus LTA, 1 μM CpG DNA, or media alone for 24 hours and tested for the amount of NO formed in the supernatant as estimated from the accumulation of the stable NO metabolite nitrite with the Griess reagent as described previously [Scott, et al].
The data shown is the average of three experiments ± standard error.
E. coli O111:B4 LPS (100 ng/ml) increased the expression of many polynucleotides in A549 cells as studied by polynucleotide microarrays. LPS was incubated with the A549 cells for 4 h and the RNA was isolated. 5 μg total RNA was used to make Cy3/Cy5 labelled cDNA probes and hybridised onto Human Operon arrays (PRHU04). The examples of polynucleotide
The Salmonella Typhimurium strain SL1344 was obtained from the American Type Culture Collection (ATCC; Manassas, Va.) and grown in Luria-Bertani (LB) broth. For macrophage infections, 10 ml LB in a 125 mL flask was inoculated from a frozen glycerol stock and cultured overnight with shaking at 37° C. to stationary phase. RAW 264.7 cells (1×105 cells/well) were seeded in 24 well plates. Bacteria were diluted in culture medium to give a nominal multiplicity of infection (MOI) of approximately 100, bacteria were centrifuged onto the monolayer at 1000 rpm for 10 minutes to synchronize infection, and the infection was allowed to proceed for 20 min in a 37° C., 5% CO2 incubator. Cells were washed 3 times with PBS to remove extracellular bacteria and then incubated in DMEM+10% FBS containing 100 μg/ml gentamicin (Sigma, St. Louis, Mo.) to kill any remaining extracellular bacteria and prevent re-infection. After 2 h, the gentamicin concentration was lowered to 10 μg/ml and maintained throughout the assay. Cells were pretreated with inhibitors for 30 min prior to infection at the following concentrations: 50 μM PD 98059 (Calbiochem), 50 μM U 0126 (Promega), 2 mM diphenyliodonium (DPI), 250 μM acetovanillone (apocynin, Aldrich), 1 mM ascorbic acid (Sigma), 30 mM N-acetyl cysteine (Sigma), and 2 mM Ng-L-monomethyl arginine (L-NMMA, Molecular Probes) or 2 mM NG-D-monomethyl arginine (D-NMMA, Molecular Probes). Fresh inhibitors were added immediately after infection, at 2 h, and 6-8 h post-infection to ensure potency. Control cells were treated with equivalent volumes of dimethylsulfoxide (DMSO) per mL of media. Intracellular survival/replication of S. Typhimurium SL1344 was determined using the gentamicin-resistance assay, as previously described. Briefly, cells were washed twice with PBS to remove gentamicin, lysed with 1% Triton X-100/0.1% SDS in PBS at 2 h and 24 h post-infection, and numbers of intracellular bacteria calculated from colony counts on LB agar plates. Under these infection conditions, macrophages contained an average of 1 bacterium per cell as assessed by standard plate counts, which permitted analysis of macrophages at 24 h post-infection. Bacterial filiamnentation is related to bacterial stress. NADPH oxidase and iNOS can be activated by MEK/ERK signaling. The results (Table 61) clearly demonstrate that the alteration of cell signaling is a method whereby intracellular Salmonella infections can be resolved. Thus since bacteria to up-regulate multiple genes in human cells, this strategy of blocking signaling represents a general method of therapy against infection.
SDF-1, a C-X-C chemokine is a natural ligand for HIV-1 coreceptor-CXCR4. The chemokine receptors CXCR4 and CCR5 are considered to be potential targets for the inhibition of HIV-1 replication. The crystal structure of SDF-1 exhibits antiparallel β-sheets and a positively charged surface, features that are critical in binding to the negatively charged extracellular loops of CXCR4. These findings suggest that chemokine derivatives, small-size CXCR4 antagonists, or agonists mimicking the structure or ionic property of chemokines may be useful agents for the treatment of X4 HIV-1 infection. It was found that the cationic peptides inhibited SDF-1 induced T-cell migration suggesting that the peptides may act as CXCR4 antagonists. The migration assays were performed as follows. Human Jurkat T cells were resuspended to 5×106/ml in chemotaxis medium (RPMI 1640/10 mM Hepes/0.5% BSA). Migration assays were performed in 24 well plates using 5 pm polycarbonate Transwell inserts (Costar). Briefly, peptide or controls were diluted in chemotaxis medium and placed in the lower chamber while 0.1 ml cells (5×106/ml) was added to the upper chamber. After 3 hr at 37° C., the number of cells that had migrated into the lower chamber was determined using flow cytometry. The medium from the lower chamber was passed through a FACscan for 30 seconds, gating on forward and side scatter to exclude cell debris. The number of live cells was compared to a “100% migration control” in which 5×105/ml cells had been pipetted directly into the lower chamber and then counted on the FACscan for 30 seconds. The results demonstrate that the addition of peptide results in an inhibition of the migration of Human Jurkat T-cells (Table 62) probably by influencing CXCR4 expression (Tables 63 and 64).
Methods and Materials
S. aureus was prepared in phosphate buffered solution (PBS) and 5% porcine mucin (Sigma) to a final expected concentration of 1-4×107 CFU/ml. 100 μl of S. aureus (mixed with 5% porcine, mucin) was injected intraperitoneally (IP) into each CD-1 mouse (6-8 weeks female weighing 20-25 g (Charles River)). Six hours after the onset of infection, 100 μl of the peptide was injected (50-200 μg total) IP along with 0.1 mg/kg Cefepime. After 24 hours, animals were sacrificed and heart puncture was performed to remove 100 μl of blood. The blood was diluted into 1 ml PBS containing Heparin. This was then ftrther diluted and plated for viable colony counts on Mueller-Hinton agar plates (10−1, 10−2, 10−3 & 10−4). Viable colonies, colony-forming units (CFU), were counted after 24 hours. Each experiment was carried out a minimum of three times. Data is presented as the average CFU+standard error per treatment group (8-10 mice/group).
Experiments were carried out with peptide and sub-optimal Cefepime given 6 hours after the onset of systemic S. aureus infection (
SEQ ID NO: 1, as an example, induced phosphorylation and activation of the mitogen activated protein kinases, ERK1/2 and p38 in human peripheral blood-derived monocytes and a human bronchial epithelial cell line but not in B- or T-lymphocytes. Phosphorylation was not dependent on the G-protein coupled receptor, FPRL-1, which was previously proposed to be the receptor for SEQ ID NO: 1-induced chemotaxis on human monocytes and T cells. Activation of ERK1/2 and p38 was markedly increased by the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), but not macrophage-colony stimulating factor (M-CSF). Exposure to SEQ ID NO: 1 also led to the activation of Elk1/2, a transcription factor that is downstream of and activated by phosphorylated ERK1/2, as well as the up-regulation of various Elk-1 controlled genes. The ability of SEQ ID NO: 1 to signal through these pathways has broad implications in immunity, monocyte activation, proliferation and differentiation.
SEQ ID NO: 1 (sequence LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), was synthesized by Fmoc [(N-(9-fluorenyl) methoxycarbonyl)] chemistry at the Nucleic Acid/Protein Synthesis (NAPS) Unit at UBC. Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and macrophage colony-stimulating factor (M-CSF) were purchased from Research Diagnostics Inc. (Flanders, N.J., USA). Pertussis toxin was supplied by List Biological Laboratories Inc. (Campbell, Calif., USA).
Blood monocytes were prepared using standard techniques. Briefly, 100 ml of fresh human venous blood was collected in sodium heparin Vacutainer collection tubes (Becton Dickinson, Mississauga, ON, Canada) from volunteers according to UBC Clinical Research Ethics Board protocol C02-0091. The blood was mixed, at a 1:1 ratio, with RPMI 1640 media [supplemented with 10% v/v fetal calf serum (FBS), 1% L-glutamine, 1 nM sodium pyruvate] in an E-toxa-clean (Sigma-Aldrich, Oakville, ON, Canada) washed, endotoxin-free bottle. PBMC were separated using Ficoll-Paque Plus (Amersham Pharmacia Biotech, Baie D'Urfé, PQ, Canada) at room temperature and washed with phosphate buffered saline (PBS). Monocytes were enriched with the removal of T-cells by rosetting with fresh sheep red blood cells (UBC animal care unit) pre-treated with Vibrio cholerae neuraminidase (Calbiochem Biosciences Inc., La Jolla, Calif., USA) and repeat separation by Ficoll Paque Plus. The enriched monocytes were washed with PBS, then cultured (approximately 2-3×106 per well) for 1 hour at 37° C. followed by the removal of non-adherent cells; monocytes were >95% pure as determined by flow cytometry (data not shown). B-lymphocytes were isolated by removing non-adherent cells and adding them to a new plate for one hour at 37° C. This was repeated a total of three times. Any remaining monocytes adhered to the plates, and residual non-adherent cells were primarily B cells. Cells were cultured in Falcon tissue culture 6-well plates (Becton Dickinson, Mississauga, ON, Canada). The adherent monocytes were cultured in 1 ml media at 37° C. in which SEQ ID NO: 1 and/or cytokines dissolved in endotoxin-free water (Sigma-Aldrich, Oakville, ON, Canada) were added. Endotoxin-free water was added as a vehicle control. For studies using pertussis toxin the media was replaced with 1 ml of fresh media containing 100 ng/ml of toxin and incubated for 60 min at 37° C. SEQ ID NO: 1 and cytokines were added directly to the media containing pertussis toxin. For the isolation of T lymphocytes, the rosetted T cells and sheep red blood cells were resuspended in 20 ml PBS and 10 ml of distilled water was added to lyse the latter. The cells were then centrifuged at 1000 rpm for 5 min after which the supernatant was removed. The pelleted T cells were promptly washed in PBS and increasing amounts of water were added until all sheep red blood cells had lysed. The remaining T cells were washed once in PBS, and viability was confirmed using a 0.4% Trypan blue solution. Primary human blood monocytes and T cells were cultured in RPMI 1640 supplemented with 10% v/v heat-inactivated FBS, 1% v/v L-glutamine, 1 nM sodium pyruvate (GIBCO Invitrogen Corporation, Burlington, ON, Canada). For each experiment between two and eight donors were used.
The simian virus 40-transformed, immortalized 16HBE4o-bronchial epithelial cell line was a generous gift of Dr. D. Gruenert (University of California, San Francisco, Calif.). Cells were routinely cultured to confluence in 100% humidity and 5% CO2 at 37° C. They were grown in Minimal Essential media with Earles' salts (GIBCO Invitrogen Corporation, Burlington, ON, Canada) containing 10% FBS (Hyclone), 2mM L-glutamine. For experiments, cells were grown on Costar Transwell inserts (3-μm pore size, Fischer Scientific) in 24-well plates. Cells were seeded at 5×104 cells per 0.25 ml of media on the top of the inserts while 0.95 ml of media was added to the bottom of the well and cultured at 37° C. and 5% CO2. Transmembrane resistance was measured daily with a Millipore voltohmeter and inserts were used for experiments typically after 8 to 10 days, when the resistance was 500-700 ohms. The cells were used between passages 8 and 20.
Western Immunoblotting—After stimulation, cells were washed with ice-cold PBS containing 1 mM vanadate (Sigma). Next 125 μl of RIPA buffer (50 mM Tris-HCI, pH 7.4, NP-40 1%, sodium deoxycholate 0.25%, NaCl 150 mM, EDTA 1 mM, PMSF 1 mM, Aprotinin, leupeptin, pepstatin 1 μg/ml each, sodium orthovanadate 1 mM, NaF 1 mM) was added and the cells were incubated on ice until they were completely lysed as assessed by visual inspection. The lysates were quantitated using a BCA assay (Pierce). 30 μg of lysate was loaded onto 1.5 mm thick gels, which were run at 100 volts for approximately 2 hours. Proteins were transferred to nitrocellulose filters for 75 min at 70 V. The filters were blocked for 2 hours at room temperature with 5% skim milk in TBST (10 mM Tris-HCI pH 8, 150 mM NaCl, 0.1% Tween-20). The filters were then incubated overnight at 4° C. with the anti-ERK1/2-P or anti-p38-P (Cell Signaling Technology, Ma) monoclonal antibodies. Immunoreactive bands were detected using horseradish peroxidase-conjugated sheep anti-mouse IgG antibodies (Amersham Pharmacia, New Jersey) and chemiluminescence detection (Sigma, Mo). To quantify bands, the films were scanned and then quantified by densitometry using the software program, ImageJ. The blots were reprobed with β-actin antibody (ICN Biomedical Incorporated, Ohio) and densitometry was performed to allow correction for protein loading.
Kinase Assay—An ERK1/2 activity assay was performed using a non-radioactive kit (Cell Signaling Technology). Briefly, cells were treated for 15 min and lysed in lysis buffer. Equal amounts of proteins were immunoprecipitated with an immobilized phospho-ERK1/2 antibody that reacts only with the phosphorylated (i.e. active) form of ERK1/2. The immobilized precipitated enzymes were then used for the kinase assay using Elk-1 followed by Western blot analysis with antibodies that allow detection and quantitation of phosphorylated substrates.
Quantification of IL-8—Human IL-8 from supernatants of 16HBE40-cells was measured by using the commercially available enzyme-linked immunosorbent assay kit (Biosource) according to the manufacturer's instructions.
Semiquantitative RT-PCR—Total RNA from two independent experiments was isolated from 16HBE4o-cells using RNaqueous (Ambion) as described by the manufacturer. The samples were DNase treated, and then cDNA synthesis was accomplished by using a first-strand cDNA synthesis kit (Gibco). The resultant cDNAs were used as a template in PCRs for various cytokine genes:
Each RT-PCR reaction was performed in at least duplicate. Results were analysed in the linear phase of amplification and normalized to the housekeeping control, glyceraldehyde-3-phosphate dehydrogenase. Reactions were verified for RNA amplification by including controls without reverse transcriptase.
Peptides induce ERK1/2 and p38 phosphorylation in peripheral blood derived monocytes. To determine if peptide induced the activation of the MAP kinases, ERK1/2 and/or p38, peripheral blood derived monocytes were treated with 50 μg/ml SEQ ID NO: 1 or water (as a vehicle control) for 15 min. To visualize the activated (phosphorylated) form of the kinases, Western blots were performed with antibodies specific for the dually phosphorylated form of the kinases (phosphorylation on Thr202+Tyr204 and Thr180+Tyr182 for ERK1/2 and p38 respectively). The gels were re-probed with an antibody for β-actin to normalize for loading differences. In all, an increase in phosphorylation of ERK1/2 (n=8) and p38 (n=4) was observed in response to SEQ ID NO: 1 treatment (
Peptide induced activation of ERK1/2 is greater in human serum than in fetal bovine serum. It was demonstrated that SEQ ID NO: 1 induced phosphorylation of ERK1/2 did not occur in the absence of serum and the magnitude of phosphorylation was dependent upon the type of serum present such that activation of ERK1/2 was far superior in human serum (HS) than in fetal bovine serum (FBS).
Peptide induced activation of ERK1/2 and p38 is dose dependent and demonstrates synergy with GM-CSF. GM-CSF, IL-4, or M-CSF (each at 100 ng/ml) was added concurrently with SEQ ID NO: 1 and phosphorylation of ERK1/2 was measured in freshly isolated human blood monocytes. ERK1/2 phosphorylation was evident when cells were treated with 50 μg/ml of SEQ ID NO: 1 (8.3 fold increase over untreated, n=9) but not at lower concentrations (n=2). In the presence of 100 ng/ml GM-CSF, SEQ ID NO: 1-induced ERK1/2 phosphorylation increased markedly (58 fold greater than untreated, n=5). Furthermore, in the presence of GM-CSF, activation of ERK1/2 occurred in response to concentrations of 5 and 10 μg/ml of SEQ ID NO: 1, respectively, in the two donors tested (
Activation of ERK1/2 leads to transcription of Elk-1 controlled genes and secretion of IL-8. IL-8 release is governed, at least in part, by activation of the ERK1/2 and p38 kinases. In order to determine if peptide could induce IL-8 secretion the human bronchial cell line, 16HBE4o-, was grown to confluency in Transwell filters, which allows for cellular polarization with the creation of distinct apical and basal surfaces. When the cells were stimulated with 50 μg/ml of SEQ ID NO: 1 on the apical surface for four hours a statistically significant increase in the amount of IL-8 released into the apical supernatant was detected (
The innate immune response is a dynamic system since it can be triggered by receptor. recognition of conserved bacterial components, initiating a broad inflammatory response to infectious agents, but must be able maintain homeostasis in the presence of commensal organisms, which contain many of these same conserved components. A delicate balance of pro- and anti-inflammatory mediators is vital for efficient functioning of the immune system under these disparate circumstances. In recent years, there has been speculation and some evidence implicating the sole human cathelicidin, SEQ ID NO: 1, in maintaining homeostasis, combating pathogenic challenge, and protecting against endotoxemia, an extreme inflammation-like condition (Devine D A, et al. Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 2002; 8:703-14; Ciornei C D, et al. Antimicrobial and chemoattractant activity, Lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37. Antimicrob Agents Chemother 2005; 49:2845-50). The data presented herein demonstrate that SEQ ID NO: 1 is an important component of human immunity that regulates the balance of pro- and anti-inflammatory molecules both under homeostatic conditions and during endotoxin challenge (i.e., infection situations).
Materials and Methods
Cell Isolation and Cell Lines—Human monocytic cells, THP-1 (Tsuchiya S, et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980; 26:171-6), were obtained from American type culture collection, ATCC® (TIB-202) and were grown in suspension in RPMI-1640 media (Gibco®, Invitrogen™ Life technologies, Burlington, ON), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen Life Technologies). Cultures were maintained at 37° C. in a humidified 5% (v/v) CO2 incubator up to a maximum of six passages. THP-1 cells at a density of 1×106 cells/ml were treated with 0.3 μg/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich Canada, Oakville ON) for 24 hr (Tsuchiya S, et al. Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res 1982; 42:1530-6), inducing plastic-adherent cells that were further rested in complete RPMI-1640 medium for an additional 24 hr prior to stimulations with various treatments. Venous blood (20 ml) from healthy volunteers was collected in Vacutainer collection tubes containing sodium heparin as an anticoagulant (Becton Dickinson, Mississauga, ON) in accordance with UBC ethical approval and guidelines. Blood was diluted 1:1 with complete RPMI 1640 medium and separated by centrifugation over a Ficoll-Paqueg Plus (Amersham Biosciences, Piscataway, N.J., USA) density gradient. White blood cells were isolated from the buffy coat, washed twice in RPMI 1640 complete medium, and the number of peripheral blood mononuclear cells (PBMC) was determined by trypan blue exclusion. PBMC (5×105) were seeded into 12-well tissue culture dishes (Falcon; Becton Dickinson) at 1×106 cells/ml at 37° C. in 5% CO2. All experiments using human THP-1 cells or PBMCs involved at least three biological replicates.
Stimulants, Reagents and Antibodies—LPS was isolated from P. aeruginosa H103 using the Darveau-Hancock method as previously described (Darveau RP, et al. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 1983; 155:831-8). Briefly, P. aeruginosa was grown overnight in LB broth at 37° C. Cells were collected and washed and the isolated LPS pellets were extracted with a 2:1 chloroform:methanol solution to remove contaminating lipids. Purified LPS samples were quantitated using an assay for the specific sugar 2-keto-3-deoxyoctosonic acid (KDO assay) and then resuspended in endotoxin-free water (Sigma-Aldrich).
TLR2 agonists lipoteichoic acid (LTA) from S. aureus and a synthetic tripalmitoylated lipopeptide, Pam3CSK4, were purchased from InvivoGen (San Diego, Calif., USA). TLR9 agonist CpG oligodeoxynucleotide #2007 (Krieg AM. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374:546-9) was a gift from Dr. Lorne Babuik (Vaccine and Infectious Disease org., SK, Canada). Recombinant human TNFα and recombinant human ILL1β were obtained from Research Diagnostics Inc., (Flanders, N.J., USA). All reagents were tested for endotoxin and reconstituted in endotoxin-free water. LTA from S. aureus used in this study had 1.25 EU of endotoxin/μg of LTA. Polymyxin B was purchased from InvivoGen, Actinomycin D (transcriptional inhibitor) was purchased from Calbiochem-Novabiochem Corporation (La Jolla, Calif.) and Monensin (inhibitor of protein secretion) was purchased from eBiosciences., CA, USA. A cationic peptide, SEQ ID NO: 1, was synthesized using F-moc chemistry at the Nucleic Acid/Protein Synthesis Unit, University of British Columbia (Vancouver, BC, Canada). The synthetic peptide was re-suspended in endotoxin-free water and stored at −20° C. until flurther use.
Rabbit polyclonal antibodies against the NFκB subunits p105/p50, p65 and RelB were purchased from Cell Signaling Technologies (Mississauga, ON, Canada). Rabbit polyclonal antibody against the NFκB subunit c-Rel was purchased from Chemicon International (Temecula, CA, USA) and mouse IgG2a monoclonal antibody against NFκB subunit p100/p52 was purchased from Upstate Cell Signaling Solutions (Lake Placid, N.Y., USA). HRP-conjugated goat anti-rabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Technologies and Amersham Biosciences respectively.
Treatment with inflammatory stimuli, peptide or inhibitors—THP-1 cells or PBMC were stimulated with LPS (10 or 100 ng/ml), LTA (1 μg/ml), Pam3CSK4 (100 ng/ml), CpG-ODN 2007 (2 μg/ml), recombinant human TNFα (50 ng/ml) or recombinant human IL1β (50 ng/ml) for 1, 2, 4, or 24 hours. SEQ ID NO: 1 (0.5-50 lg/ml) was added simultaneously or 30 min after addition of the stimulants. Alternatively, cells were stimulated with SEQ ID NO: 1 (20 μg/ml) for 30 min, washed with RPMI complete media to remove the peptide and then stimulated with LPS (100 ng/ml). Polymyxin B (0.1 mg/ml), actinomycin D (4 μg/ml), or monensin (working concentration as per the manufacturer's instructions) were added to the THP-1 cells 30 min prior to stimulants.
Detection ofcytokines—Following incubation of the cells under various treatment regimens, the tissue culture supernatants were centrifuged at 1000× g for 5 min, then at 10,000×g for 2 min to obtain cell-free samples. Supernatants were aliquoted and then stored at −20° C. prior to assay for various cytokines. TNFα and IL-8 secretion were detected with a capture ELISA (eBioscience and BioSource International Inc., CA, USA respectively) using either tissue culture supernatants or the nuclear and cytoplasmic extracts (see below) as per the experimental design. All assays were performed in triplicate. The concentration of the cytokines in the culture medium was quantified by establishing a standard curve with serial dilutions of the recombinant human TNFα or IL-8 respectively. Alternatively, five cytokines (GM-CSF, IL-1β, IL-6, IL-8 and TNFα) were measured simultaneously using the Human Cytokine 5-Plex kit from Biosource International Inc., (Medicorp Inc., Montreal, Canada) as per the manufacturer's instructions. The multiplex bead immunoassays were analyzed using Luminex 100™ StarStation software (Applied Cytometry Systems, Sacramento, CA, USA).
RNA extraction, amplification and hybridization to DNA microarrays—RNA was isolated from THP-1 cells with RNeasy Mini kit, treated with RNase-Free DNase (Qiagen Inc., Canada) and eluted in RNase-free water (Ambion Inc., Austin, Tex., USA) as per the manufacturer's instructions. RNA concentration, integrity and purity were assessed by Agilent 2100 Bioanalyzer using RNA 6000 Nano kits (Agilent Technologies; USA). RNA was (reverse) transcribed with incorporation of amino-allyl-UTP (aa-UTP) using the MessageAmpII™ amplification kit, according to the manufacturer's instructions, then column purified and eluted in nuclease-free water. Column purified samples were labeled with mono-functional dyes, Cyanine-3 and Cyanine-5 (Amersham Biosciences), according to manufacturer's instructions, and then purified using the Mega Clear kit (Ambion). Yield and fluorophore incorporation was measured using Lambda 35 UV/VIS fluorimeter (PerkinElmer Life and Analytical Sciences, Inc., USA). Microarray slides were printed with the human genome 21K Array-Ready Oligo Set™ (Qiagen Inc., USA) at The Jack Bell Research Center (Vancouver, BC, Canada). The slides were pre-hybridized for 45 min at 48° C. in pre-hybridization buffer containing 5×SSC (Ambion), 0.1% (w/v) SDS and 0.2% (w/v) BSA. Equivalent (20 pmol) cyanine labeled samples from control and treated cells were then mixed and hybridized on the array slides, in Ambion SlideHyb™ buffer#2 (Ambion) for 18 hr at 37° C. in a hybridization oven. Following hybridization, the slides were washed twice in 1×SSC/0.1% sodium dodecyl sulphate (SDS) for 5 min at 65° C., then twice in 1×SSC and 0.1×SSC for 3 min each at 42° C. Slides were centrifugated for 5 min at 1000×g, dried and scanned using ScanArray™ Express software/scanner (scanner and software by Packard BioScience BioChip Technologies) and the images were quantified using ImaGene™ (BioDiscovery Inc., El Segundo, Calif., USA).
Analysis of DNA Microarrays—Assessment of slide quality, normalization, detection of differential gene expression and statistical analysis was carried out with ArrayPipe (version 1.6), a web-based, semi-automated software specifically designed for processing of microarray data (Hokamp K, et al. ArrayPipe: a flexible processing pipeline for microarray data. Nucleic Acids Res 2004; 32(Web Server issue):W457-9) (www.pathogenomics.ca/arraypipe). The following processing steps were applied: 1) flagging of markers, 2) subgrid-wise background correction, using the median of the lower 10% foreground intensity as an estimate for the background noise, 3) data-shifting, to rescue negative spots, 4) printTip LOESS normalization, 5) merging of technical replicates, 6) two-sided one-sample Student t-test on the log2-ratios within each treatment group, 7) averaging of biological replicates to yield overall fold-changes for each treatment group. Further, the gene expression data was overlaid on molecular interaction networks using Cytoscape (Shannon P, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003; 13:2498-504). Interactions networks were custom built from manually curated data and information contained within the Transpath pathway database (Krull M, et al. TRANSPATH: an integrated database on signal transduction and a tool for array analysis. Nucleic Acids Res 2003; 31:97-100). The false discovery rate of selecting differentially expressed genes from microarray analysis was estimated at 35%, based on Beta Uniform Mixture model (Pounds S, et al. Estimating the occurrence of false positives and false negatives in microarray studies by approximating and partitioning the empirical distribution of p-values. Bioinformatics 2003; 19:1236-42) and Q-Value model (Storey JD. A direct approach to false discovery rates. Journal of the Royal Statistical Society 2002; 64:479-498). This was consistent with the confirmation, using qPCR, at 4 different time points, of array results for 14 of 20 genes (70%) selected for follow-up.
Quantitative real-time PCR (qPCR)—Differential gene expression identified by microarray analysis was validated using quantitative real-time PCR (qPCR) using SuperScriptTM III Platinum® Two-Step qRT-PCR Kit with SYBR® Green (Invitrogen Life Technologies), as per the manufacturer's instructions, in the ABI PRISMS® 7000 sequence detection system (Applied Biosystems, Foster city, Calif., USA). Briefly, 1 μg of total RNA was reverse transcribed in a 20 μl reaction volume for 50 min at 42° C., the reaction was terminated by incubating for 5 min at 85° C. and then digested for 30 min at 37° C. with RNAse H. The PCR reaction was carried out in a 12.5 μl reaction volume containing 2.5 μl of 1/10 diluted cDNA template. A melting curve was performed to ensure that any product detected was specific to the desired amplicon. Fold changes were calculated after normalization to endogenous GAPDH and using the comparative Ct method (Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29:No. 9 e45). The primers used for qRT-PCR are reported in Table 65.
Nuclear and Cytoplasmic Extracts—THP-1 cells (3×106) seeded into 60 mm2 petri dishes (VWR International, Mississauga, ON) were pre-treated with inhibitors for 30 min, and then stimulated with agonists or peptide for 30 min or 60 min. Cells were subsequently treated with Versene for 10 min at 37° C. in 5% CO2 (to detach adherent cells) then washed twice with ice-cold phosphate buffered saline. Cytoplasmic and nuclear extracts were isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology, Rockford, Ill., USA) according to the manufacturer's instructions. The protein concentration of the extracts was quantified using a Bicinchoninic Acid (BCA) Protein Assay (Pierce Biotechnology) and the extracts were stored at −80° C. until fturther use.
Translocation of NFκB subunits—Equivalent nuclear extracts (5-10 μg) were resolved on a 7.5% SDS-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) Immobilon-P membranes (Millipore Canada Ltd., Mississauga, ON). Equivalent protein loading was verified by staining PVDF membranes with Blot-Fast-Stain™ (Chemicon International) according to the manufacturer's instructions. Subsequently, the PVDF membranes were incubated with anti-p105/p50, anti-p65, anti-c-Rel, anti-Rel B or anti-p100/p52 antibodies at 1/1000 dilution in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% skimmed milk powder (TBST/milk) for 1 hr. Membranes were washed for 1 hour in TBST and then incubated with a 1/5000 dilution of HRP-conjugated goat anti-mouse or anti-rabbit Ab (in TBST/milk) for 30 min. The membranes were incubated for 30 to 60 min in TBST and developed with chemiluminescence peroxidase substrate (Sigma-Aldrich), according to manufacturer's instructions. Alternatively, equivalent nuclear extracts (2.5-10 ug) were analyzed for NFκB subunits p50 or p65 by StressXpress NFκB p50 or p65 ELISA kits (Stressgen Bioreagents, Victoria, BC, Canada) according to manufacturer's instructions. Luminescence was detected with SpectraFluor Plus Multifunction Microplate Reader (Tecan Systems Inc., SJ, USA).
Results
Low, physiological concentrations of SEQ ID NO: 1 suppress LPS-indauced secretion of the pro-inflarmnnatoiry cytokine TNFα. SEQ ID NO: 1 is found at mucosal surfaces at concentrations of around 2.5 to 5 μg/ml in adults and up to 20 μg/ml in infants (Schaller-Bals S, et al. Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection. Am J Respir Crit Care Med 2002; 165:992-5). Previous studies indicated that it has the ability to down-regulate pro-inflammatory cytokines in isolated monocytic cells (Bowdish D M, et al. Immunomodulatory activity of small host defense peptides. Antimicrob Agents Chemother 2005; 49:1727-32). To determine the lowest dose of SEQ ID NO: 1 that exhibited anti-endotoxin activity, THP-1 cells were stimulated with LPS (10 and 100 ng/ml) in the absence or presence of SEQ ID NO: 1 added simultaneously at concentrations ranging from 0.5 to 50 μg/ml for a period of 4 hours in complete RPMI cell culture media (i.e., which contains physiological salt concentrations). Tissue culture supernatants were assayed by ELISA for the presence of the pro-inflammatory cytokine TNFα (
To gain further insight into the mode of inhibition exerted by SEQ ID NO: 1, TNFα production and release was monitored in the supernatants of LPS-stimulated THP-1 cells treated with the transcriptional inhibitor actinomycin D. Four μg/ml of actinomycin D was used since this concentration was required for inhibition, by more than 96% within 1 hour of treatment, of LPS-induced transcription of the genes for both the cytokine TNFα and the pro-inflammatory TNFα-inducible protein 2 (TNFAIP2) (monitored by qPCR, data not shown). Actinomycin D reduced the level of TNFα release by 97.6% (
The sustained presence of SEQ ID NO: 1 inhibits TNFα release. To determine the kinetics of the anti-endotoxin effect, the supernatant from THP-1 cells was monitored for TNFα after 1, 2, 4 and 24 hr of stimulation with LPS (100 ng/ml) in absence or presence of SEQ ID NO: 1 (20 μg/ml). When the peptide and LPS were added simultaneously, the release of TNFα was substantially inhibited (90 to 97%) by SEQ ID NO: 1 at all time points (
SEQ ID NO: 1 suppresses TLR-induced cytokine secretion by PBMC. PBMC were treated with agonists of TLR2 (LTA, PAM3CSK4), TLR4 (LPS), TLR9 (CpG), and the inflammatory cytokines TNFα and IL-1β, to determine if SEQ ID NO: 1 could suppress cytokine secretion induced by inflammatory stimuli LPS and other agonists in primary cells. Cytokine production was analyzed by Luminex 100™ StarSystem using the human 5-Plex cytokine kit to monitor IL-1β, IL-6, IL-8 and TNFα in the culture supernatants. The cytokine profile of stimulated PBMC in the presence or absence of SEQ ID NO: 1 was monitored after 4 or 24 hours of treatment. The release of all 4 cytokines was significantly reduced by SEQ ID NO: 1 in both LPS- and LTA-stimulated cells after 4 hr of treatment, and this anti-inflammatory activity was sustained over 24 hr (
In contrast, SEQ ID NO: 1 enhanced TNFα and IL-6 production by CpG-stimulated PBMC and IL-6, IL-8 and (modestly) TNFα by PBMC stimulated with IL-1β (
Table 66 lists percent inhibition or enhancement of agonist-induced cytokine production by SEQ ID NO: 1. PBMC were incubated alone or with TLR agonists (LPS, LTA, CpG) or inflammatory cytokines (TNFα, IL-1β) for 4 or 24 hr in the presence or absence of SEQ ID NO: 1. The concentration of IL-1β, IL-6, IL-8 and TNFα released in the tissue culture supernatant is reported. The percent inhibition of IL-1β, IL-6, IL-8 and TNFα in the presence of SEQ ID NO: 1±the standard deviation of 3 biological repeats is reported, as well as the fold enhancement of cytokine production in the presence of SEQ ID NO: 1±the standard deviation of 3 biological repeats.
LPS-induced gene expression profile is altered by SEQ ID NO: 1. Human 21K oligo-based DNA microarrays were probed to elucidate the impact of SEQ ID NO: 1 on LPS stimulation of gene responses in human monocytic cells. Transcriptional responses were analyzed following 1, 2, 4 and 24 hr of stimulation to provide a temporal profile of gene expression in monocytes equivalent to the early, intermediate and late stages of innate immune responses. Microarray analyses were performed in duplicate from three independent biological replicates. Statistically significant, differentially expressed genes were defined as those with a fold change of at least 1.5 with a Student's t-test p-value≦0.05 (MIAME compliant data was deposited to ArrayExpress). The number of differentially expressed genes was greatest at the 2 and 4 hr time points. Over the monitored time period, 561 and 410 genes were differentially regulated in the presence of LPS, without or with SEQ ID NO: 1 respectively. Of the 561 genes that were differentially expressed in LPS-stimulated cells, only 39 (˜7%) were identified as being up-regulated in cells stimulated with LPS in the presence of SEQ ID NO: 1 (Table 67). At least 163 genes that were upregulated in cells stimulated with LPS (i.e., proinflaninatory genes) were suppressed in the presence of SEQ ID NO: 1 (Table 68). This indicates that SEQ ID NO: 1 effectively suppressed the induction of a large subset of LPS-responsive genes, but maintained a modest subset of genes that function in promoting some aspects of inflammation or anti-inflammatory response.
Drosophila Bicaudal D Homolog 1
Given that LPS has been known to induce inflammatory responses via the TLR4 to NFκB pathway (Chow JC, et al. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999; 274:10689-92) and the product of certain differentially expressed genes in the microarray analysis were associated with this pathway, we analyzed in more detail the NFκB-regulated genes and the TLR4 pathway. This pathway was first mapped by integrating protein:protein interaction, signal transduction and regulatory data from the literature into Cytoscape (www.cytoscape.org), an open-source bioinformatics software platform for visualizing molecular interaction networks and integrating these interactions with other data. The microarray expression data was then overlaid onto this signal transduction protein network by colour coding the individual nodes (equivalent to specific genes/proteins) according to the extent of regulation (ranging from red to green, where the intensity of colour demonstrated the extent of up- to down regulation respectively). This then provided a graphic illustration of the genes with altered expression in response to LPS in the absence or presence of SEQ ID NO: 1 at each of the time points (
To investigate further whether a defined portion of the LPS-responsive genes were likely co-regulated by NFκB, LPS-responsive, differentially-expressed genes with similar temporal expression profiles were clustered using the K-means procedure, a non-hierarchical algorithm, with an affinity threshold of 85% (
SEQ ID NO: 1 selectively modulates the transcription of specific LPS-induced inflammatory genes. Using qPCR, the expression profiles were validated for 14 of 20 selected genes differentially expressed according to the microarray analysis (
From the temporal transcriptional profiling of LPS-induced genes, it was concluded that SEQ ID NO: 1 did not substantially affect the LPS-induced expression of selected genes that are required for cell recruitment and movement (chemokines) or negative regulators of NFκB. In contrast, SEQ ID NO: 1 neutralized the expression of genes coding for inflammatory cytokines, NFκB1 (p105/p50) and TNFα-induced pro-inflarnmatory genes such as TNFAIP2.
SEQ ID NO: 1 significantly inhibits LPS-induced translocation of the NFκB subunits p50 and p65. The above data indicated that although LL-37 reduced TNFα secretion by more than 95% at all time points, it had a lesser effect (58-87%) in reducing TNFα transcription. To study this in more detail we investigated the key transcription factor NFκB. TLR activation results in nuclear translocation of NFκB, the key transcription factor required for expression of many innate immunity and inflammatory genes (Bonizzi G, et al. The two NF-B activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004; 25:280-8; Li Z W, et al. Genetic dissection of antigen receptor induced-NF-kappaB activation. Mol Immunol 2004; 41:701-14). Although NFκB has a number of subunits with different primary transcriptional regulatory functions, the p50/p65 NFκB heterodimer is most commonly implicated in the regulation of immunity genes. Nevertheless, transcriptionally active NFκB heterodimers other than p50/p65 have important functions as it has been shown that they can influence gene responses to bacterial molecules as well as susceptibility to a variety of infections (Tato CM, et al. Host-Pathogen interactions: Subversion and utilization of the NF-κB pathway during infection. Infect Immunity 2002; 70:3311-7; Mason N, et al. Cutting edge: identification of c-Rel-dependent and -independent pathways of IL-12 production during infectious and inflammatory stimuli. J Immunol 2002;168:25904). To determine if SEQ ID NO: 1 suppressed LPS-induced changes in gene expression by affecting NFκB translocation into the nucleus, the nuclear localization of five NFκB subunits was assessed by Western blots. All monitored subunits of NF-κB (p105/50, p65, c-Rel, Rel B and p100/52) were detected in the nuclear extracts of THP-1 cells (
To more accurately quantify the translocation of p50 or p65, the nuclear extracts were analyzed by ELISA-based immunoassays specific for these subunits (
To evaluate the anti-endotoxic activity of SEQ ID NO: 1, two different concentrations of LPS, 10 ng/ml and 100 ng/ml respectively, were used to stimulate human monocytic cells in the presence or absence of this host defense peptide, in an attempt to reflect concentrations of endotoxin ranging from the presumably low concentrations secreted by the normal flora (homeostatic conditions) and early in infection, to those observed in septic infections. To date there has been considerable controversy concerning the role of SEQ ID NO: 1 in human infections, particularly at physiological concentrations. Direct antimicrobial action will certainly occur at low salt concentrations but in the presence of more physiological concentrations of Na+(130 mM) and Mg2+/Ca2+(1-2 mM) found in tissues and in tissue culture medium (as employed here), SEQ ID NO: 1 has weak or no direct antimicrobial action at the peptide concentrations (1-5 μg/ml) apparently present at mucosal surfaces (Bowdish DM, et al. Impact of SEQ ID NO: 1 on anti-infective immunity. J Leukoc Biol 2005; 77:451-9). Nevertheless there is clear evidence of an anti-infective role (Scott MG, et al. The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J Immunol 2002; 169:3883-91; Bowdish DM, et al. Impact of LL-37 on anti-infective immunity. J Leukoc Biol 2005; 77:451-9; Kirikae T, et al. Protective effects of human 18-kilodalton cationic antimicrobial protein (CAP-18)-derived peptide against murine endotoxemia. Infect Immun 1998; 66:1861-8; Fukumoto K, et al. Effect of antibacterial cathelicidin peptide CAP 18/LL-37 on sepsis in neonatal rats. Pediatr Surg Int 2005; 21:20-4; Ciornei CD, et al. Antimicrobial and chemoattractant activity, Lipopolysaccharide neutralization, cytotoxicity, and inhibition by serum of analogs of human cathelicidin LL-37. Antimicrob Agents Chemother 2005; 49:2845-50), which could be explained if SEQ ID NO: 1 has a role in modulating innate immunity. Consistent with this concept, at physiological concentrations SEQ ID NO: 1 is able to mediate chemotaxis (Agerberth B, et al. The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 2000; 96:3086-93; Yang D, et al. Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37). J Leukoc Biol 2001; 69:691-7; Niyonsaba F, et al. A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis. Immunology 2002;106:20-6), MAP kinase phosphorylation (Scott MG, et al. The human antimicrobial peptide LL-37 is a multiftuctional modulator of innate immune responses. J Immunol 2002; 169:3883-91; Tjabringa G S, et al. The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 2003; 171:6690-6; Bowdish D, et al. The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004;. 172:3758-65; Lau YE, et al. Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells. Infect Immun 2005; 73:583-91), Ca2+mobilization (Niyonsaba F, et al. Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells. Eur J Immunol; 2001; 31:1066-75) and IL-8 release in GM-CSF treated monocytes (Bowdish D M, et al. Impact of LL-37 on anti-infective immunity. J Leukoc Biol 2005; 77:451-9), and as shown herein, anti-endotoxic activity.
The sole human cathelicidin peptide, SEQ ID NO: 1, has been shown to protect animals against endotoxemia/sepsis. Low, physiological concentrations of SEQ ID NO: 1 (≦1 μg/ml) are able to modulate inflammatory responses by inhibiting the release of the pro-inflammatory cytokine TNFα in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile, and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of SEQ ID NO: 1. SEQ ID NO: 1 significantly inhibited the expression of specific pro-inflammatory genes upregulated by NFκB in the presence of LPS, including NFκB1 (p105/p50) and TNFα-induced protein 2 (TNFAIP2). In contrast, SEQ ID NO: 1 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNFα-induced protein 3 (TNFAIP3) and the NFκB inhibitor, NFκBIA, or certain chemokine genes that are classically considered pro-inflammatory. Nuclear translocation, in LPS-treated cells, of the NFκB subunits p50 and p65 was reduced >50% in the presence of SEQ ID NO: 1, demonstrating that the peptide altered gene expression in part by acting directly on the TLR to NFκB pathway. SEQ ID NO: 1 almost completely prevented the release of TNFα and other cytokines by human peripheral blood mononuclear cells (PBMC) following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNFα or IL1β. Biochemical and inhibitor studies were consistent with a model whereby SEQ ID NO: 1 modulated the inflammatory response to LPS/endotoxin and other agonists of TLRs by a complex mechanism involving multiple points of intervention.
The data presented herein conclusively demonstrates that endotoxin-induced inflammatory gene responses and cytokine secretion in monocytes were suppressed by low, physiological concentrations of SEQ ID NO: 1, implicating SEQ ID NO: 1 in the regulation and control of pro-inflammatory responses associated with pathogenic assault and, by extension, with homeostatic levels of TLR agonists secreted by commensals. The data further demonstrates that SEQ ID NO: 1 can suppress LPS-induced NFκB translocation, and exert an anti-inflammatory effect that is not restricted to endotoxin-induced inflammation. In the human THP-1 monocytic cell line as well as in human PBMC, SEQ ID NO: 1 suppressed pro-inflammatory cytokine production induced by LPS as well as other agonists of TLR2 (LTA, PAM3CSK4) and in part TLR9 (CpG), but selectively enhanced responses to the pro-inflammatory cytokines IL 1β and TNFα. To gain mechanistic insight, transcriptional responses were profiled using microarrays and real time PCR over the course of 1 to 24 hr to study the effects of SEQ ID NO: 1 on LPS-stimulated monocytes. While the transcription of LPS-induced pro-inflammatory cytokines peaked at 2-4 hr and waned by 24 hr, a single, low dose of SEQ ID NO: 1 suppressed pro-inflammatory cytokine secretion by 1 hr, and this effect was sustained for 24 hr.
Overall, the data provides evidence that SEQ ID NO: 1 can manipulate both pre- and post-transcriptional events to modulate the TLR-induced inflammatory response in monocytes. A model consistent with the data in this manuscript is outlined in
LPS-induced activation of NFκB is mediated by TLR4, a receptor containing TIR domain. It is known that receptors with TIR domains are potent activators of NFκB, as well as several other transcription factors such as AP-1, NF-IL6 and IRF3/7 (Takeda K, et al. Toll receptors and pathogen resistance. Cell Microbiol 2003;5:143-53). Mice deficient in TLR4 or MD2 are hyposensitive to LPS, moreover expression of some NFκB target genes is defective without MD2 (Poltorak A, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998; 282:2085-8; Hoshino K, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749-52; Nagai Y, et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol 2002;3:667-72). NFκB is known to play a central role in pathogenesis resulting in sepsis (Brown M A, et al. NF-kappaB action in sepsis: the innate immune system and the heart. Front Biosci 2004; 9:1201-17; Xiao C, et al. NF-kappaB, an evolutionarily conserved mediator of immune and inflammatory responses. Adv Exp Med Biol 2005; 560:41-5) as well as innate immunity to infections (Alcamo E, et al. Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment. J Immunol 2001; 167:1592-600; Senftleben U, et al. IKKbeta is essential for protecting T cells from TNFalpha-induced apoptosis. Immunity 2001;14:217-30). NFκB transcription factor is a dimeric complex of various subunits that belong to the Rel family; p105/50 (NFκB1), p100/52 (NFκB2), p65 (RelA), RelB, and c-Rel. NFκB proteins share a 300-amino acid Rel homology domain (RHD) that contains a nuclear localization sequence (NLS) and is involved in dimerization, sequence-specific DNA binding and interaction with the inhibitory IkB proteins (Ghosh S, et al. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16:225-26). The NFκB proteins form numerous homo- and hetero-dimers that are associated with specific biological responses that stem from their ability to regulate target gene transcription differentially, e.g., p50/p52 dimers function as repressors, whereas Rel A or c-Rel dimers are transcriptional activators. In contrast, RelB does not form homodimers, but instead forms stable heterodimers with either p50 or p52 to exhibit a greater regulatory flexibility, and can be either an activator (Ryseck R P, et al. RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B. Mol Cell Biol 1992; 12:674-84) or a repressor (Ruben S M, et al. I-Rel: a novel rel-related protein that inhibits NF-kappa B transcriptional activity. Genes Dev 1992; 6:745-60). Many inflammatory stimuli trigger signal transduction pathways that result in nuclear localization of NFκB and subsequent transcription of inflammatory and immunity genes encoding for cytokines, chemokines, acute phase reactants, and cell adhesion molecules. The NFκB heterodimer comprising of p50 and p65 subunits has been strongly implicated in transcriptional events triggered by the activation of pro-inflammatory cytokine receptors or TLRs (Ghosh S, et al. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16:225-260; Wang T, et al. NF-kappa B and SpI elements are necessary for maximal transcription of toll-like receptor 2 induced by Mycobacterium avium. J Immunol 2001; 167:6924-32). The activation and nuclear translocation of NFκB p50/p65 heterodimer is associated with increased transcription of genes encoding chemokines, cytokines, adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule 1 (ELAM), as well as enzymes that produce secondary inflammatory mediators and inhibitors of apoptosis (Ghosh S, et al. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998; 16:225-260). These molecules are important components of the innate immune responses to invading pathogens and are required for migration of inflammatory mediators and phagocytic cells to tissues where NFκB has been activated in response to infection or injury (Pande V, et al. NF-kappaB in human disease: current inhibitors and prospects for de novo structure based design of inhibitors. Curr Med Chem 2005; 12:357-74).
The present invention provides evidence that the host defense peptide, SEQ ID NO: 1, can partially (˜50%) reduce LPS-induced p50/p65 translocation to the nucleus, indicating that this is one mechanism whereby SEQ ID NO: 1 suppressed LPS-induced gene transcription and exerted an anti-endotoxin effect. However if SEQ ID NO: 1 were merely blocking the binding of LPS to the TLR4 receptor through inhibiting its interaction with LBP and/or the LPS receptor complex (Scott M G, et al. Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein. J Immunol 2000; 164, 549-53), it would be expected that NFκB translocation, and all NFκB-dependent transcriptional events would be inhibited to the same extent as TNFα release, that is>95%; however, this was not observed here. Instead, the effects of SEQ ID NO: 1 on NFκB subunit translocation were selective and relatively modest, and effects on LPS-stimulated transcription of NFκB-regulated genes ranged from very high, e.g., >95% for TNFαIP2 and p105/p50, to moderate (˜80%) for TNFα itself, through to almost no inhibition for other NFκB-regulated genes like TNFAI3. Similarly SEQ ID NO: 1 can protect against sepsis in animal models when administered shortly after endotoxin (Bowdish D, et al. The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004; 172:3758-65). In unpublished mouse model experiments (K. Lee, M. G. Scott and R. E. W. Hancock), it was demonstrated that 200 μg of SEQ ID NO: 1 could protect against an 80% lethal dose (400 μg) of E. coli LPS administered peritoneally. Under such circumstances, the LPS would be in 5-fold molar excess and it seems unlikely that in this situation LPS neutralization alone could explain the protection exhibited by SEQ ID NO: 1.
The data presented herein indicates that the host defense peptide SEQ ID NO: 1 can selectively regulate genes that modulate inflammatory responses by suppressing NFκB translocation leading to dysregulation (modulation) of TLR-triggered transcriptional responses. SEQ ID NO: 1 caused inhibition of LPS-triggered pro-inflammatory gene TNFAIP2, but did not neutralize the LPS-induced expression of some of the known negative regulators of NFκB such as TNFAIP3, TNIP3 and NFκBIA (IκBα). Conversely, the transcription of known LPS-induced genes that are regulated by p50/p65 (
Accordingly, the data demonstrates that SEQ ID NO: 1 selectively suppresses the pro-inflammatory response in monocytes, particularly the TLR-induced secretion of pro-inflammatory cytokines. The ability of SEQ ID NO: 1 to dampen pro-inflammatory (septic) responses would be valuable for maintaining hormeostasis in the face of natural shedding of microflora-associated TLR agonist molecules, as well as limiting the induction of systemic inflammatory syndrome/septic shock in response to moderate pathogen challenge. The anti-inflammatory effects of SEQ ID NO: 1 were observed at physiologically relevant concentrations of the peptide, and small changes in peptide concentration led to substantial impact on the cellular response to bacterial components such as LPS. SEQ ID NO: 1 thus appears to manifest multiple, complex mechanisms of action, including direct and indirect inhibition of TLR activation and transcription. The improved understanding of the mechanism(s) utilized by SEQ ID NO: 1 to selectively modulate inflammation, and thereby balance the TLR response to commensal or pathogenic bacteria indicates that endogenous cationic host defense peptides are important players in limiting over-active inflammation.
Endogenous host defense peptides are widely distributed in nature, are essentially amphipathic in nature, 12-50 amino acids in length with a net positive charge of +2 to +7 and are about 50% hydrophobic (Hancock, et al. 1999. Peptide antibiotics. Antimicrob Agents Chemother. 43:1317-1323). As shown above, certain natural host defense peptides expressed in mammalian epithelial and a variety of myeloid cells have been demonstrated to be selectively anti-inflammatory, and are able to maintain the expression of genes that are key players in innate immunity such as certain chemokines, as well as others that antagonize inflammation. In these activities, the peptides are able to act synergistically with other immune mediators such as GM-CSF (
The overall effects of synthetic peptide SEQ ID NO: 7 on CD 14+monocytes isolated from human peripheral blood mononuclear cells (PBMC) were studied by using a functional genomics and bioinformatics approach. The global transcriptional responses elicited by the peptide, and its effect on bacterial endotoxin-stimulated cells were elucidated in human monocytic cells. Gene profiling technology using DNA microarrays, followed by other transcriptional analysis, e.g., Real-time PCR, and functional analyses applied by the investigators have provided new insight into the molecular events underlying the mechanism elicited by synthetic cationic host defense peptides.
Materials and Methods
The venous blood from healthy volunteers was collected in Vacutainere collection tubes containing sodium heparin as an anticoagulant (Becton Dickinson, Mississauga, ON). White blood cells were isolated from-the buffy coat, and the PBMC was seeded maintained in physiologically relevant RPMI-1640 media (Gibco®, Invitrogen™ Life technologies, Burlington, ON), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen Life Technologies) at 37° C. in a humidified 5% (v/v) C02 incubator. Human monocytic cells, THP-1 (ATCC TIB-202) were grown in suspension in RPMI-1640 complete media as described above. They were treated with 0.3 μg/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich Canada, Oakville ON) for 24 hr, inducing plastic-adherent cells that were further rested in complete RPMI-1640 medium for an additional 24 hr prior to stimulations with various treatments.
The cells were stimulated with synthetic host defense peptide SEQ ID NO: 7 (200 μg/ml), as well as purified bacterial LPS/endotoxin (2 ng/ml) in the presence and absence of synthetic peptide SEQ ID NO: 7 for 4 hours. The cells were treated with the synthetic peptide 45 mins prior to stimulation with bacterial LPS. Highly purified LPS free of proteins and lipids was obtained from P. aeruginosa strain H103 using the Darveau-Hancock method as previously described (Darveau, et al. 1983. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa, Salmonella enterica ssp., and Typhimurium strains. J Bacteriol. 155: 831-838). The synthetic peptide SEQ IID NO: 7 was synthesized using F-moc chemistry.
Following incubation of the cells, the tissue culture supernatants were centrifuged at 1000×g for 5 min, then at 10,000×g for 2 min to obtain cell-free samples, aliquoted and stored at −20° C. prior to assay for various cytokines. Protein secretion were detected with a capture ELISA (eBioscience and BioSource International Inc., CA, USA respectively) using the tissue culture supernatants. In addition, CD14+ monocytes were isolated from the stimulated PBMC after incubation using magnetic Dynal® bead-based separation technology (Invitrogen™). RNA was isolated from the purified monocytes using RNeasy Mini kit, treated with RNase-Free DNase (Qiagen Inc., Canada) and eluted in RNase-free water (Ambion Inc., Austin, Tex., USA) as per the manufacturer's instructions. RNA concentration, integrity and purity were assessed by Agilent 2100 Bioanalyzer using RNA 6000 Nano kits (Agilent Technologies, USA). RNA was (reverse) transcribed with incorporation of amino-allyl-UTP (aa-UTP) using the MessageAmpII™ amplification kit, column purified and eluted in nuclease-free water, and labeled with mono-functional dyes, Cyanine-3 and Cyanine-5 (Amersham Biosciences), according to manufacturer's instructions. Yield and fluorophore incorporation was measured using Lambda 35 UV/VIS fluorimeter (PerkinElmer Life and Analytical Sciences, Inc., USA).
Microarray slides were printed with the human genome 21K Array-Ready Oligo Set™ (Qiagen Inc., USA) at The Jack Bell Research Center (Vancouver, BC, Canada). The slides were pre-hybridized and scanned as described (Mookherjee, et al. 2006. Modulation of the TLR-Mediated Inflammatory Response by the Endogenous Human Host Defense Peptide LL-37. J Immunol. 176: 2455-2464). Assessment of slide quality, normalization, detection of differential gene expression and statistical analysis was carried out with ArrayPipe (version 1.6), a web-based, semi-automated software specifically designed for processing of microarray data (www.pathogenomics.ca/arraypipe). Differentially expressed and statistically significant genes were selected from the miocroarray analysis as genes that induced an absolute fold change of at least 1.5 with a p-student value of ≦0.06.
Differential gene expression identified by microarray analysis was validated using quantitative real-time PCR (qPCR) using SuperScript™ III Platinum® Two-Step qRT-PCR Kit with SYBR® Green (Invitrogen Life Technologies), as described (Mookherjee, et al.). Fold changes were calculated after normalization to endogenous human GAPDH and using the comparative Ct method (Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: No. 9 e45).
Results and Discussion
Gene profiling using human 21K human DNA microarrays revealed that there were 566 genes differentially expressed in human monocytes in presence of SEQ ID NO: 7 (Table 69). Of these genes those that were significantly up-regulated by the peptide included G-coupled protein receptors, other transmembrane receptors and co-activators, genes associated with the plasma membrane including integrin, adhesion molecules such as ICAM, NCAM, genes that regulate various tyrosine-protein kinases, critical transcription factors that mediate key pathways in immune responses such as MAPK, JAK-STAT and NFκB. The peptide also up-regulated the expression of various chemotatic factors that play a central role in attracting immune cells to the site of infection and/or trauma. Other significant genes induced by the peptide were those encoding transport proteins including those involved in metal transport, several zinc finger proteins fimctioning as transcription factors, and several genes that are attributed to anti-viral activity.
The genes that were significantly upregulated by the peptide included:
(i) Genes involved in antiviral activity—representative genes include, but are not limited to Interferons e.g., IFNA2, STAT1 that activates gamma interferon, transcriptional activator MNDA, Interferons that exhibit anti-viral activities-representative genes include, but are not limited to IFNA2.
Bioinformatic analysis of the microarray data by supervised clustering of the differentially expressed genes induced by the peptide, followed by mapping of the responses to signal transduction pathways indicated that the synthetic peptide can potentially induce signaling, and activate JAK (Janus Kinases) and the STAT (signal transducers and activators of transcription) family of transcription factors. It had been previously described that the human host defense peptide LL-37 activates MAP kinases (
Transcriptional analysis of responses induced by the synthetic peptide using qPCR showed that the peptide was able to induce the expression of chemokines and interleukins such as MCP-3, MCP-1, CXCL-1, and IL-6, IL-19 (IL-10 superfamily) and IL-8, all of which are critical in cell recruitment and movement (
The peptide (SEQ ID NO: 7), as well as LL-37, also induced the expression of IL-19 (
These genes are thus markers for the biological effects of SEQ ID NO: 7. As JAK-STAT signaling is mediated by members of the IL-10 superfamily, the observation that the synthetic peptide up-regulates the expression of IL-19 further supports the conclusion that one of the signal transduction pathways activated by host defense peptides is the JAK-STAT pathway, resulting in the up-regulation of genes in the SOCS family. The peptide also appears to regulate the NFκB pathway which can in addition lead to the expression of some of the members of the SOCS family. Thus there appears to be subtle changes in the regulation of critical pathways in immune responses (JAK-STAT, NFκB and MAPK) in the presence of the peptide which potentially suggests the mechanism by which the peptide functions in combating pathogenesis. Further analysis of protein production induced by the peptide in human PBMC by ELISA confirmed that SEQ ID NO: 7 is able to induce the secretion of interleukins such as IL-6 and IL-8 in human PBMC within 4 hours of stimulation, but did not induce the expression of the pro-inflammatory cytokine TNFα (
In response to bacterial endotoxin (LPS) there were 836 differentially expressed genes in human monocytes (Table 70), and 1012 genes were differentially expressed in response to LPS in the presence of the peptide SEQ ID NO: 7 (Table 71). The peptide appeared to induce the expression of several genes synergistically only in the presence of LPS. These genes are either induced only in the presence of the peptide on LPS stimulation (not with LPS alone) and/or the LPS-induced expression is synergistically enhanced in the presence of the peptide, but not with the peptide alone. These genes included several genes that are critical in immune response in presence of pathogenic challenge such as chemotactic factor CCL20, and CCL23; cytokine receptor EBI3, factors involved in lymphocyte activation such as SLAMF1, CD58, and IL32; regulators of signal transduction such as MAP2K2 (activator of ERK1), DUSP5 (ERK phopsphatase), MAPK8IP3, RIN2 (RAS-effector), RANBP9 (GTP-binding protein that affects Ras-signaling pathway), IP3 3-kinase A,-B3ATF, IRAK3, NM1 (augments cytokine-mediated STAT transcription), SP3, RAP2C, PNRC1, NEK1, CHC1, ZNF219, ZNF593, WIF1, PIM2, CD79A, and LATS2; substrate transporters such as SLC23A3, and SLC17A5; apoptosis regulators such as BOK (Bc1-2 like inhibitor), BIRC3, TNFRSF6, and CASP9; genes associated with plasma membrane such as STIM1, BPAG1, PTPN4, TRIM36, SDK1, and FNDC5 (fibronectin type III); genes involved in selective ion transport and in mediating selective ion-channel such as VGCNL1, TRPC5, CACNA1B, KCNA6, KCNJ2, KCNA10, and AQP9; growth modulating genes and/or those that play a role in wound healing such as FGF10, and AREG; inflammatory mediators such as PTGS2, SOD2, TNFAIP8 and anti-inflammatory gene TNIP3. This indicates that one of the mechanism by which cationic host defense peptides exhibit their protective mechanism is by delicately modulating specific signal pathways only in presence of agonists such as bacterial endotoxin, thus trigger just sufficient amount of inflammation and/or immune responses that is necessary to combat pathogenesis, while also maintaining the anti-inflammatory checks in place in order to prevent excessive inflammation that can lead to sepsis.
Of the 596 genes that were up-regulated by LPS stimulation, 33 genes were suppressed in the presence of the peptide (Table 72), which included the genes for pro-inflammatory TNF-α and NF-κB2 (p52). Interestingly, the peptide alone was able to up-regulate NF-κB2 as analyzed by DNA microarrays, an observation that also has mechanistic implications as individual subunits of NFκB are known to be involved in expression of only a subset of NFκB-regulated genes (the major pair of NFκB subunits p50 and p65 are responsible for much of the pro-inflammatory gene expression). Further transcriptional analysis using qRT-PCR revealed that the peptide exhibited the ability to suppress LPS-induced gene expression of cytokines such as IL-8, IL-6 as well as chemokines such as CXCL-1 within 4 hours of stimulation (
C. albicans and is also active against E. coli and
The data in
The overall data extrapolated from this study indicates that the efficacy of host defense peptides can be improved significantly and specifically in order to obtain novel therapeutics that not only have anti-inflammatory properties, but also can specifically modulate responses in critical pathways that are involved in host defenses in pathogenesis.
LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. It is a multifimctional modulator of innate immune responses demonstrating an ability to modulate gene regulation in certain cells, to alter cytokine expression in macrophages, to demonstrate chemotactic activity for neutrophils, monocytes and T cells, and to neutralize the endotoxic effects of lipopolysaccharide (LPS). However, the underlying mechanisms determining these effects of LL-37 were not clear. The general aim of this example is to determine how LL-37 affects initial cell signaling and to link cell signaling to some of these observed biological functions that are described herein such as the anti-endotoxin property and the ability to modulate cytokine and chemokine production in a human monocytic THP-1 cell line and human peripheral blood mononuclear cells (PBMCs).
To meet these goals, a variety of specific approaches were applied: (1) Assessing, by RT-PCR and ELISA, the ability of LL-37 to block the LPS-induced upregulation of transcription of certain genes and production of pro-inflammatory cytokines, respectively, in THP-1 cells and PBMCs; (2) Degradation of IκBα (known to be an NF-κB negative regulator) and translocation of the transcription factor NFκB were examined in the LPS-stimulated human THP-1 cell line in the presence of LL-37; (3) Since LPS and cytokine interleukin-1α (IL-1α) share similar signaling transduction pathways, it was interesting to compare the effects of LL-37 in modulating cytokine (IL-6) and chemokine (MCP-3) production in LPS- or IL-1α-stimualted human PBMCs; (4) Inhibitors (including oxidized ATP, an agonist of the P2X7 receptor, pertussis toxin, which inhibits G-protein coupled receptors, and LY294002, a PI3 Kinase inhibitor) were used to test if the synergistic effects of LL-37 were mediated by these receptors or pathways; (5) Western blots were performed to evaluate protein kinase B (Akt) phosphorylation and the activation of downstream transcription factors e.g., assessing the phosphorylation of cAMP-responsive element binding protein (CREB) and translocation of NFκB subunit p50 in human PBMCs. These analyses are designed to test if the PI3K-Akt-IκBα-NFκB and PI3K-Akt-CREB pathways are involved in LL-37-induced modulation of cytokine and chemokine release.
Methods
Western blotting—THP-1 cells (1×106 cells per condition) or PBMCs (5×106 cells per condition) were stimulated by adding LPS, LL-37 or endotoxin-free water as a vehicle control, and incubated for 20 min at 37° C., 5% CO2. After stimulation, the cells were centrifuged, washed once with ice-cold PBS with 1 mM vanadate and nuclear extracts were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the manufacturer's instructions. The lysates were assayed for protein concentration using a BCA assay (Pierce). 7.5 μg nuclear lysate and 15 μg cytoplasm lysate were loaded per lane, resolved on a 8-10% SDS-PAGE at 120 V for 1 hr, and transferred to Immuno-blot PVDF membranes (Bio-Rad) for 75 min at 100 V. Immunoblot was performed using 1/1000 dilution of anti-p50, anti-total IκBα, anti-p-IκBα and anti-Akt (cell signaling) monoclonal antibodies in TBST/milk for 1 hour at room temperature. Membranes were washed for 30 min in TBST and then incubated with a 1/5000 dilution of HRP-conjugated goat anti-mouse or anti-rabbit Ab (in TBST/milk) for 1 hour. The membranes were washed for 30 min in TBST and developed with chemiluminescence peroxidase substrate (Sigma-Aldrich), according to manufacturer's instructions. The blots were reprobed with anti-total Akt or anti-GAPDH antibody as loading control.
Detection of cytokines—Fresh human PBMCs were plated at 8×105 cells in 1 ml of RPMI 1640 media (supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) L-glutamine, 1 nM sodium pyruvate) respectively in 24 well plates. Cells were then incubated in media for periods of 24 hours in the presence of Pseudomonas aeruginosa PAO1 strain H103 LPS, LL-37, IL-1α (at the concentrations stated above), or endotoxin-free water as a vehicle control, in at least triplicate. Supernatants were collected and stored at −20° C. until use. The concentrations of IL-6 (eBioscience) and MCP-3 (R&D system) in the supernatants were measured using commercially prepared ELISA plates in accordance to the manufacturer's suggestion.
Results
LL-37 alone (20 μg/ml) caused IκBα degradation: protein levels of total IκBα diminished within 30 min and returned to control levels by 60 min in THP-1 cells, indicating that LL-37 may directly modulate elements of the LPS signaling pathway. Results are representative of three independent experiments (
Fresh isolated human PBMCs were incubated with IL-1β (10 ng/ml) or LPS (100 ng/ml) in absence or presence of LL-37 (20 ug/ml) for 24 hours. IL-6 and MCP-3 ELISA were performed to measure the level of protein release (
Cells were incubated with LL-37 alone or IL-1β (10 ng/ml) with or without LL-37 (20 μg/ml). Western blots were performed for cytoplasm protein and nuclear protein, showing that combined treatment of LL-37 and IL-1β showed higher IκBα phosphorylation after 30 min and p50 nuclear translocation after 60 min than LL-37 or IL-1β treatment alone in human PBMCs (
The immunomodulatory effects of LL-37 have been proposed to be dependent on signaling through a number of receptors, including the G-protein coupled receptor FPRL-1 and the purinergic receptor P2X7. Human PBMCs were pre-incubated with pertussis toxin (PTx, a G-protein coupled receptor inhibitor), oxidised ATP (OATP, a P2X7 receptor inhibitor) before exposure to LL-37 with or without IL-1β. MCP-3 production that resulted from combined treatment with LL-37 and IL-1β was dramatically inhibited by pre-treatment of PTx and partially blocked by oATP preincubation.
Since activated G-protein coupled receptor can lead to the elevated signaling of phosphatidylinositol 3-kinase (PI3 kinase) pathway, the role of PI3 kinase signaling in the above effects was further examined. Human PBMCs were pre-treated with PI3 kinase inhibitor, LY294002 (25 μM) for lh and following incubation with IL-1β (10 ng/ml) in presence or absence of LL-37 (20 μg/ml) for 24 hours (FIGS. 22A-22D). The LL-37 plus IL-1β mediated production of IL-6 and MCP-3 was significantly inhibited by LY294002 pre-incubation, indicating that PI3 kinase plays a role in LL-37-induced modulation of cytokine and chemokine production. Results are two representatives of three donors for human PBMCs.
Activation of PI3 kinase causes activation of a number of intracellular signal transduction pathways, including phosphorylation of the downstream target protein kinase B (Akt). Cytoplasm protein was isolated from human PBMCs treated with LL-37 with or without IL-1β. Western blot analysis showed that phosphorylation of Akt was observed in human PBMCs after exposure to LL-37 for 30 min (
The transcription factor CREB is downstream of the PI3 kinase→Akt pathway that leads to phosphorylation and activation of CREB and resulting in transcriptional up-regulation of a broad array of cellular responses. Western blot analysis showed that phosphorylation of CREB was observed in human PBMCs after exposure to LL-37 for 30 min and 60 min (
Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
This application claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 11/241,882, filed Sep. 29, 2005, which is a continuation-in-part of U.S. patent application Ser. No. 10/661,471, filed Sep. 12, 2003, which is a continuation-in-part of U.S. patent application Ser. No. 10/308,905, filed Dec. 2, 2002, which claims priority under 35 U.S.C. §119(e) to U.S. patent application Ser. No. 60/336,632, filed Dec. 3, 2001, herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60336632 | Dec 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11241882 | Sep 2005 | US |
| Child | 11400411 | Apr 2006 | US |
| Parent | 10661471 | Sep 2003 | US |
| Child | 11241882 | Sep 2005 | US |
| Parent | 10308905 | Dec 2002 | US |
| Child | 10661471 | Sep 2003 | US |
