Patent Grant
10799520
Provided herein are compositions comprising lipotechoic acid (LTA) and methods of use thereof for immune modulation. In particular, compositions comprising S. epidermidis lipotechoic Acid (SELTA) are provided, as well as method of use thereof for the treatment of immune- (e.g., autoimmune) and inflammation-related conditions and diseases (e.g., chronic pelvic pain syndrome (CPPS), arthritis, etc.).
Staphylococcus epidermidis (SE) is a commensal bacterium of the skin (ref. 7; incorporated by reference in its entirety) that has a role in mediating local immune homeostasis (ref 11; incorporated by reference in its entirety). During local immune activation it can dampen inflammation signals (ref. 27; incorporated by reference in its entirety) and prevent pathogenic superinfection by species such as S. aureus (refs. 1-2; incorporated by reference in their entireties). SE also acts as a facultative pathogen forming biofilms on surgically implanted materials (refs. 5, 21, 49; incorporated by reference in their entireties). Recently, a more distinct method of immune interaction via specific sub-dermal dendritic cells has been described (ref. 26; incorporated by reference in its entirety) involving activation of the IL1 and IL17 signaling pathways. The cell wall LTA is an immunogenic pathogen associated molecular pattern (PAMP) and signals via TLR2 (refs. 16, 29; incorporated by reference in their entireties). TLR2 mediates signaling responses by forming homo or hetero dimers with TLR1 and/or TLR6. Resulting downstream signaling is determined by which dimer is engaged and which signaling intermediaries are utilized.
Chronic pelvic pain syndrome (CPPS) is a disorder characterized by pelvic pain and frequently lower urinary tract symptoms (LUTS). CPPS is classically not associated with bacterial infection but prostate specific bacterial strains have been isolated from the expressed prostatic secretions (EPS) of both CPPS patients and controls. Patient-derived gram-negative uropathogenic E. coli (UPEC) bacteria has been shown to establish tactile allodynia in male NOD mouse by initiating a Th17 mediated prostate immune activation (ref. 32; incorporated by reference in its entirety). These effects were restricted to the genetically susceptible NOD mice compared to C57BL/6 mice. Pelvic tactile allodynia and immune activation persisted even after bacterial clearance (refs. 32, 37; incorporated by reference in their entireties).
Provided herein are compositions comprising lipotechoic acid (LTA) and methods of use thereof for immune modulation. In particular, compositions comprising S. epidermidis lipotechoic acid (SELTA) are provided, as well as method of use thereof for the treatment of immune- (e.g., autoimmune) and inflammation-related conditions and diseases (e.g., chronic pelvic pain syndrome (CPPS), arthritis, etc.).
In some embodiments, provided herein are compositions formulated for administration to a subject comprising Staphylococcus epidermidis lipotechoic acid (LTA). In some embodiments, compositions comprise a probiotic comprising live S. epidermidis bacteria. In some embodiments, the S. epidermidis bacteria comprise the NPI strain. In some embodiments, compositions comprise isolated LTA. In some embodiments, the isolated LTA is derived from the cell of S. epidermidis bacteria. In some embodiments, the isolated LTA is synthesized (e.g., chemical synthesis of LTA molecules) and/or formulated synthetically (e.g., combination of LTA molecules to generate an LTA composition of specific LTA make-up). In some embodiments, the LTA comprises one or more chemical species of Formula I:
wherein n is 2-100; wherein each R is any suitable amino acid, saccharide, or H; wherein S1 and S2 are monosaccharides connected by any suitable linker; wherein G is a glyceride moiety; and wherein FA1 and FA2 are fatty acids. In some embodiments, one or more R is D-alanine, a monosaccharide, or H. In some embodiments, S1 and S2 are independently D-glucose, fructose, mannose, galactose, glucosamine, xylopyranose, or rhamnose, connected by linker selected from α(1→2), α(1→3), α(1→6), β(1→2)β, β (1→2), β (1→3), and β (1→6). In some embodiments, FA1 and FA2 are independently saturated or unsaturated fatty acids of any suitable length.
In some embodiments, provided herein are methods of treating an inflammation- and/or immune-related disease or condition in a subject, the method comprising administering to the subject a composition comprising Staphylococcus epidermidis lipotechoic acid (LTA) described herein. In some embodiments, provided herein is the use of a composition comprising Staphylococcus epidermidis lipotechoic acid (LTA) described herein for the treatment of an inflammation- and/or immune-related disease or condition in a subject. In some embodiments, an immune response of the subject is modulated. In some embodiments, the disease is an autoimmune disease. In some embodiments, inflammation is reduced in the subject. In some embodiments, the disease is chronic pelvic pain syndrome (CPPS). In some embodiments, the disease is arthritis (e.g., rheumatoid arthritis). In some embodiments, the composition is administered orally. In some embodiments, the composition is administered rectally.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a probiotic agent” is a reference to one or more probiotic agents and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “and/or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and/or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and/or C.” As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry (e.g., chickens), fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a human subject that is being treated for a disease or condition.
As used herein, the term “effective amount” refers to the amount of a composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) (e.g., a pharmaceutical composition comprising beneficial bacteria, and/or additional therapeutics) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety.
As used herein, a “prebiotic” refers to an ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that may (or may not) confer benefits upon the host. In some embodiments, a prebiotic is a comestible food or beverage or ingredient thereof. In some embodiments, a prebiotic is a selectively fermented ingredient. Prebiotics may include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition. Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carregenaan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides.
The term “species” is defined as collection of closely related organisms with greater than 97% 16S ribosomal RNA sequence homology and greater than 70% genomic hybridization and sufficiently different from all other organisms so as to be recognized as a distinct unit (e.g., an operational taxonomic unit).
The term “strain” as used herein in reference to a microorganism describes an isolate of a microorganism considered to be of the same species but with a unique genome and, if nucleotide changes are non-synonymous, a unique proteome differing from other strains of the same organism. Strains may differ in their non-chromosomal genetic complement. Typically, strains are the result of isolation from a different host or at a different location and time, but multiple strains of the same organism may be isolated from the same host.
As used herein, the term “microbiota” refers to an assemblage of microorganisms localized to a distinct environment. Microbiota may include, for example, populations of various bacteria, eukaryotes (e.g., fungi), and/or archaea that inhabit a particular environment. For example, “gut microbiota,” “vaginal microbiota,” and “oral microbiota” refer to an assemblage of one or more species of microorganisms that are localized to, or found in, the gut, vagina, or mouth, respectively.
“Normal microbiota” refers to a population of microorganisms that localize in a particular environment in a normal, non-pathological state (e.g., a sample of gut microbiota from a subject without sepsis). A “normal microbiota” has normal membership and normal relative abundance.
“Abnormal microbiota” refers to a population of various microorganisms that localize in a particular environment in a subject suffering from or at risk of a pathological condition (e.g., a sample of gut microbiota from a subject with sepsis). Abnormal microbiota differs from normal microbiota in terms of identity (e.g., membership), absolute amount, or relative amount (e.g., relative abundance) of the various microbes.
As used herein, the term “commensal microbe” refers to a microorganism that is non-pathogenic to a host and is part of the normal microbiota of the host.
As used herein, the term “relative abundance” relates to the abundance of microorganisms of a particular taxonomic unit or OTU in a test biological sample compared to the abundance of microorganisms of the corresponding taxonomic unit or OTU in one or more non-diseased control samples. The “relative abundance” may be reflected in e.g., the number of isolated species corresponding to a taxonomic unit or OTU or the degree to which a biomarker specific for the taxonomic unit or OTU is present or expressed in a given sample. The relative abundance of a particular taxonomic unit or OTU in a sample can be determined using culture-based methods or non-culture-based methods well known in the art. Non-culture based methods include sequence analysis of amplified polynucleotides specific for a taxonomic unit or OTU or a comparison of proteomics-based profiles in a sample reflecting the number and degree of polypeptide-based, lipid-based, polyssacharide-based or carbohydrate-based biomarkers characteristic of one or more taxonomic units or OTUs present in the samples. Relative abundance or abundance of a taxon or OTU can be calculated with reference to all taxa/OTUs detected, or with reference to some set of invariant taxa/OTUs.
Methods for profiling the relative abundances of microbial taxa in biological samples, including biological samples of gut microbiota, are well known in the art. Suitable methods may be sequencing-based or array-based. For example, the microbial component of a gut microbiota sample is characterized by sequencing a nucleic acid suitable for taxonomic classification and assigning the sequencing reads to operational taxonomic units (OTUs) with a defined (e.g., >97%) nucleotide sequence identity to a database of annotated and representative sequences. An example of such a database is Greengenes version of May 2013; however, any suitable database may be used. After OTUs are defined, a representative sequence from each OTU can be selected and compared to a reference set. If a match is identified in the reference set, that OTU can be given an identity. Relative abundance of a bacterial taxon may be defined by the number of sequencing reads that can be unambiguously assigned to each taxon after adjusting for genome uniqueness. Other methods of profiling the relative abundances of microbial taxa in biological samples are known within the field and within the scope herein.
In some embodiments, a suitable nucleic acid for taxonomic classification is universally distributed among the gut microbial population being queried allowing for the analysis of phylogenetic relationships among distant taxa, and has both a conserved region and at least one region subject to variation. The presence of at least one variable region allows sufficient diversification to provide a tool for classification, while the presence of conserved regions enables the design of suitable primers for amplification (if needed) and/or probes for hybridization for various taxa at different taxonomic levels ranging from individual strains to whole phyla. While any suitable nucleic acid known in the art may be used, one skilled in the art will appreciate that selection of a nucleic acid or region of a nucleic acid to amplify may differ by environment. In some embodiments, a nucleic acid queried is a small subunit ribosomal RNA gene. For bacterial and archaeal populations, at least the V1, V2, V3, V4, V5, V6, V7, V8, and/or V9 regions of the 16S rRNA gene are suitable, though other suitable regions are known in the art. Guidance for selecting a suitable 16S rRNA region to amplify can be found throughout the art, including Guo et al. PLOS One 8(10) e76185, 2013; Soergel D A W et al. ISME Journal 6: 1440, 2012; and Hamady M et al. Genome Res. 19:1 141, 2009, each hereby incorporated by reference in its entirety.
Provided herein are compositions comprising lipotechoic acid (LTA) and methods of use thereof for immune modulation. In particular, compositions comprising S. epidermidis lipotechoic Acid (SELTA) are provided, as well as method of use thereof for the treatment of immune- (e.g., autoimmune) and inflammation-related conditions and diseases (e.g., chronic pelvic pain syndrome (CPPS), arthritis, etc.).
CPPS, for example, is a debilitating syndrome with a wide range of symptoms, the severity of which varies significantly between patients. Lack of understanding of the complex etiology of the disorder has hampered effective treatment development. The data presented herein describe the use of a commensal bacterial strain, or compositions (e.g., LTA) derived therefrom, to ameliorate tactile allodynia in an autoimmune murine model of CPPS. These data indicate that the bacterial flora of the prostate facilitate resetting of the immune milieu of diseased prostates and thus counteract EAP induced tactile allodynia responses. Additionally, these data indicate that in some embodiments, the commensal bacterial strains described herein, or compositions (e.g., LTA) derived therefrom, find use in the treatment of a variety immune- (e.g., autoimmune) and inflammation-related conditions and diseases.
Aged male NOD mice develop spontaneous prostatitis with incidences matching the pancreatitis observed in female mice (40-60%) (refs. 10, 18, 34; incorporated by reference in their entireties). Induction of prostatitis can be hastened in these mice by intraurethral infection with a specific strain of E. coli isolated from human EPS (CP-1 model) (ref. 37; incorporated by reference in its entirety). Increases in CD4+ve T-cell derived IL17A is necessary for initiation of pelvic tactile allodynia in the EAP murine model (refs. 32, 39; incorporated by reference in its entirety). Analysis of human EPS samples demonstrated that IL7, a cytokine involved in maintenance of activated CD4 T-cells (ref. 25; incorporated by reference in its entirety) is increased in CPPS patients and correlated with pain severity. Development of tactile allodynia responses in the CP-1 infection model is dependent on host genetic background and specific to certain bacterial strains (refs. 37-38; incorporated by reference in their entireties). Compared to C57BL/6 mice, NOD mice are susceptible to induction of tactile allodynia by CP1 (refs. 37-38; incorporated by reference in their entireties). NOD mice develop spontaneous prostatitis with age (refs. 8, 10, 18; incorporated by reference in their entireties). Data presented here demonstrated that NPI instillation does not induce referred allodynia in this background. Furthermore, it did not show increases in expression of CD4 T-cell markers associated with activation of Th1, Th2, or Th17 immune responses that are associated with CP1 instillation. These data demonstrate the commensal properties of the NPI strain, indicating that the local immune response can tolerate chronic colonization with the bacteria, even in a susceptible mouse background.
The EAP autoimmune model of CPPS induces tactile allodynia in NOD and C57BL/6 mice which is mediated, at least in part, by increases in levels of CD4+ve T-cells expressing IL17 (ref. 25; incorporated by reference in its entirety). Instillation of NPI into C57BL/6 mice with EAP reverses both tactile allodynia responses and associated increases in IL17 expressing cells at the level of the prostate and iliac lymph nodes. The effect of bacterial instillation on referred allodynia was detectable as early as one-day post treatment and remained for seven days. Modulation of T-cell immunity was concomitant with reduction in tactile allodynia. S. epidermidis species have been shown to prevent super-infection (ref 1; incorporated by reference in its entirety) of the skin via modulation of the local innate immune response (ref. 2; incorporated by reference in its entirety) and additional studies have focused on the direct interaction between bacteria and neurons (refs. 6, 22; incorporated by reference in its entirety). These indicate that bacterial components can directly interact with neural pathways to influence pain in acute infection, in the absence of classical pathogen-associated molecular pattern (PAMP) activation (refs. 6, 22; incorporated by reference in its entirety). Reduction of IL17 alone in the EAP model is ineffective at reducing tactile allodynia response (ref 25; incorporated by reference in its entirety).
Numerous studies that have detected bacteria and bacterial components in EPS and post-prostate massage voided bladder samples of patients (refs. 4, 13, 19, 28, 46; incorporated by reference in their entireties). The exact nature of this flora, whether it is transient or permanent and its role in diseases of the male urogenital tract has not been previously evaluated. Experiments conducted during development of embodiments herein demonstrate that the effects of NPI instillation are highly specific compared to another S. epidermidis strain isolated from a patient with CPPS. This bacterium, designated 7244, showed no capacity to reduce tactile allodynia in EAP treated C57BL/6 mice but could colonize the prostate as effectively as NPI. Infection with 7244 did not increase referred pain responses above EAP induced levels.
The LTA component of the gram-positive cell wall is highly immunogenic and has been shown to activate members of the TLR family of receptors (refs. 16, 20, 29, 33, 36; incorporated by reference in their entireties). Experiments conducted during development of embodiments herein demonstrate the specificity of SELTA to reduce tactile allodynia compared to SALTA and BSLTA. This effect may dependent on differences in superstructure of the various LTA, which have been shown to vary significantly between bacterial strains resulting in unique downstream signaling effects and receptor utilization (ref. 20; incorporated by reference in its entirety); although embodiments herein are not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice such embodiments. SELTA increases expression of the CTLA-4-like ligands PDL1 and PDL2 on the surface of CD11b+ve monocytes.
IL10 secretion and FoxP3 expressing T-regulatory cells have been shown in multiple studies and disease models to dampen the effects of CD4+ve IL17 expressing activated T-cells (refs. 15, 17, 23-24, 40, 50; incorporated by reference in their entireties). Experiments conducted during development of embodiments herein demonstrate that IL10 is necessary for the effect of NPI instillation on tactile allodynia, using IL10 knockout mice, correlates neatly with the observed decreases of IL17 in these mice. Blockade of either CD25 (used here as a surrogate marker for T-regulatory cells) or the PD1 receptor, by antibody treatment, prevents SELTAs amelioration of the tactile allodynia induced by EAP. This data further indicates PD1-PDL1/2 pathway involvement SELTA activity, while also further implicating the role of T-regulatory cells in mediating these effects.
Experiments conducted during development of embodiments herein demonstrate the capacity of a commensal bacteria (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) and cell wall components thereof (e.g., LTA) to reduce referred allodynia in a murine model of CPPS. Experiments conducted during development of embodiments herein demonstrate not only the specificity of bacterial isolates from the human prostate in modulating behavioral and immune responses but highlight also the role of the host immune response in these effects.
Experiments conducted during development of embodiments herein indicated that certain strains and species of bacteria, for example, Staphylococcus epidermidis, and strains thereof, for example, the NPI strain, are useful for modulating host immune responses and for treating/preventing immune-related and/or inflammation-related diseases and conditions in a subject.
Experiments conducted during development of embodiments herein demonstrate the capacity of a LTA (or a commensal bacteria that produces LTA (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) or cell wall components thereof (e.g., LTA)) to reduce referred allodynia and arthritis in a murine collagen induced arthritis (CIA) model (e.g., an accepted model of rheumatoid arthritis).
Experiments conducted during development of embodiments herein indicated that certain strains and species of bacteria, for example, Staphylococcus epidermidis, and strains thereof, for example, the NPI strain, are useful for modulating host immune responses and for treating/preventing immune-related and/or inflammation-related diseases and conditions in a subject.
In some embodiments, an immune response (e.g., autoimmune response, etc.) is dependent upon conditions (e.g. local conditions (e.g., organ or tissue specific conditions), systemic conditions) within the subject (e.g., microbiota). In particular, the identity or characteristics (e.g., concentration or level) of the microflora within a subject affects a subject's own immune response. In some embodiments, the presence or level of one or more microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) in a subject modulates immune response. In some embodiments, the absence or a decreased level of one or more microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) in a subject results in increased immune response (e.g., autoimmune response). In some embodiments, the absence and/or decreased level of one or more microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) causes one or more diseases of medical conditions. In some embodiments, the presence or increased level of one or more microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) in a subject promotes evasion of immune response (e.g., an autoimmune response, a detrimentally severe immune response, etc.). In some embodiments, the presence or increased level of one or more microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) in a subject facilitates treatment of a disease or condition related to inflammation, immune response, and/or autoimmunity.
In some embodiments, the presence of beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) in a subject (e.g., in a local environment within a subject) creates an environment or microenvironment (e.g., metabolome) that is conducive to the treatment of immune-related, autoimmune-related, and/or inflammation-related diseases and/or conditions. In some embodiments, the presence of detrimental microbes in a subject (e.g., in a local environment within a subject) creates an environment or microenvironment (e.g., metabolome) that is conducive to immune-related, autoimmune-related, and/or inflammation-related diseases and/or conditions and/or inhibits treatment thereof. In some embodiments, modulation of levels and/or identity of the microflora (e.g., reduce levels of detrimental microbes, increase levels of S. epidermidis, etc.) in a subject (e.g., in a local environment within a subject) facilitates treatment of immune-related, autoimmune-related, and/or inflammation-related diseases and/or conditions.
Modulation of microflora levels and/or identity may comprise encouraging or facilitating growth of one or more types of beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics), discouraging or inhibiting growth of one or more types of detrimental microbes (e.g., microbes that facilitate immune-related, autoimmune-related, and/or inflammation-related diseases and/or conditions), administering one or more types of beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) to the subject, and/or combinations thereof. Embodiments within the scope herein are not limited by the mechanisms for introducing one or more microbes (e.g., fecal transplant, probiotic administration, etc.), encouraging growth of beneficial microbes (e.g., administering agents that skew the environment within the subject toward growth conditions for the beneficial microbes), discouraging or inhibiting growth of detrimental microbes (e.g., administering agents that skew the environment within the subject away from growth conditions for the detrimental microbes, administration of antimicrobial(s), etc.), and combinations thereof.
In some embodiments, methods are provided for the treatment or prevention of immune-related, autoimmune-related, and/or inflammation-related diseases by the manipulation of the presence, amount, or relative ratio of commensal microflora (e.g., increasing the absolute or relative amount of S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics). In some embodiments, the presence, amount, or relative ratio of S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics within a subject is manipulated.
In some embodiments, microflora-modulation utilizes prepared probiotic compositions for administration to/by a subject. Probiotic compositions comprise one or more beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) formulated such that administration of the probiotic (e.g., orally, rectally, by inhalation, by injection, etc.) results in population of the subject by the beneficial microbes.
In some embodiments, probiotic compositions comprise cultured microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) that are combined and/or formulated for administration to a subject. In some embodiments, probiotics contain microbes of known genera, species, etc. and/or at known concentrations (cfus). Probiotic compositions may be in the form of a pharmaceutical-type composition (e.g., capsule, tables, liquid, aerosol, etc.) or in the form of a food supplement.
In some embodiments, probiotic microbes (e.g., bacteria including S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) are formulated in a pharmaceutically acceptable composition for delivery to a subject. In some embodiments, probiotics are formulated with a pharmaceutically acceptable carrier suitable for a solid or semi-solid formulation. In some embodiments, probiotic microbes are formulated with a pharmaceutically acceptable carrier suitable for a liquid or gel formulation. Probiotic formulations may be formulated for enteral delivery, e.g., oral delivery, or delivery as a suppository, but can also be formulated for parenteral delivery, e.g., vaginal delivery, inhalational delivery (e.g., oral delivery, nasal delivery, and intrapulmonary delivery), injection, and the like.
The probiotic compositions that find use in embodiments described herein may be formulated in a wide variety of oral administration dosage forms, with one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is a finely divided solid which is a mixture with the probiotic microbes. In tablets, the microbes are mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Other forms suitable for oral administration include liquid form preparations such as emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Aqueous suspensions can be prepared by dispersing the probiotic microbes in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
The probiotic compositions (e.g., microbes (e.g., bacteria)) may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the probiotic microbes are dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into conveniently sized molds, allowed to cool, and to solidify.
The probiotic compositions (e.g., microbes (e.g., bacteria)) may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays, may contain agents in addition to the bacteria, such carriers, known in the art to be appropriate.
In some embodiments, probiotic compositions (e.g., microbes (e.g., bacteria)) may be formulated for delivery by inhalation. As used herein, the term “aerosol” is used in its conventional sense as referring to very fine liquid or solid particles carries by a propellant gas under pressure to a site of therapeutic application. The term “liquid formulation for delivery to respiratory tissue” and the like, as used herein, describe compositions comprising probiotic microbes with a pharmaceutically acceptable carrier in flowable liquid form. Such formulations, when used for delivery to a respiratory tissue, are generally solutions, e.g. aqueous solutions, ethanolic solutions, aqueous/ethanolic solutions, saline solutions and colloidal suspensions.
Rather than pharmaceutical-type formulation, probiotic compositions may be formulated as food additive and/or food product and incorporated into a variety of foods and beverages. Suitable foods and beverages include, but are not limited to, yogurts, ice creams, cheeses, baked products such as bread, biscuits and cakes, dairy and dairy substitute foods, soy-based food products, grain-based food products, starch-based food products, confectionery products, edible oil compositions, spreads, breakfast cereals, infant formulas, juices, power drinks, and the like.
In some embodiments, a probiotic composition is administered over a dosing time period (e.g., <1 minute, <1 hour, <2 hours, <4 hours, <6 hours, <12 hours, <24 hours, etc.) in an amount that is sufficient to provide a desired therapeutic benefit (e.g., as a single dose, in combination with other doses, in combination with a co-administered therapeutic, etc.) In some embodiments, the dose of the probiotic composition administered for the dosing time period is concentration of from about 10 to about 1×1014 colony forming units (cfu) of the commensal microbial agent(s) (e.g., 10 cfu, 100 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, or any suitable ranges therein (e.g., from about 102 cfu to about 1013 cfu, about 1×104 to about 1×1011 cfu, about 1×106 to about 1×109 cfu, about 1×1010 to about 1×1012 cf, etc.), etc.).
In some embodiments, the microbial make-up of a probiotic composition consists or consists essentially of one or more beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics). In some embodiments, the microbial make-up of a probiotic composition consists or consists essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or any ranges therein (e.g., 1-4, 5-10, 8-20, etc.) strains and/or species of microbes. In some embodiments, fewer than 50 microbial strains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or any ranges therein (e.g., 1-4, 5-10, 8-20, etc.) are at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. 99.9%, 99.99%) of the microbial population (e.g., by mass, by cfu, etc.) of a probiotic composition. For example, in some embodiments, a single species or strain of S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics is at least 95% of the microbial population, as measured by colony forming units, of a particular probiotic composition. As another example, in some embodiments, a single species or strain of S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics is at least 40% of the microbial population, as measured by mass, of a particular probiotic composition. These examples are not limiting.
In some embodiments, microflora in a subject (e.g., a subject suffering from an immune-related, autoimmune-related and/or inflammation-related disease) are modulated by transplantation of microbiota from a subject with favorable characteristics (e.g., a healthy subject) into the recipient subject. In some embodiments, donor microflora are obtained sampling microflora from the desired region of the donor subject body (e.g., colon, oral cavity, vagina, etc.). In particular embodiments, fecal material (e.g., 100 g-500 g) is obtained from a donor. The material may be administered to a recipient subject with or without subsequent preparation steps (e.g., diluting, mixing, oxygenating, filtering, supplementing (e.g., with prebiotics, with growth media, etc.), testing (e.g., for pathogens or detrimental microbes), etc.). The donor microflora (e.g., fecal material) may be administered without preservation (e.g., administered within 12 hours (e.g., <6 hours, <4 hours, <2 hours, <1 hour, etc.)) or may be preserved (e.g., frozen, freeze dried, etc.), for example, to allow for delay (e.g., 1 day, 2, days, 1 week, 1 month, or more) before delivery to the subject. In some embodiments, donor microflora are proceed to remove one or more components. For example, parasitic of detrimental microbes may be removed or killed. Contaminants within the donor sample may be removed. In some embodiments, donor microflora is enriched (e.g., 2-fold, 3-fold, 4 fold, 10-fold, 20-fold, or more enrichment) for one or more specific microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics). In some embodiments, donor microflora is enriched such that at least 1% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of the microbes in the population are the desired beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics). In some embodiments, donor microflora are doped with one or more cultured beneficial microbes.
In particular embodiments, transplanted microflora may be administered to the recipient subject by any suitable delivery mechanism, including but not limited to enema, colonoscope, nasogastric or nasoduodenal tube, lavage or irrigation, or orally (e.g., in the form of a capsule).
In some embodiments, a commensal microbial agent or population of microbial agents is administered (e.g., via probiotic composition or microflora transplant) over a dosing time period (e.g., <1 minute, <1 hour, <2 hours, <4 hours, <6 hours, <12 hours, <24 hours, etc.) in an amount that is sufficient to provide a desired therapeutic benefit (e.g., as a single dose, in combination with other doses, in combination with a co-administered therapeutic, etc.) In some embodiments, the dose of commensal microbial agent(s) administered for the dosing time period is concentration of from about 10 to about 1×1014 colony forming units (cfu) of the commensal microbial agent(s) (e.g., 10 cfu, 100 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, 1013 cfu, or any suitable ranges therein (e.g., from about 102 cfu to about 1013 cfu, about 1×104 to about 1×1011 cfu, about 1×106 to about 1×109 cfu, about 1×1010 to about 1×1012 cf, etc.), etc.).
The dose can be administered in a single unit dose administered at any time during a day. Alternatively, the loading dose can be administered in two or more doses administered at a single time of day or at two or more separate times of day. Over the course of multiple dosing periods, the dose can be tapered from an initial dose to a higher dose (or increased from an initial dose to a higher dose), on predetermined timing or by the when the subject and/or clinician based on the results of the treatment. The appropriate dosage amount will vary by, for example, an individual subject's age, weight, condition or disease, severity of disease, etc.
In some embodiments, microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) for probiotic compositions are obtained from culture. In some embodiments, strains of beneficial microbes are genetically engineered to enhance one or more of production (e.g., at scale), formulation, delivery, or the biological effect of the microbe. In some embodiments, microbes are engineered to express a detectable marker that allows tracking of the microbes within a subject, or confirmation that the microbe has integrated into a subject's microflora. In some embodiments, microbes are engineered to express therapeutic agents (e.g., anti-inflammatory agent or other drug).
In some embodiments, one or more prebiotics are administered to a subject as an independent treatment (e.g., to increase the level of S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics) or in conjunction with other treatments described herein. Prebiotics are agents that increase the in vivo growth rate or activity of commensal microbes, such as S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics. In some embodiments, prebiotics are soluble fiber sources. In some embodiments, when prebiotics are administered (e.g., fed) to a subject they are not digested or are not fully digested by the subject's digestive enzymes, but rather support the intestinal health of the subject and provide an energy source for the beneficial microbes and enhance the growth thereof. Prebiotics include, for example, naturally occurring lecithins and/or oleic acid, and are described, for example in U.S. Pat. No. 8,449,878 which is herein incorporated by reference in its entirety.
In some embodiments, the level or presence of one or more detrimental microbes (e.g., microbes that facilitate autoimmunity, inflammation, etc.) is modulated, for example, by the administration of one or more antimicrobial agents to a subject or modulation of conditions within the subject to disfavor growth of the detrimental microbes. In some embodiments, antimicrobial agents are administered.
In some embodiments, the antimicrobial agent is an antibiotic. Exemplary antibiotics that may find use in some embodiments include, but are not limited to: amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem, meropenem, cefaclor, cefamandole, cefotoxin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobirprole, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, aztreonam, amoxicillin, ampicillin, azociling, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, peperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, clavulanic acid, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nonfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, AL-15469A, AL-38905, OP-145, afenide, prontosil, sulfacetamide, sulfamethiazole, sulfanamide, sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetraycline, linezolid, arsogebanubem chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, rifampicin, thamphenicol, tinidazole, amoxicillin+clavulanic acid, Maximin H5, Dermcidin, Cecropins, andropin, moricin, ceratotoxin, melittin, Magainin, dermaseptin, bombinin, brevinin-1, esculentins and buforin II, CAP 18, LL37, abaecin, apidaecins, prophenin, indolicidin, brevinins, protegrin, tachyplesins, defensins, drosomycin, alamethicin, pexiganan or MSI-78, MSI-843, MSI-594, polyphemusin, colicin, pyocin, klebicin, subtilin, epidermin, herbicolacin, brevicin, halocin, agrocin, alveicin, carnocin, curvaticin, divercin, enterocin, enterolysin, erwiniocin, glycinecin, lactococin, lacticin, leucoccin, mesentericin, pediocin, plantaricin, sakacin, sulfolobicin, vibriocin, warnerinand, nisin, or a salt or cocrystal, or prodrug or solvate thereof, or a combination thereof.
In some embodiments, the antimicrobial is an antifungal agent. Exemplary antifungals that may find use in some embodiments include, but are not limited to: amrolfine, utenafine, naftifine, terbinafine, flucytosine, fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole, clotrimazole, econazole, miconazole, oxiconazole, sulconazole, terconazole, tioconazole, nikkomycin Z, caspofungin, micafungin, anidulafungin, amphotericin B, liposomal nystastin, pimaricin, griseofulvin, ciclopirox olamine, haloprogin, tolnaftate, undecylenate, clioquinol, and combinations thereof.
In some embodiments, the antimicrobial is an antiparasitic. Exemplary antiparasitics that may find use in some embodiments include, but are not limited to: amitraz, amoscanate, avermectin, carbadox, diethylcarbamizine, dimetridazole, diminazene, ivermectin, macrofilaricide, malathion, mitaban, oxamniquine, permethrin, praziquantel, prantel pamoate, selamectin, sodium stibogluconate, thiabendazole, and combinations thereof.
In some embodiments, methods and compositions for reduction of detrimental microbe levels are co-administered (e.g., serially, concurrently, etc.) with methods and compositions for increasing beneficial microbe levels. In some embodiments, by reducing overall microbe levels or by reducing the levels of specific microbes (e.g., detrimental microbes, high population microbes, etc.), the population of beneficial microbes can more effectively be modulated (e.g., increased).
In some embodiments, in order to develop a microflora population within a subject that inhibits inflammation/autoimmunity and/or facilitates treatment of inflammation/autoimmunity, antimicrobial agents are first administered to eliminate or reduce the microflora within the subject, and then the microflora population is reestablished using the methods and compositions described herein (e.g., administration of beneficial microbes (e.g., S. epidermidis, specific strains thereof (e.g., NPI), taxonomically-related bacteria, and/or bacteria with similar LTA characteristics). In some embodiments, antimicrobials (e.g., antibiotics) that reduce the microbe (e.g., bacteria) population generally are employed. In some embodiments, antimicrobials that target detrimental microbes preferentially are employed.
In certain embodiments, microflora composition is manipulated along with one or more other therapies (e.g., anti-inflammatory therapies, treatment of autoimmune disease, etc.). In some embodiments, manipulation of the microflora composition (e.g., identity and/or level) treats inflammation/autoimmunity by a mechanism independent of one or more additional therapeutics. In other embodiments, modulation of microflora composition facilitates (e.g., increases the effectiveness of) co-administered therapeutics/therapies. In some embodiments, one or more therapeutics/therapies enhance the effectiveness of the modulation of microflora composition. Embodiments herein are not limited by the types of additional therapeutics/therapies unless specifically noted.
Experiments conducted during development of embodiments herein indicated that certain compositions comprising lipoteichoic acid (LTA), for example, LTA derived from the cell will of Staphylococcus epidermidis, LTA derived from certain strains thereof (e.g., the NPI strain), and/or LTA comprising similar or the same structural/functional characteristics as the aforementioned are useful for modulating host immune responses and for treating/preventing immune-related and/or inflammation-related diseases and conditions in a subject.
Lipoteichoic acid (LTA) is a major constituent of the cell wall of gram-positive bacteria. The structure of LTA varies between the different species of Gram positive bacteria. LTA comprises an oligomer of glycerophosphates linked to a glycolipid anchor by a disaccharide. The LTA is anchored to the cell membrane via a diacylglycerol. In some embodiments, LTA comprises formula I:
wherein n is 2-100 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or ranges therebetween; wherein each R is any suitable amino acid (e.g., D-alanine), saccharide (e.g., N-acetylglucosamine, glucose, galactose, etc.), or H; wherein S1 and S2 are monosaccharides (e.g., D-glucose, fructose, mannose, galactose, glucosamine, xylopyranose, rhamnose, etc.) connected by any suitable linker (e.g., α(1→2), α(1→3), α(1→6), β(1→2)β, β (1→2), β (1→3), β (1→6), etc.), wherein G is a glyceride moiety and FA1 and FA2 are fatty acids (e.g., saturated or unsaturated) of any suitable length (e.g., C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or ranges therebetween). In some embodiments, LTA of Formula I is administered to a subject.
In some embodiments, saccharides of the LTA described herein (e.g., R, S1 and S2 groups) are monosaccharides. In some embodiments, monosaccharides comprise include trioses, such as glycerose and dihydroxyacetone; textroses such as erythrose and erythrulose; pentoses, such as xylose, arabinose, ribose, xylulose ribulose; methyl pentoses (6-deoxyhexoses), such as rhamnose and fucose; hexoses, such as ascarylose, glucose, mannose, galactose, fructose, and sorbose; and heptoses, such as glucoheptose, galamannoheptose, sedoheptulose, and mannoheptulose. Exemplary monosaccharides embrace radicals of allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, L-fucitol, fucosamine, fucose, fuculose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose glyceraldehyde, L-glycero-D-mannos-heptose, glycerol, glycerone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, quinovose, quinovasamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose. The monosaccharide can be in D- or L-configuration.
In some embodiments, S1 and S2 are present in an LTA as a disaccharide. Exemplary disaccharides present in the LTA herein include: sucrose, lactulose, lactose, maltose, trehalose, cellobiose, chitobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiulose, mannobiose, melibiose, melibiulose, rutinose, rutinulose, and xylobiose. In particular embodiments, S1 and S2 are present in an LTA as gentiobiose.
In some embodiments, R of Formula I is any suitable natural amino acid, unnatural amino acid, or amino acid analog. In some embodiments, the amino acid may be present in the D or L stereoisomer, unless otherwise indicated, if their structures allow such stereoisomeric forms. Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). Unnatural amino acids include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine (“naph”), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine (“tBuG”), 2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline (“hPro” or “homoP”), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline (“3Hyp”), 4-hydroxyproline (“4Hyp”), isodesmosine, allo-isoleucine, N-methylalanine (“MeAla” or “Nime”), N-alkylglycine (“NAG”) including N-methylglycine, N-methylisoleucine, N-alkylpentylglycine (“NAPG”) including N-methylpentylglycine. N-methylvaline, naphthylalanine, norvaline (“Norval”), norleucine (“Norleu”), octylglycine (“OctG”), ornithine (“Orn”), pentylglycine (“pG” or “PGly”), pipecolic acid, thioproline (“ThioP” or “tPro”), homoLysine (“hLys”), and homoArginine (“hArg”). Amino acid analogs include to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain functional group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.
In some embodiments, FA1 and FA2 of Formula I are any suitable fatty acid. For example, FA1 and FA2 independently comprise carboxylic acids with long aliphatic chains, which are either saturated or unsaturated. Examples of fatty acids that find use in embodiments herein, include:
In some embodiments, LTA comprises the chemical make-up (e.g., one or more species comprising specific chemical formulas or chemical genera (e.g., in appropriate ratios), etc.) of the LTA of S. epidermidis. In some embodiments, LTA is prepared from S. epidermidis (e.g., a culture of S. epidermidis). In other embodiments, an LTA composition is prepared (e.g., synthetically), to match the natural make-up of LTA obtained from S. epidermidis (e.g., or sufficiently similar thereto to elicit a beneficial effect when administered to a subject). In some embodiments, an LTA composition comprises one or more LTA chemical species. In some embodiments, an LTa composition comprises two or more LTA species present in a ration that (i) simulates the make-up of natural S. epidermidis LTA, and/or (ii) elicits the immune-modulatory/anti-inflammatory effects of S. epidermidis LTA.
In some embodiments, the compositions and methods herein find use the treatment, prevention, and/or symptom reduction of diseases related to autoimmunity, inflammation, improper immune response, and/or pain. Autoimmune diseases are those which are characterized as having a component of self-recognition. Examples of autoimmune diseases include, but are not limited to, autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, type i diabetes, rheumatoid arthritis, psoriasis, hashimoto's thyroiditis, grave's disease, ankylosing spondylitis sjogrens disease, CREST syndrome, scleroderma, etc. Most autoimmune diseases are also chronic inflammatory diseases. This is defined as a disease process associated with long-term (>6 months) activation of inflammatory cells (e.g., leukocytes). The chronic inflammation leads to damage of patient organs or tissues. Many other diseases are inflammatory disorders, but are not necessarily known to have an autoimmune basis. For example, chronic pelvic pain syndrome, atherosclerosis, congestive heart failure, Crohn's disease, ulcerative colitis, polyarteritis nodosa, Whipple's Disease, Primary Sclerosing Cholangitis, etc. The clinical manifestations of autoimmune and inflammatory diseases range from mild to severe. Mild disease encompasses symptoms that may be function-altering and/or comfort-altering, but are neither immediately organ-threatening nor life-threatening. Severe disease entails organ-threatening and/or life-threatening symptoms. For example, severe autoimmune disease is often associated with clinical manifestations such as nephritis, vasculitis, central nervous system disease, premature atherosclerosis or lung disease, or combinations thereof, which require aggressive treatment and may be associated with premature death. In some embodiments, the compositions and methods described herein find use in treatment, prevention (when applicable), and/or symptom reduction of the aforementioned diseases and conditions.
In some embodiments, compositions and methods herein comprise multiple modes for modulating host immune responses and for treating/preventing immune-related and/or inflammation-related diseases and conditions in a subject. In some embodiments, the treatments described herein are co-administered with prebiotics and/or other agents that facilitate the growth of the beneficial microbes. In some embodiments, beneficial microbes are provided/administered (e.g., by a probiotic composition, fecal transplant, etc.) with antimicrobial(s) (e.g., antibiotics) directed to kill or inhibit the growth of detrimental microbes. In some embodiments, prebiotics and/or other agents that facilitate the growth of the beneficial microbes are provided/administered with antimicrobial(s) (e.g., antibiotics) directed to kill or inhibit the growth of detrimental microbes. In some embodiments, beneficial microbes, prebiotics and/or other agents that facilitate the growth of the beneficial microbes, and an antimicrobial(s) (e.g., antibiotics) directed to kill or inhibit the growth of detrimental microbes are all co-administered. In some embodiments, LTA is administered with one or more agents that further treat inflammation and/or the diseases/conditions caused by or underlying the inflammation.
In some embodiments, the co-administered agents are formulated into a single dose and/or composition. In some embodiments, the co-administered agents are in separate doses and/or compositions. In some embodiments in which separate doses and/or compositions are administered, the doses and/or compositions are administered simultaneously, consecutively, or spaced over a time span (e.g., <30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or more, or any suitable ranges therebetween).
In some embodiments, compositions described herein are co-administered with one or more therapeutics useful for the treatment of rheumatoid arthritis, such as disease-modifying antirheumatic drugs (DMARDs) (e.g., methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, TNF-alpha inhibitors (e.g., certolizumab, infliximab and etanercept), abatacept, anakinra, rituximab, tocilizumab, etc.), anti-inflammatory and analgesic agents (e.g., Glucocorticoids, non-NSAID drugs to relieve pain (e.g., paracetamol), NSAIDs, etc.
In some embodiments, compositions described herein are co-administered with one or more therapeutics useful for the treatment of CPPS, such as alpha blockers (e.g., tamsulosin, alfuzosin, etc.), antibiotics (e.g., fluoroquinolones, tetracyclines, macrolides, etc.), NSAIDS, estrogen reabsorption inhibitors (e.g., mepartricin, etc.), etc.
Additional description of the experiments and results of Example 1 are described in Murphy et al. (2017), which is herein incorporated by reference in its entirety.
Materials and Methods
Animal Use
Male 5-7 week old C57BL/6 (B6), NOD/ShiLtJ (NOD) and B6.129P2-Il10tm1Ggn (IL10KO) mice were purchased from Jackson Laboratory (Bar Harbor, Me.). All comparisons were made directly between genetically identical commercially obtained animals. Experiments were conducted in male mice, as the disease condition being studied is restricted to men. EAP was induced in specific mice by subcutaneous injection of rat prostate homogenate at a 1:1 ratio with TITERMAX adjuvant. Following this injection referred pain responses were measured by Von Frey force filament behavioral testing for tactile allodynia, as described (ref. 15; incorporated by reference in its entirety). Testing was performed prior to EAP induction (baseline) and then every 7 days unless otherwise stated. Results were calculated as a percentage change above baseline and for each Von Frey filament of increasing force per group individually, values were averaged across experimental groups. Behavioral testing was performed following treatments (with bacteria or LTA) in a blinded manner with no knowledge of treated versus untreated groups. After 28 days of EAP (unless otherwise stated) mice were sacrificed and relevant tissues removed. Prostate and bladder tissues were dissociated using collagenase/DNase buffer (1640-RPMI (Corning) containing 10% FCS (Hyclone), 0.1 ul/ml DNase (Thermo-scientific) and 1 ug/ml collagenase D (Roche)) and shaking for 2 hours at 37° C. Tissues were then made into single cell suspensions by passing through 0.2 μm mesh filter membranes and washing with 2% FCS (HyClone) in PBS (Gibco). For splenic and iliac lymph node tissues, single cell suspensions were generated without collagenase treatment.
Bacterial Isolation, Growth and Instillation
Human expressed prostatic secretion (EPS) and voided bladder 1, 2 and 3 samples were collected from the urology clinic at Northwestern Memorial Hospital. S. epidermidis was isolated from both the VB3 (post-massage urine) and EPS of a healthy control sample. The bacterial isolate was grown in Luria Broth, as previously described for UPEC strains (ref. 13; incorporated by reference in its entirety). Bacteria were grown in 3 ml at 37° C. overnight followed by transfer of 100 μl of this culture to 40 ml and growing for 37° C. without shaking. Following this bacterial was pelleted and re-suspended in ice cold sterile PBS, the solution was then made up to an OD 420 value of 1.00, corresponding to 2×10^10 bacteria per ml. 10 μl of the NPI strain preparation was introduced into anesthetized male mice by catheterization. This correlates with 2×10^8 bacteria per infection per mouse.
LTA Isolation and Treatment
The NPI strain was shipped on dry ice to a commercial biofermentation lab (University of Maryland, Rockville, Md.) followed by purification by HPLC at FinoBiolabs (Rockville Md.). Purified LTA was returned (3.01 lmg from a 10 L culture) as a desiccated powder. This was reconstituted using DNA grade water (Gibco) and stored at lmg/ml at −80° C., until use. 10 μl of a 1:100 dilution of this solution (100 ng) was used per mouse per treatment, this was calculated based on an estimation of the LTA content of the dry weight of the 2×10^8 bacteria normally used in infections. S. aureus and B. subtilis LTA were purchased from Invivogen and equal doses were administered as above. Mice were treated by intraurethral instillation under anesthesia in a similar manner to the infection protocol.
In Vivo Antibody Treatment
Blockade of CD25 and PD1 was performed, therapeutically; by intraperitoneal injection with 100 μg anti-CD25 (PC61) (BioLegend 102002) or 100 μg anti-PD1 (BxCell BE0146) or relevant IgG isotype control antibody at Day 19 post-EAP treatment and repeated every two days with pain/tactile allodynia assessed every alternate day until Day 28.
QRTPCR
RNA isolation was performed from respective tissues (collagenase treated prostate and bladder homogenates) using Trizol™ (Invitrogen). RNA concentration was measured using a NANODROP spectrophotometer (Thermo Scientific) and normalized to 1 μg per cDNA synthesis reaction. cDNA was synthesized using qScript Supermix (Quanta) according to manufacturer instructions. QRTPCR was performed using perfeCTa qPCR Supermix (Quanta) with 50 ng of template per 25 μl reaction and run on the CFX CONNECT (Biorad) platform. Primers were designed using the online NIH curated primer blast tool. A full table of primer sequences is below. ddCT calculations were performed in EXCEL, mouse genes were normalized first against the L19 housekeeping gene and finally by expression of CD4, human genes normalized to GAPDH and both analyzed using GraphPad PRISM software.
Flow Cytometry Analyses.
Flow cytometry was performed on single cell suspensions using mouse antibodies: PerCp-CD4, Alexa-488-CD4, Alexa-647-FoxP3, PE-IL17A, FITC-CD25, PE-PD1 (Biolegend), PE-FoxP3, PC-IL17, FITC-CD25, APC-CD11b, FITC-PDL2, PE-PDL1 (eBiosciences). Flow was run on an Accuri benchtop C6 cytometer and analysed using FLOWJO software. Unless otherwise stated, samples were gated on lymphocyte populations based on size, as assessed by SSC and FSC, followed by gating for CD4 positivity. Intracellular staining was performed by fixation and permeabilization using eBioscience Fix-Perm Intracellular staining buffers (Cat. Num 8222-49 and 8333-56). Staining was performed for 1 hour at room temperature followed by washing in FACS buffer (2% FCS (Hyclone), in PBS (Gibco)) Analyses were performed using FLOWJO and data statistically tested using GraphPad PRISM software, with tests described in respective figure legends.
Immunofluorescent Staining
10% formalin-fixed tissues were mounted and embedded in paraffin and sectioned (5 μm) onto slides. These were then rehydrated in xylene (VWR), followed by increasing dilutions in ethanol (100%, 75%, 50% and 25%). Antigen retrieval was performed by boiling for 25 mins in 10 mM Sodium Citrate Buffer at a of pH 6.0, sections were then transferred to PBS (Gibco), excess liquid removed and staining performed using rabbit anti-mouse PDL1 antibody (AbCam ab58810) or PDL2 antibody (AbCam ab21239). Secondary staining with Alexa-488-anti-rabbit was then performed, sections mounted in Anti-fade mounting media containing DAPI and imaged using a Leica DMLA microscope.
Statistical Analyses
Statistical analyses were performed using GraphPad PRISM software. Flow cytometric samples were compared using one-way ANOVA with Tukey's multiple comparison test across samples. Datasets were treated as normal, unless otherwise stated, as assessed by median to mean agreement.
Results
NOD Mice do not Develop Tactile Allodynia in Response to S. epidermidis Instillation.
A strain of Staphylococcus epidermidis, NPI, was isolated from the expressed prostatic secretions (EPS) of a healthy male subject who reported no pelvic pain symptoms or symptoms related to prostate dysfunction. Experiments were conducted during development of embodiments herein to determine the consequences of NPI instillation in mice genetically predisposed to the development of pelvic tactile allodynia through altered T cell immune responses. NPI strain was administered intraurethrally into NOD mice (refs. 32, 37; incorporated by reference in their entireties) and analyzed tactile allodynia responses every 7 days for 28 days and immune activation by QRTPCR. Tactile allodynia responses as assessed by behavioral testing for referred pelvic pain by tactile allodynia (ref. 31; incorporated by reference in its entirety) showed no increase in NPI treated mice compared to naïve counterparts,
Intra-Urethral NPI Instillation in EAP Mice Reduces Referred Pelvic Pain.
Experiments were conducted during development of embodiments herein to examine what effect instillation of NPI would have on tactile allodynia responses during ongoing inflammation using the xenogeneic model of prostatitis. EAP was induced in immunocompetent C57BL/6 mice (refs. 35, 39; incorporated by reference in their entireties) and symptom development was monitored by behavioral testing every seven days for 21 days. At day 21, when prostatitis induction is deemed chronic as characterized by the presence of chronic tactile allodynia and alterations in immune response, NPI was intraurethrally instilled and the experiment continued for a further 7 days. Increased tactile allodynia responses upon EAP induction were observed (
NPI's Effects on Tactile Allodynia are Strain Specific but not Dependent on Levels of Prostate Bacterial Colonization.
To examine whether the effects on tactile allodynia and immune modulation were specific to the commensal Staphylococcus species isolated from the normal prostate, experiments were conducted during development of embodiments herein to directly compare the ability of NPI to reduce tactile allodynia in EAP mice with that of an S. epidermidis 7244 strain isolated from the EPS of a CPPS patient with active disease. Mice were given EAP for 28 days and behavioral testing was performed at both Day 0 and Day 28. Following this, mice were instilled with either the NPI or the 7244 strains. Behavioral testing was performed at Day 29 (1-day post treatment), Day 31 (3 days post treatment) and Day 35 (7 days post treatment). At each time point (days 1, 3 & 7) mice were sacrificed (4-5 per group) and colony formation assays performed on both prostate and bladder tissues. The NPI strain showed reduced tactile allodynia responses in C57BL/6 mice when compared to 7244,
LTA is the Immunogenic Component of NPI
Due to the apparent strain specific nature of NPI to counteract EAP induced tactile allodynia response, experiments were conducted during development of embodiments herein to determine the bacterial component responsible. LTA was isolated from the NPI bacterial strain by HPLC and intra-urethrally instilled into naïve and EAP mice. LTA treatment significantly decreased tactile allodynia responses (
LTA Increases Expression of CD274/CD273 (PDL1/2) on CD11b+Ve Cells.
TLR2 is expressed on a wide variety of tissues including macrophages and dendritic cells (refs. 3, 41; incorporated by reference in their entireties). Given the ability of these monocytic cell types to modulate CD4 T-cell activation and differentiation, experiments were conducted during development of embodiments herein to examine the effect of LTA on CD11b+ve monocytes in the prostate. Findings indicate that this specific LTA dampens pro-inflammatory Th17 responses; therefore, experiments focused on the expression of the negative CTLA4-like co-stimulatory molecules PDL1 and PDL2 in response to EAP and/or LTA treatment in prostate tissues. Flow cytometry revealed that although EAP induction had no effect on PDL1/2 expression on CD11b+ve cells at day 28, EAP followed by LTA treatment significantly increased expression level of these ligands,
Modulations in Referred Pain are Dependent on IL10, PD1 and CD25 Expressing Cells.
Given the increased level of PDL1 and PDL2 expression associated with LTA treatment, experiments were conducted during development of embodiments herein to determine whether IL10 secreted by CD11b+ve cells may serve to dampen tactile allodynia responses in the prostate. Such increases in IL10 secretion could also account for the decreased levels of IL17 expressing CD4+ve T-cells observed when EAP mice are treated with either NPI or NPI-LTA. To interrogate this, EAP was induced in IL10KO mice and subsequently one group was instilled with NPI at day 21. As demonstrated in
Collagen Induced Arthritis (CIA) is a murine model of autoimmune driven arthritis (e.g., rheumatoid arthritis) induced by intradermal tail vein injection of collagen antigen and Complete Freud's Adjuvant (CFA). The model induces joint destruction and immune cell infiltration into the joints of the hind limbs and paws of affected mice. This model was used as it has been shown to be mediated by IL17 expression and also has technical similarities to the experimental autoimmune prostatitis (EAP) model where pelvic tactile allodynia is induced by subcutaneous injection of prostate antigen and an adjuvant. In order to assess arthritis onset in our C57BL/6 animals, paw withdrawal testing was performed, which uses Von Frey filaments to examine the force necessary for mouse paw withdrawal from filaments with varying hardness. The lower the force necessary for paw retraction indicating a hyper-response to the filaments and thus paw discomfort. This acted as a non-invasive metric for extent of allodynia and arthritis in affected mice. Experiments conducted during development of embodiments herein demonstrate effective induction of the model in both paws of treated mice (CIA B and CIA C above) compared to Naïve control animals (
Treatment with the Lipotechoic acid (LTA) moiety was performed at day 104 and repeated every 3 days to allow for mouse recovery following injection. LTA was applied by direction injection into the mouse foot-pad. Paw-withdrawal measurements post-treatment from day 0 and continued until day 21 (
The following references, some of which are cited above by number, are herein incorporated by reference in their entireties.
The present invention claims priority to U.S. Provisional Patent Application 62/473,022, filed Mar. 17, 2017, which is incorporated by reference in its entirety.
This invention was made with government support under DK094898 02 awarded by the National Institutes of Health. The government has certain rights in the invention
| Number | Name | Date | Kind |
|---|---|---|---|
| 8449878 | Yonak | May 2013 | B2 |
| 20100166708 | Gallo | Jul 2010 | A1 |
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| Number | Date | Country | |
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