Patent Application
20230024893
The present invention relates to use of Akkermansia muciniphila-derived threonyl-tRNA synthetase (TARS) or a fragment thereof as an active ingredient for prevention, alleviation, and treatment of an inflammatory disease.
Toll-like receptors (TLRs), cell membrane receptors that have an important role in human innate immunity, induce an inflammatory or anti-inflammatory immune response by recognizing a pathogen-associated molecular pattern (PAMP) derived from microbes and activating cell signaling through adaptor proteins. TLRs are expressed in various species and various cells, and 10 types of TLRs have been known to be expressed in humans. Of these, TLR2 perform functions by forming heterodimers with TLR1, TLR6, and TLR10. These mainly recognize cell wall components (lipoproteins, lipoteichoic acid, zymosan, etc.) of pathogens. When a ligand is recognized by TLR2, inflammatory transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and activator protein 1 (AP-1), are activated by myeloid differentiation primary response gene 88 (MyD88) and MyD88 adapter-like (MAL)/Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP)-dependent signaling and the production of anti-inflammatory cytokines are introduced through phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling. TLR2 is also known to have an important role in the regulation of macrophage differentiation.
Macrophages initiate inflammatory responses in response to pathogens or harmful stimulations caused by apoptosis and necrosis. Macrophages are activated through downstream signaling of TLR and cytokine receptors. The activated macrophages cause the production of inflammatory cytokines and chemokines, migration of cells, and removal of harmful substances by transcriptional and epigenetic changes. In the late stage of inflammation, macrophages contribute to the prevention of sustained inflammatory responses causing tissue injury and eliminate inflammation.
Macrophages are classified into functional phenotypes M1 and M2 macrophages according to the intracellular microenvironment, wherein M1 macrophage polarization is accelerated by interferon-γ, lipopolysaccharide (LPS), inflammatory cytokines, or the like, and M2 macrophage polarization is accelerated by interleukin (IL)-4, IL-13 cytokines, or the like. M1 macrophages mainly secrete inflammatory cytokines and chemokines, such as IL-6, IL-12, and tumor necrosis factor (TNF)-α, while M2 macrophages secrete anti-inflammatory cytokines, such as arginase-I, IL-10, and transforming growth factor (TGF)-β, to reduce inflammation and inhibit immune responses. In particular, IL-10 secreted by M2 macrophages is known to be a very important cytokine in the inhibition of inflammatory responses and is also reported to increase the proliferation and viability of B cells. B cells are very well known to have a core role in the regulation of immune responses through antibody production, and a previous study has reported that B cells, together with regulatory T cells, are essential for inhibiting the inflammatory response in inflammatory bowel disease (IBD) (L Wang, et al., T regulatory cells and B cells cooperate to form a regulatory loop that maintains gut homeostasis and suppresses dextran sulfate sodium-induced colitis, Mucosal Immunol. 2015,8(6):1297-312).
Aminoacyl-tRNA synthetase (AARS) is an enzyme that attaches an amino acid to transport RNA (tRNA) in the early stages of protein synthesis. Some of the human-derived AARS proteins are known to be secreted from cells to perform various functions. Typically, it is known that in a specific environment, human tyrosyl-tRNA synthetase (YARS) is secreted from cells to perform a function as a cytokine, and human lysyl-tRNA synthetase (KARS) stimulates immune cells to promote the inflammatory response. In particular, it has been reported that human threonyl-tRNA synthetase (TARS), when secreted from cells, induces angiogenesis via an inflammatory signal and, in pancreatic cancer cells, increases the synthesis of mucin 1 (MUC1), one of the major proteins of mucin, containing a large amount of threonine. Additionally, TARS has been reported as a key component of vertebrate-specific protein synthesis initiation complexes needed for protein synthesis of genes necessary for development of blood vessels, the nervous system, and the like during the evolution from invertebrates to vertebrates.
On the basis of the functions of human AARSs after being secreted from cells, the present inventors confirmed that Akkermansia muciniphila, a gut microbe that has been reported to have an important role in metabolic diseases through human immune regulation, specifically secretes TARS, wherein the TARS activates B cells by promoting IL-10 secretion of macrophages, thereby inducing anti-inflammatory immune homeostasis, and thus confirmed that TARS has efficacy of preventing, alleviating, and treating inflammatory diseases via macrophages.
An aspect of the present invention is to provide a pharmaceutical composition for prevention or treatment of an inflammatory disease, the pharmaceutical composition containing Akkermansia muciniphila-derived threonyl-tRNA synthetase (TARS) or a fragment thereof as an active ingredient.
Another aspect of the present invention is to provide a method for preventing or treating an inflammatory disease, the method including administering to a subject a composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
Still another aspect of the present invention is to provide a composition for proliferation of B cells, the composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
Still another aspect of the present invention is to provide a composition for proliferation of macrophages, the composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
Still another aspect of the present invention is to provide a composition for differentiation of M2 macrophages, the composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
Still another aspect of the present invention is to provide a quasi-drug for prevention or alleviation of an inflammatory disease, the quasi-drug containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The Akkermansia muciniphila TARS or a fragment thereof according to the present invention increases the secretion of the anti-inflammatory cytokine IL-10 by promoting the differentiation of M2 macrophages, anti-inflammatory macrophages, to induce the proliferation of macrophages, and thus exhibits an anti-inflammatory effect through the proliferation of B cells, and therefore exhibits an effect of alleviating not only an inflammatory disease but also an immune disease, an infectious diseases, and a metabolic disease, which are accompanied by inflammation, and especially, has an excellent effect in the prevention or treatment of inflammatory bowel disease (IBD) or the like.
The present invention will be specifically described as follows. Each description and exemplary embodiment disclosed herein may also be applied to other descriptions and exemplary embodiments. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific description below.
In accordance with an aspect of the present invention, there is provided a pharmaceutical composition for prevention or treatment of an inflammatory disease, the pharmaceutical composition containing Akkermansia muciniphila-derived threonyl-tRNA synthetase (TARS) or a fragment thereof as an active ingredient.
As used herein, the term “threonyl-tRNA synthetase (TARS)” is one of aminoacyl-tRNA synthetases (AARSs), enzymes that attach amino acids to transport RNA (tRNA) in the initial stage of protein synthesis, and some of human-derived AARS proteins are known to be secreted from cells to perform various functions. Typically, it is known that in a specific environment, human tyrosyl-tRNA synthetase (YARS) is secreted from cells to perform a function as a cytokine and human lysyl-tRNA synthetase (KARS) stimulates immune cells to promote the inflammatory response. Particularly, it has been reported that human threonyl-tRNA synthetase (TARS), when secreted from cells, induces angiogenesis via an inflammatory signal and, in pancreatic cancer cells, increases the synthesis of mucin 1 (MUC1), one of the major proteins of mucin containing a large amount of threonine. Additionally, TARS has been reported as a key component of vertebrate-specific protein synthesis initiation complexes needed for protein synthesis of genes necessary for development of blood vessels, the nervous system, and the like during the evolution from invertebrates to vertebrates.
In the present invention, the TARS may be derived from human gut bacteria, specifically Akkermansia sp., and more specifically, Akkermansia muciniphila, but is not limited thereto. The TARS may include, for example, the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. The TARS comprises a protein comprising the amino acid sequence of SEQ ID NO: 1, a protein having the amino acid sequence of SEQ ID NO: 1, and a protein consisting of the amino acid sequence of SEQ ID NO: 1.
In the present invention, SEQ ID NO: 1 means an amino acid sequence having TARS activity. Specifically, SEQ ID NO: 1 may be a sequence of a protein having have TARS activity encoded by a gene encoding TARS. According to an aspect of the present invention, the protein may be derived from specifically, Akkermansia sp., and more specifically Akkermansia muciniphila, but is not limited thereto, and any sequence that has the same activity as in the amino acid may be included without limitation. The amino acid sequence of SEQ ID NO: 1 may be obtained from GenBank, a known database (GenBank: ACD05550.1). The TARS may be used interchangeably with Akkermansia muciniphila-derived TARS (AmTARS).
Although the protein having TARS activity in the present invention is defined as a protein including the amino acid sequence of SEQ ID NO: 1, the addition of a meaningless sequence upstream or downstream of the nucleotide sequence of SEQ ID NO: 1, a mutation that may occur naturally, or a silent mutation thereof is not excluded. It is obvious to a person skilled in the art that any protein that has an activity identical or corresponding to that of the protein including the amino acid sequence of SEQ ID NO: 1 corresponds to the protein having TARS activity of the present invention.
Specifically, the protein having TARS activity of the present invention may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 1. In addition, it is obvious that any protein that consists of an amino acid sequence with a deletion, a modification, a substitution, or an addition in a part thereof may also fall within the scope of the protein of the present invention as long as the amino acid sequence has such a homology or identity and shows efficacy corresponding to that of the above protein.
That is, although described as “protein or polypeptide consisting of an amino acid sequence set forth in a particular sequence number” herein, any protein that consists of an amino acid sequence having a deletion, a modification, a substitution, or an addition in a part thereof may also be used in the present invention as long as the peptide has an activity identical or corresponding to that of the polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it is obvious that any polypeptide that has an activity identical or corresponding to that of the “polypeptide consisting of the amino acid sequence of SEQ ID NO: 1” may also fall within the “polypeptide consisting of the amino acid sequence of SEQ ID NO: 1”.
As used herein, the term “homology” or “identity” refers to a percentage of identity between two polynucleotide or polypeptide moieties. The homology between sequences from one moiety to another moiety may be determined by a known technique in the art. For example, the homology between two moieties may be determined by directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules by using an easily accessible computer program capable of aligning sequence information. Additionally, the homology between polynucleotides may be determined by hybridizing the polynucleotides under conditions in which a stable double strand is formed between homologous regions and then decomposing the hybridized polynucleotides via a single-strand-specific nuclease to deciding the size of the decomposed fragments.
In the present invention, the gene encoding TARS may be derived from bacteria, specifically Akkermansia sp., and more specifically, Akkermansia muciniphila, but any Akkermansia sp. microbe that can express the gene encoding TARS may not be particularly limited. The gene encoding TARS may include a nucleotide sequence encoding the amino acid sequences of SEQ ID NO: 1, and more specifically, the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 2 may be obtained from GenBank, a known database (GenBank: CP001071.1).
As used herein, the term “polynucleotide” refers to a nucleotide polymer composed of nucleotide monomers covalently bonded in a chain, and examples thereof are a DNA or RNA strand having a predetermined length or longer. More specifically, the term refers to a polynucleotide fragment encoding the variant.
Specifically, in the polynucleotide of the present invention, various modifications may be made in a coding region within the range in which the amino acid sequence of the polypeptide is not changed, due to codon degeneracy or in consideration of the codons preferred by an organism in which the polypeptide is to be expressed. Specifically, any polynucleotide that encodes TARS of the present invention may be included without limitation.
In addition, any probe that can be prepared from a known gene sequence, for example, any sequence that can hybridize with a complementary sequence to a part or the entirety of the nucleotide sequence under stringent conditions to encode the protein having TARS activity of the present invention may be included without limitation. The term “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. Such conditions are well known in the art. For example, the conditions may include conditions under which genes having high homology or identity, such as, genes having at least 40%, specifically at least 90%, more specifically at least 95%, still more specifically at least 97%, and still more specifically at least 99% homology or identity hybridize with each other but genes having lower homology or identity do not hybridize with each other; or typical washing conditions for southern hybridization, i.e., washing is conducted one time, specifically, two or three times at a salt concentration and temperature corresponding to 60° C., 1×SSC, and 0.1% SDS, specifically 60° C., 0.1×SSC, and 0.1% SDS, and more specifically 68° C., 0.1×SSC, and 0.1% SDS.
Hybridization requires that two nucleic acids have complementary sequences, although mismatches between nucleotides may be possible depending on hybridization stringency. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each another. For example, as for DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the present invention may include not only substantially similar nucleic acid sequences but also isolated nucleic acid fragments complementary to the entire sequence.
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. under the above-described conditions. In addition, the Tm value may be 60° C., 63° C., or 65° C., but is not limited thereto, and may be appropriately controlled by a person skilled in the art according to the purpose.
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity thereof, and variables thereof are well known in the art.
As for the amino acid or nucleotide sequence in the present invention, the term “fragment” refers to a part of a parent sequence. For example, the fragment may be a polypeptide in the form in which one or more amino acids are removed from the C or N terminus of the parent sequence.
In the present application, “fragment” of an enzyme may refer to “functional fragment”. The “functional fragment” may also be referred to as an active ingredient, and means a polypeptide that is a part of a parent enzyme and has enzymatic activity of the parent enzyme. For example, the functional fragment of the enzyme may include a catalytic site of the enzyme.
The fragment of the enzyme may include a part of the full length of the parent enzyme. For example, the fragment of the enzyme may include amino acids of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%, or less than 100% of the full length of the parent enzyme, but is not limited thereto.
In the present invention, the fragment of TARS may mean the amino acid sequence of the above-described Akkermansia muciniphila-derived TARS or a part of the polynucleotide sequence encoding the same, and any sequence that is derived from Akkermansia muciniphila-derived TARS or a part of the polynucleotide sequence encoding the same may be included without limitation.
Specifically, the fragment of TARS may be derived from the amino acid sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 2, and more specifically, may include at least one amino acid sequence selected from SEQ ID NO: 3, which is the sequence corresponding to 99th to 113rd amino acids of SEQ ID NO: 1, and SEQ ID NO: 4, which is the sequence corresponding to 290th to 312nd amino acids of SEQ ID NO: 1. Alternatively, polynucleotied sequences encoding a fragment of TARS may include at least one polynucleotied sequence selected from SEQ ID NO: 5, which is the sequence corresponding to the 295th to 339th nucleotides of SEQ ID NO: 2, and SEQ ID NO: 6, which is the sequence corresponding to the 868th to 936th nucleotides of SEQ ID NO: 2.
Specifically, TARS or a fragment thereof of the present invention may be a peptide consisting of 14 to 630 consecutive amino acids in the amino acid sequence of SEQ ID NO: 1, including SEQ ID NO: 3 and/or SEQ ID NO: 4. As one example, the peptide may be a peptide including the amino acid sequence of SEQ ID NO: 3 or 4. As another example, the TARS of the subject application or a fragment thereof comprises the amino acid sequences of SEQ ID NOs: 3 and 4, and may comprise the structural sequence of a TARS derived from other microorganisms. If the structural sequence of a TARS is derived from a microorganism, the sequence thereof is not particularly limited. Further, any structural sequence of the TARS of a microorganism known in the art may be included in the scope of the subject application. For example, the TARS of the subject application or a fragment thereof may comprise the amino acid sequence of SEQ ID NOs: 3 and/or 4, and may comprise the structural sequence of the TARS derived from E. coli, but is not limited thereto.
As used herein, the term “corresponding to” refers to an amino acid residue at a position recited in a polypeptide, or an amino acid residue similar, identical, or homologous to a residue recited in a polypeptide. Identifying an amino acid at a corresponding position may be determining a particular amino acid of a sequence referring to a particular sequence. As used herein, the term “corresponding region” generally refers to a similar or corresponding site in a related protein or reference protein.
For example, any amino acid sequence is aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence may be numbered with reference to an amino acid residue numerical position corresponding to an amino acid residue of SEQ ID NO: 1. For example, a sequence alignment algorithm as described in the present invention may identify the position of an amino acid or a position of occurrence of a modification, such as a substitution, an insertion, or a deletion, compared with a query sequence (also referred to as a “reference sequence”).
For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) may be used, but without limitation thereto, a sequence alignment program, a pairwise sequence comparison algorithm, or the like, which are known in the art, may be used as appropriate.
TARS or a fragment thereof of the present invention exhibits an anti-inflammatory effect and can be used for prevention or treatment of an inflammatory disease.
As used herein, the term “inflammatory disease” collectively refers to a disease having inflammation as a major lesion.
Examples of the inflammatory disease may include inflammatory bowel disease (IBD), edema, dermatitis, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gout, sacrificial spondylitis, gastritis, psoriatic arthritis, osteoarthritis, periarthritis, tendinitis, tenosynovitis, myositis, hepatitis, lymphangeitis, felon, urinary tract infection, peritonitis, cystitis, nephritis, respiratory disease, and sepsis, and specifically, may be inflammatory bowel disease, and more specifically, may be at least one selected from the group consisting of colitis, ulcerative colitis, Crohn's disease, and Behçet's enteritis, but is not limited thereto.
The inflammatory disease of the present invention may include an immune disease, a metabolic disease, and an infectious disease, which are accompanied by inflammation.
Examples of the “immune disease accompanied by inflammation” are autoimmune disease, transplant rejection, graft-versus-host disease, and the like. Specifically, examples of the autoimmune disease may include atopy, allergy, rheumatoid arthritis, Hashimoto's thyroiditis, multiple sclerosis, endometriosis, psoriasis, asthma, hypothyroidism, hyperthyroidism, Behçet's disease, myasthenia gravis, Meniere's syndrome, Guillain-Barre syndrome, Sjögren's syndrome, vitiligo, systemic scleroderma, and the like, and specifically may be inflammatory bowel disease, and more specifically may be at least one selected from the group consisting of atopy, allergy, rheumatoid arthritis, Hashimoto's thyroiditis, multiple sclerosis, endometriosis, psoriasis, hypothyroidism, hyperthyroidism, and asthma, but is not limited thereto.
Search results have been recently reported that a metabolic disease is caused by increased expression of inflammatory factors, and thus the metabolic disease may be caused by inflammation or accompanied by inflammation. Examples of the “metabolic disease accompanied by inflammation” may include obesity, diabetes, insulin resistance, abnormal lipid metabolism, hypertriglyceridemia, increased free fatty acids, reduced high-density cholesterol, hypertension, and various complications resulting therefrom, and the like, and specifically may be at least one selected from the group consisting of obesity and diabetes, but is not limited thereto.
The “infectious disease accompanied by inflammation” refers to an inflammatory disease caused by the spread or invasion of a disease-causing pathogen, such as virus, bacterium, fungus, and parasite, to animals or humans. The inflammatory disease is as described above.
In the present invention, the TARS or a fragment thereof can increase the secretion of IL-10.
In addition, the TARS or a fragment thereof can reduce the secretion of IL-6 and/or TNF-α.
In an embodiment of the present invention, when bone marrow-derived macrophages (BMDM) as mouse-derived immune cells and human macrophages THP1 as human-derived immune cells were treated with TARS in a non-inflammatory or inflammatory environment, the inflammatory cytokines IL-6 and TNF-α were not or less secreted, while the inflammatory cytokine IL-10 was increasingly secreted in a dose-dependent manner based on AmTARS (
These results confirmed that TARS induced an inflammatory disease alleviating effect by increasing the secretion of IL-10 and reducing the secretion of IL-6 and/or TNF-α.
The above-described effect of increasing IL-10 secretion and reducing the secretion of IL-6 and/or TNF-α may be exerted specifically by Akkermansia muciniphila-derived TARS.
In an embodiment of the present invention, to determine whether TARS from other bacteria in the human microbiome and TARS secreted from human cells perform similar functions to Akkermansia muciniphila-derived TARS, TARSs (respectively, BfTARS, eTARS, RbTARS, and hTARS) derived from Bacteroides fragilis, Escherichia coli, and Ruminococcus bromii, which are strains in the human microbiome, and Homo sapiens were purified and applied to BMDM in a non-inflammatory or inflammatory environment, and as a result, the secretion of the inflammatory cytokine IL-10 was specific to Akkermansia muciniphila-derived TARS (
In addition, the above-described anti-inflammatory effect of TARS may be exerted from a fragment of TARS.
In an embodiment of the present invention, as a result of investigating the immune cytokine secretion by treatment with TARS, functional site 1 (R1, SEQ ID NO: 3), functional site 2 (R2, SEQ ID NO: 4), and Tat-function site 1 (Tat-R1) and Tat-function site 2 (Tat-R2) obtained by adding, to the functional sites, Tat tag, which is a tag for favorable delivery into cells, TARS, R2, and Tat-R2 reduced the secretion of IL-6 and increased the secretion of IL-10 in the created inflammatory environment (
When the concentrations of functional site 2 were 100 μM, 200 μM, 300 μM in the same conditions, the secretion of IL-6 was reduced and the secretion of IL-10 was increased by all of R2 100 μM and 300 μM and Tat-R2 100 μM, 200 μM, and 300 μM.
Of these, the secretion of IL-10 was increased by 2-fold in the Tat-R2 treatment groups compared with TARS.
These results confirmed that the TARS-specific regulation of immune cytokine secretion was exerted by a fragment of TARS.
In the present invention, the TARS or a fragment thereof can inhibit inflammatory responses and increase inflammatory cytokine production by regulating the phosphorylation of intracellular signal transducers.
In an embodiment of the present invention, the signal transducers affected by TARS treatment were p38, c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), while extracellular signal-regulated kinase (ERK) was not affected thereby. JNK was affected through the regulation of phosphorylation from 30 min after TARS treatment, and p38 and NF-kB showed distinctive differences in the level of phosphorylation compared with a control from 2 h after TARS treatment (
In the present invention, the TARS or a fragment thereof can affect the regulation of metabolic disease-associated intracellular signaling.
In an embodiment of the present invention, the treatment of mouse adipocyte 3T3-L1 with insulin resulted in the phosphorylation of protein kinase B (AKT), and the treatment with insulin and PMA reduced the phosphorylation compared with the treatment with insulin alone. However, the treatment with ARS after the treatment with insulin and PMA again increased the phosphorylation (
In addition, when insulin resistance was induced by TNF-α treatment, the phosphorylation of AKT was reduced by even the treatment with insulin, and the phosphorylation was increased by the further treatment with TARS. In the same experiment, glycogen synthase kinase (GSK)-3β, a downstream signal transducer of AKT, was phosphorylated and inhibited, and S6 kinase (S6K), affected by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), was phosphorylated and activated (
In the present invention, the TARS or a fragment thereof can increase B cells.
In an embodiment of the present invention, as a result of administering TARS to an inflammatory bowel disease mouse model, the secretion of the cytokine IL-10 in the plasma was increased and the amount of IgA increased due to an inflammation response was reduced. Additionally, the distribution of B cells that have been reported to play an important role in alleviating inflammatory bowel disease, was significantly increased by TARS administration (
However, a B cell-deficient inflammatory bowel disease mouse model, unlike normal mice with no deficiency of B cells, did not show a reduction in the intestine length and an alleviation of the colon tissue damage, an increase in B cells by TARS, and the expression of IgA secreted from B cells (
These results confirmed that TARS increased the distribution of B cells in the colon to induce an inflammatory bowel disease alleviation effect.
In addition, the anti-inflammatory function of the TARS or a fragment thereof was exerted through the interaction with Toll-like receptor 2 (TLR2) (
In the present invention, the TARS or a fragment thereof can increase macrophages.
In addition, the TARS and the fragment thereof can promote the differentiation of macrophages into M2 microphages.
In an embodiment of the present invention, TARS bound to macrophages but did not bind to the other cells including B cells, and on the basis of this, as a result of, on the basis of the results, determining whether the anti-inflammatory efficacy of TARS was exhibited via macrophages in a macrophage-deficient inflammatory bowel disease mouse model, the distribution of B cells in the colon was not increased by TARS in the macrophage-deficient mice, and the proportion of M2 macrophages, anti-inflammatory macrophages, reduced by an inflammatory response, was increased by TARS.
In another embodiment of the present invention, the proportion of macrophage differentiating into anti-inflammatory macrophages was significantly increased by TARS.
These results confirmed that TARS promoted the differentiation of M2 macrophages, anti-inflammatory macrophages, to result in IL-10 secretion, which was then used as a signal to increase the distribution of B cells, thereby systemically inducing the anti-inflammatory function.
The present invention has significance for first confirming that among AARS of Akkermansia muciniphila strains, especially TARS has an effect of preventing, alleviating, or treating various inflammatory diseases.
In particular, TARSs derived from Bacteroides fragilis, Escherichia coli, and Ruminococcus bromii, which are strains in the human microbiome, and Homo sapiens are proteins having the same function as Akkermansia muciniphila-derived TARS, but exhibited a different effect on the secretion of inflammatory or anti-inflammatory cytokines from Akkermansia muciniphila-derived TARS, and therefore Akkermansia muciniphila-derived TARS according to the present invention had a significant effect on the prevention, alleviation, or treatment of inflammatory diseases.
As used herein, the term “prevention” refers to all actions that inhibit or delay the outbreak of an inflammatory disease by administration of the composition of the present invention, and the term “treatment” refers to all actions that alleviates or favorably change the symptoms of an inflammatory disease by administration of the composition.
The pharmaceutical composition containing Akkermansia muciniphila-derived TARS or a fragment thereof of the present invention may further contain an appropriate carrier, excipient, or diluent that is commonly used in the preparation of a pharmaceutical composition. The content of Akkermansia muciniphila-derived TARS or a fragment thereof contained in the composition may include, but is not particularly limited to, 0.0001 wt % to 10 wt %, and preferably 0.001 wt % to 1 wt %, relative to the total weight of the composition.
The pharmaceutical composition may have any one formulation selected from the group consisting of a tablet, pills, a powder, granules, a capsule, an oral liquid preparation, a syrup, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository, or may have several oral or parenteral formulations. The composition, when formulated as a preparation, may be formulated using a diluent or an excipient, such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant, which are commonly used. Exemplary solid preparations for oral administration include a tablet, pills, a powder, granules, a capsule, and the like, and such solid preparations may be prepared by mixing at least one compound with at least one excipient, for example, starch, sucrose, or lactose, gelatin, or the like. Lubricants, such as magnesium stearate and talc, may also be used in addition to simple excipients. Exemplary liquid preparations for oral administration correspond to a suspension, an oral liquid preparation, an emulsion, a syrup, and the like, and may contain simple diluents that are frequently used, such as water and liquid paraffin, as well as several excipients, such as a wetting agent, a sweetener, an aroma, and a preservative. Exemplary preparations for parenteral administration include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and a suppository. Examples of the non-aqueous solvent and suspension may include propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethylolate, and the like. Exemplary base materials for the suppository may include Witepsol, Macrogol, Tween 61, cocoa butter, laurin butter, glycerogelatin, and the like.
The composition of the present invention may be administered at a pharmaceutically effective amount.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the level of the effective dose may be determined depending on the subject's type, the disease severity, the subject's age and sex, the type of the disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, excretion rate, the duration of treatment, factors including drugs used in combination with the composition, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be sequentially or simultaneously administered together with a conventional therapeutic agent. In addition, the composition may be administered once or multiple times. It is important to administer the composition at an amount to obtain the maximum effect with a minimum amount and without side effects considering all of the above factors, and such an amount may be easily determined by a person skilled in the art. A preferable dose of the composition of the present invention varies depending on the condition and body weight of a patient, severity of the disease, the dosage form of the drug, and the route and period of administration; however, for a preferred effect, the Akkermansia muciniphila-derived TARS or fragment thereof of the present invention may be administered daily in an amount of 0.0001 μg/kg to 100 μg/kg, and preferably 0.001 μg/kg to 100 μg/kg. The administration may be carried out once or several times in divided doses a day. The composition may be administered to various mammals, such as rats, livestock, and humans, through various routes, and the manner of administration is included without limitation as long as the manner is commonly used in the art, and for example, the composition may be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebroventricular injection.
The pharmaceutical composition of the present invention may be used in the form of veterinary medicines as well as pharmaceuticals applied to humans.
In accordance with another aspect of the present invention, there is provided a method for preventing or treating an inflammatory disease, the method including administering to a subject a composition including Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
The term “subject” used herein refers to any animal that has or is likely to have an inflammatory disease, and the administration of the pharmaceutical composition of the present invention to a subject suspected of having the inflammatory disease can efficiently treat the subject.
As used herein, the term “administration” refers to an introduction of the pharmaceutical composition of the present invention into a subject suspected of having an inflammatory disease by any suitable method, and the pharmaceutical composition may be administered through various oral or parenteral routes as long as the pharmaceutical composition can reach a target tissue.
The pharmaceutical composition of the present invention may be administered at a pharmaceutically effective amount, and the pharmaceutically effective amount is as described above.
The pharmaceutical composition of the present invention may be applied to any subject without particular limitation as long as the pharmaceutical composition has a purpose of preventing or treating an inflammatory disease of the subject. For example, the pharmaceutical composition may be applied to non-human animals, such as monkeys, dogs, cats, rabbits, marmots, rats, mice, cows, sheep, pigs, and goats, birds, fishes, and the like, and the pharmaceutical composition may be administered through parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes, and for topical treatment, if necessary, the pharmaceutical composition may be administered by any suitable method including intralesional administration. A preferable dose of the pharmaceutical composition of the present invention may vary depending on condition and body weight of a subject, severity of the disease, the dosage form of the drug, and the manner and period of administration, but may be appropriately selected by a person skilled in the art. For example, the pharmaceutical composition may be administered by oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebrovascular injection, but is not limited thereto.
In accordance with still another aspect of the present invention, there is provided a composition for differentiation or proliferation of B cells, the composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
The composition for differentiation or proliferation of B cells is an inducer for differentiation or proliferation of B cells and may further contain a substance known in the art in addition to the Akkermansia mucimphila-derived TARS or a fragment thereof, but is not limited thereto.
The effect of the TARS or a fragment thereof for the differentiation and proliferation of B cells is as described above.
In accordance with still another aspect of the present invention, there is provided a composition for differentiation or proliferation of M2 macrophages, the composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
The composition for differentiation or proliferation of M2 macrophages is an inducer for differentiation or proliferation of M2 macrophages and may further contain a substance known in the art in addition to the Akkermansia muciniphila-derived TARS or fragment thereof, but is not limited thereto.
The effect of the TARS or fragment thereof for the differentiation and proliferation of M2 macrophages is as described above.
In accordance with still another aspect of the present invention, there is provided a food composition for prevention or alleviation of an inflammatory disease, the food composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
As used herein, the “alleviation” refers to any action that alleviates or advantageously changes symptoms of a subject having or suspected of having an inflammatory disease by using the composition.
As a food-acceptable salt that may be contained in the food composition of the present invention, an acid addition salt formed by a food-acceptable free acid or a metal salt formed by a base is useful. As one example, an inorganic acid and an organic acid may be used as the free acid. Examples of the inorganic acid may include hydrochloric acid, sulfuric acid, bromic acid, sulfurous acid, or phosphoric acid, and examples of the organic acid may include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, and the like. Examples of the metal salt may include an alkali metal salt or an alkaline earth metal salt, such as a sodium, potassium, or calcium salt. However, the present invention is not limited thereto.
The food composition of the present invention includes forms of pills, a powder, granules, an infusion, a tablet, a capsule, a liquid preparation, or the like, and exemplary foods that may include the composition may be various kinds of foods, for example, beverages, gums, teas, vitamin complexes, and health supplement foods.
As for ingredients that may be contained in the food composition of the present invention, the other ingredients other than the active ingredient as an essential ingredient are not particularly limited, and various herbal extracts, food supplements, or natural carbohydrates may be contained as additional ingredients, like in common foods. The content of the active ingredient in the food composition may be appropriately determined according to the purpose of use (prevention, alleviation, or therapeutic treatment). The content of the active ingredient contained in the composition is, but not particularly limited to, 0.0001 wt % to 10 wt %, and preferably 0.001 wt % to 1 wt %, relative to the total weight of the composition.
Examples of the food supplement additives include food supplement additives that are commonly used in the art, for example, flavours, savoury flavours, colorings, fillers, stabilizers, and the like.
Examples of the natural carbohydrates may include ordinary sugars, for example, monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose, and polysaccharides, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol, sorbitol, and erythritol. In addition to the above-described ingredients, natural flavours (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavours (saccharin, aspartame, etc.) may be advantageously used as the aroma.
Additionally, the food composition of the present invention may contain several types of nutrients, vitamins, minerals (electrolytes), savory flavors such as synthetic savory flavors and natural savory flavors, colorings, fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used for carbonated drinks, and the like. Additionally, the food composition of the present invention may contain fruit flesh for manufacturing natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used either alone or in combination.
In the present invention, examples of the health supplement food may include a health functional food, a health food, and the like.
The functional food, being the same term as food for special health use (FoSHU), refers to a food with high medicinal and medical effects to efficiently exhibit a bio-regulatory function in addition to a function of nutrient supply. The term “functional” refers to controlling nutrients for the structure or functions of the human body or providing beneficial effects to health purposes, such as physiological effects.
The food containing the food composition of the present invention may be manufactured by a method that is commonly used in the art, and in the manufacturing of the food, raw materials and ingredients that are commonly added in the art may be added. In addition, a formulation of the food is not limited, as long as the formulation is accepted as a food. The food composition of the present invention may be prepared as a variety of formulations, and since a food is used as raw materials, unlike general drugs, the food composition lacks side effects that may occur when a drug is taken for a long period, and may have excellent portability.
In accordance with another aspect of the present invention, there is provided a quasi-drug for prevention or alleviation of an inflammatory disease, the quasi-drug containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
As used herein, the term “quasi-drug” refers to an article that is not an instrument, machine, or device, among the articles used for diagnosing, curing, relieving, treating, preventing, or alleviating diseases of humans or animals, and an article that is not an instrument, machine, or device, among the articles used for giving pharmaceutical influences on structures and functions of humans or animals, and also encompasses an externally-applied preparation for skin and a personal hygiene product.
When the Akkermansia muciniphila-derived TARS or fragment thereof of the present invention is added to a quasi-drug composition for prevention or alleviation of an inflammatory disease, the Akkermansia muciniphila-derived TARS or fragment thereof is added as it is or may be used together with other quasi-drug ingredients, or may be appropriately used according to a conventional method. The amount of the active ingredient mixed may be appropriately determined according to the purpose of use.
The externally-applied preparation for skin is not particularly limited, but may be preferably prepared and used in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a dispersion, a gelling agent, or a gel. Examples of the personal hygiene product may preferably include soaps, cosmetics, wet tissues, toilet paper rolls, shampoos, skin creams, facial creams, toothpastes, lipsticks, make-ups, foundations, blushers, mascaras, eye shadows, sunscreen lotions, haircare products, air-freshener gels, or facial cleansing gels, but are not limited thereto. In addition, other examples of the quasi-drug composition of the present invention may include disinfectants, shower foams, mouthwash products, wet tissues, detergents, handwashes, humidifier fillers, masks, ointments, or filters.
In accordance with another aspect of the present invention, there is provided a feed composition for prevention or alleviation of an inflammatory disease, the feed composition containing Akkermansia muciniphila-derived TARS or a fragment thereof as an active ingredient.
The terms used herein are as described above.
As used herein, the term “feed composition” may refer to any natural or artificial diet, single meal, or an ingredient of the single meal for animals to eat, ingest, and digest or suitable for this purpose, and the feed composition may be prepared in various forms known in the art.
The type of feed is not particularly limited, and any feed commonly used in the art may be used. Non-limiting examples of the feed may include: vegetable feeds, such as grains, nuts, food byproducts, algae, fibers, pharmaceutical byproducts, fats and oils, starches, barks, or grain byproducts; and animal feeds, such as proteins, inorganic substances, fats and oils, minerals, single-cell proteins, animal planktons, or foods. These may be used alone or in a mixture of two or more thereof. The animals have a concept encompassing livestock and pets.
The feed composition of the present invention may further contain binders, lubricating agents, preservatives, and the like added for prevention of quality deterioration thereof; and amino acids, vitamins, enzymes, probiotics, flavors, non-protein nitrogen compounds, silicates, buffers, colorings, extracts, oligosaccharides, and the like for efficiency improvement, as well as other feed mixtures and the like, but is not limited thereto.
Hereinafter, the present invention will be described in more detail through the following exemplary embodiments. However, the exemplary embodiments according to the present invention may be modified in many different forms, and the scope of the present invention shall not be construed as being limited to the exemplary embodiments described below. The exemplary embodiments of the present invention are provided to illustrate the present invention more completely to those skilled in the art.
Akkermansia muciniphila was inoculated in basal tryptone threonine medium (BTTM), and then cultured for one day with anaerobic conditions maintained. 1 L of bacterial culture was collected, sequentially centrifuged three times (500×g, 5000×g, and 15000×g, 30 min for each), and then filtered through a 0.22 μm filter to remove bacteria and thereby obtain only the supernatant. A QuixStand system with a 10 kDa cut-off filter was used to concentrate the supernatant, and the supernatant after concentration to 100 mL was again centrifuged at 15000×g for 30 min and filtered through a 0.22 μm filter. Finally, the supernatant was centrifuged at ultra-high speed (200000×g) for 2 h to obtain pellets including vesicles.
As a result of investigating the size of the vesicles in the pellets by dynamic light scattering (DLS), the vesicles were particles with a size of 30-100 nm (average 42 nm) and had a spherical shape through transmission electron microscopy (TEM) analysis (
Thereafter, the obtained pellets were lysed using a lysis buffer (containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% Na-deoxycholate, and a protease inhibitor cocktail). The lysate and supernatant were separately centrifuged at 13,000 rpm for 15 min, and then 20 μg for each was subjected to protein quantification, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins separated on SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated with a solution of 1×TBST (137 mM NaCl, 50 mM Tris-HCl, 0.1% Tween 20) and 4% BSA for 1 h and 30 min.
Rabbit polyclonal antibodies were prepared as antibodies against threonyl-tRNA synthetase (TARS), tyrosyl-tRNA synthetase (YARS), and prolyl-tRNA synthetase (PARS), which are Akkermansia muciniphila-derived aminoacyl-tRNA synthetases (AARSs), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two rabbits for each antigen were inoculated three times with high-purity AmTARS, AmYARS, AmPARS, and AmGAPDH as antigens. After the inoculation, individuals having antigen-specific antibodies in sera were selected through ELISA, and the secured sera were affinity-purified to obtain polyclonal antibodies.
Each antibody was diluted in 1×TBST and 4% BSA according to the concentration of use, and then incubated on the membrane at 4° C. overnight. Thereafter, the patterns of constituent proteins were analyzed through Coomassie brilliant blue (CBB) staining. Each sample was divided into three and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to thereby identify proteins including AARSs.
In the pellets containing vesicles, AmTARS (Akkermansia muciniphila-derived) was the most abundant among the Akkermansia muciniphila-derived AARSs, and AmYARS (Akkermansia muciniphila-derived YARS) and μm PARS (Akkermansia muciniphila-derived PARS) were present in relatively small amounts.
In this example, all His-tagged proteins were purified in vitro using Escherichia coli (E. coli). First, pET21a-AmTARS was introduced to cells through heat shock, and then cells containing genes were selected using the antibiotic ampicillin and cultured in liquid. The cultured cells were obtained using centrifugation, and then suspended using a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The cells were lysed using sonication treatment, and then the supernatant was obtained by using high-speed centrifugation. The AmTARS-His protein was purified using Ni-NTA agarose affinity chromatography. The obtained protein was intensively dialyzed with a solution of 1×PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2K2HPO4), and then subjected to lipopolysaccharide (LPS) removal using TX-114 on the basis of the paper by Y Aida et al. (1990) to remove LPS from bacteria, thereby obtaining a high-purity protein. AmTARSΔ1, AmTARSΔ2, AmYARS, AmPARS, AmGAPDH, BfTARS, eTARS, RbTARS, and hTARS proteins were also obtained in the same manner.
As for Strep-tagged protein, HEK293T cells as mammalian cells were used, and 50-100 sheets of 150 mm culture dishes were cultured through several cell passages. For transduction of pEXPR-IBA105-AmTARS into the cells, polyethylenimine (PEI) was used. When the cells were cultured 24 h after the introduction, the cells were obtained using centrifugation. The cells were suspended in the solution of 100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1× protease inhibitor (GenDEPOT), and 1× phosphatase inhibitor (GenDEPOT), and then incubated at 4° C. for 30 min by using a rotator. A supernatant was obtained through high-speed centrifugation, and subjected to Strep-Tactin agarose affinity chromatography to purify Strep-AmTARS. The obtained protein was intensive-dialyzed using 1×PBS (pH 7.4) and then used for an experiment.
FLAG-tagged proteins were purified by affinity chromatography using FLAG M2 affinity gel.
The DNA constructs used in the present example are shown in Table 1 below.
Akkermansia muciniphila has been known to regulate immune cytokines in the gut environment, and thus to determine whether cytokines were also secreted from immune cells by AmTARS treatment, ELISA was performed using mammalian-derived immune cells.
Specifically, mouse bone marrow-derived macrophages (BMDMs) as mouse-derived immune cells were extracted from the mouse thighbone and shinbone, and then cultured in DMEM containing 10% fetal bovine serum (FBS) and antibiotics. Human macrophages (THP1) as human-derived immune cells were cultured in RPMI media containing 10% FBS and antibiotics.
The cultured cells were treated with AmTARS at 0.1 μM, 0.2 μM, and 0.5 μM for 24 h to obtain cell cultures. For cell removal, the media were centrifuged at 500×g for 10 min to obtain supernatants, which were then analyzed using an ELISA set (mouse interleukin (IL)-6, mouse IL-10, mouse tumor necrosis factor (TNF)-α, human IL-6, human IL-10, human TNF-α; BD Biosciences) to investigate the secretion of mouse- and human-derived immune cytokines.
As a result, as shown in
Similar to BMDMs, when THP1 were treated with AmTARS, IL-6 and TNF-α were not secreted while the secretion of IL-10 was increased in a dose-dependent manner based on AmTARS.
It was therefore confirmed that AmTARS induced the secretion of the inflammatory cytokine IL-10 from immune cells.
Next, To examine the involvement of commensal ARSs in immune regulation, we purified A. muciniphila ARSs (AmARSs) and tested their anti-inflammatory activity against bone marrow-derived macrophages (BMDMs). We found that AmTARS, but not other AmARSs, triggered a significant increase in production of anti-inflammatory IL-10 by BMDMs under steady state conditions (
Of note, although AmTARS induced production of anti-inflammatory IL-10 by BMDMs under normal conditions, it had no effect on production of pro-inflammatory cytokines IL-6 and TNF-α (
To determine whether AmTARS affects immune cytokine secretion when cellular inflammation was induced, mouse bone marrow-derived macrophages BMDMs as mouse-derived immune cells and human macrophages THP1 as human-derived immune cells were cultured by way of the same method as in Example 2.
Specifically, the cultured BMDMs and THP1 were treated with lipopolysaccharide (LPS) to induce an inflammatory response and were simultaneously treated with AmTARS at 0.1 μM, 0.2 μM, and 0.5 μM for 24 h to obtain cell cultures. The obtained cell cultures were analyzed for immune cytokine secretion by way of the same method as in Example 2.
As a result, AmTARS increased IL-10 levels, and suppressed IL-6 and TNF-α to an even greater extent following stimulation of BMDMs with lipopolysaccharide (LPS) (
Also, as shown in
Similar results were observed in AmTARS-treated THP1 cells under both normal and inflammatory (LPS) conditions (
In addition, as shown in
Taken together, these results suggest that the commensal bacterium A. muciniphila secretes AmTARS to maintain (under normal conditions) and induce (under inflammatory conditions) host immune homeostasis.
To determine which signaling pathway was involved in the anti-inflammatory effect by the immune cytokine secretion regulation of AmTARS, an inflammatory environment was created through LPS treatment on mouse macrophage Raw264.7 as mouse-derived immune cells, and then changes in phosphorylation of signal transducers involved in the regulation of immune cytokine secretion according to the presence or absence of AmTARS treatment and the elapsed time (0.5 to 3 hours) were investigated. Raw264.7 cells were cultured in DMEM (Hyclone) containing 10% FBS and antibiotics.
As a result, as shown in
Next, to determine whether the anti-inflammatory function of AmTARS affected the regulation of metabolic disease-associated intracellular signaling, immunoblot analysis was performed using the mouse adipocyte 3T3-L1.
Specifically, 3T3-L1 cells were cultured in DMEM containing 10% bovine calf serum (BCS) and antibiotics under conditions of 5% CO2 and 37° C. The cells were treated with 2 μM phorbol 12-myristate 13-acetate (PMA) or 20 ng/mL TNF-α to create an inflammatory environment in the cultured cells. The cells were treated with 100 nM insulin and 0.5 μM AmTARS for 24 h, and then changes in intracellular signaling were investigated.
As a result, as shown in
In addition, as shown in
It was therefore confirmed that with the induction of cellular inflammation, the treatment with AmTARS inhibited inflammatory responses and increased the production of anti-inflammatory cytokines by regulating the phosphorylation of intracellular signal transducers, and affected the metabolic disease-associated intracellular signaling.
Since the cell experiments confirmed that AmTARS had an anti-inflammatory function through immune cytokine regulation, an experiment was performed to verify the efficacy of AmTARS in an inflammatory bowel disease mouse model.
We evaluated the anti-inflammatory function of AmTARS in a mouse model of dextran sulfate sodium (DSS)-induced colitis. First, we assessed dose-dependent responses (i.e., changes in body weight and colon length) to intraperitoneal (i.p.) administration of AmTARS (0.05-0.4 mg/kg) once every other day for 10 days (
Overall, administration of AmTARS attenuated body weight loss and colon shortening in a dose-dependent manner (up to 0.2 mg/kg) (
The disease activity score was calculated by adding up scores ranging from 0 to 4 points for each item of bloody stool, diarrhea, and weight loss, and the epithelial damage score was calculated by assigning 0 to 4 points according to the degree of colon tissue damage.
To identify the cells targeted by AmTARS, mice with injected i.p. with fluorescently labeled AmTARS, and fluorescence-positive cells in peritoneal exudate cells (PECs), were analyzed by flow cytometry. Fluorescently labeled Alexa568-AmTARS was detected predominantly on macrophages (F4/80+) rather than T cells (CD3+) and B cells (CD19+) (
Since M2 macrophage polarization precedes production of anti-inflammatory cytokines such as IL-10, we assessed the effects of AmTARS on macrophage polarization. BMDMs were treated with PBS (negative control), LPS (to induce M1 polarization), or IL-4/IL-13 (to induce M2 polarization [positive control]) in the absence and presence of AmTARS, and the resulting M2 macrophage population was measured by flow cytometry using an antibody specific for the M2 macrophage surface marker CD206. Notably, AmTARS activated M2 polarization even in PBS-treated cells, and significantly re-polarized LPS-induced M1 macrophages to M2 macrophages (
To further validate the macrophage-dependent function of AmTARS in improving colitis, we investigated the anti-inflammatory effects of AmTARS in macrophage-depleted colitis mice.
A macrophage-deficient mice were prepared by administration of Clodrosome (administered at a concentration of 200 μL/animal to the abdominal cavity of mice a total of three times on Days 1, 5, and 8), and then the anti-inflammatory efficacy experiment was conducted by way of the same method as in Example 5. Normal mice injected with Encapsome were used as a control.
Consistent with the physiological data, AmTARS did not affect the numbers of inflammatory monocytes or IL-10-positive macrophages in macrophage-depleted mice (
Indeed, the therapeutic effects of AmTARS on colitis pathology were comparable with those of IL-10 in mice with colitis (
Next, to measure IgA, mouse feces were collected and diluted in PBS containing a protein inhibitor, and then a supernatant with impurities removed was obtained, and 100 μL was placed in each well of a 96-well plate to perform protein coating at 4° C. for 2 h. The plate was washed three times with a washing solution (50 mM Tris, 0.2% in PBS) and then incubated with 100 μL of blocking solution at room temperature for 1-2 h, followed by washing. HRP conjugated IgA antibody (1:30000) was added and then incubated at room temperature for 1 h, followed by washing. Thereafter, 100 μL of a substrate solution was added, followed by incubation for 30 min, and then 50 μL of a stop solution was added, followed by measurement at 405-450 nm by an ELISA reader.
As a result, as shown in
To further validate the macrophage-dependent function of AmTARS in improving colitis, we investigated the anti-inflammatory effects of AmTARS in macrophage-depleted colitis mice. Consistent with the physiological data, AmTARS did not affect the numbers of inflammatory monocytes or IL-10-positive macrophages in macrophage-depleted mice (
To determine whether the inflammatory bowel disease alleviation effect induced through the secretion of IL-10 cytokine by AmTARS corresponded to a B cell-mediated effect, DSS was administered to B cell-deficient pMut mice (p heavy chain mutation mice, B cell depletion mice) by way of the same method as in Example 5 to induce inflammatory bowel disease, and AmTARS was administered.
As a result, as shown in
As shown in
It was therefore confirmed that AmTARS increased the distribution of B cells in the colon to induce an inflammatory bowel disease alleviation effect.
To determine whether TARS derived from other bacteria in the human microbiome and TARS secreted by human cells perform similar functions to AmTARS, TARSs derived from Bacteroides fragilis, E. coli, and Ruminococcus bromii, which are strains in the human microbiome, and TARS derived from Homo sapiens (BfTARS, eTARS, RbTARS, and hTARS, respectively) were purified and subjected to ELISA using immune cells.
Specifically, AmTARS bound to pET21a vector, BfTARS bound to pET21a vector, eTARS bound to pET21a vector, RbTARS bound to pET21a vector, and hTARS bound to pET21a vector were separately introduced into E. coli through heat shock, and then cells containing genes were selected using the antibiotic ampicillin and cultured in liquid. The cells were obtained by centrifuging the cultures, and then suspended using a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The cells were lysed using sonication treatment, and then centrifuged at high speed to obtain supernatants. The supernatants were passed through Ni-NTA agarose affinity chromatography to purity AmTARS-His, BfTARS-His, eTARS-His, RbTARS-His, and hTARS-His proteins. The obtained proteins were intensively dialyzed with a solution of 1×PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2K2HPO4), and lipopolysaccharide (LPS) of E. coli was removed using TX-114 to obtain high-purity proteins (Left in
Thereafter, BMDMs were treated with each TARS (AmTARS, BfTARS, eTARS, and RbTARS) by way of the same method as in Example 2 and subjected to ELISA to investigate the secretion of IL-10.
As a result, as shown in
Next, BMDMs were treated with LPS to create an inflammatory environment by way of the same method as in Example 3, and then treated with each TARS (AmTARS, BfTARS, eTARS, and hTARS) to investigate the secretion of IL-10.
As a result, as shown in
It was therefore confirmed that as a result of comparing functions in the immune responses of TARSs derived from strains in the microbiome, the anti-inflammatory response through an increase in IL-10 secretion was shown only in the case of AmTARS treatment.
To uncover the molecular mechanism underlying the anti-inflammatory immune function of AmTARS, we compared its amino acid sequence with that of human TARS1, and with those of TARSs from bacterial species representative of the gut microbiota. AmTARS contains two additional unique regions within its N-terminal(SEQ ID NO: 3, residues 99-113 of SEQ ID NO: 1 (A region encoded by the nucleotide sequence of SEQ ID NO: 5, which is the sequence corresponding to the 295th to 339th nucleotides of SEQ ID NO: 2), hereafter referred to as U1) and catalytic(SEQ ID NO: 4, residues 290-312 of SEQ ID NO: 1 (A region encoded by the nucleotide sequence of SEQ ID NO: 6, which is the sequence corresponding to the 868th to 936th nucleotides of SEQ ID NO: 2), hereafter referred to as U2) domains, which are not found in other TARSs (
Next, we modeled the structure of AmTARS using AlphaFold (Jumper et al., 2021), and found that it showed significant structural similarity to other TARSs, including Staphylococcus aureus TARS (PDB ID 1 NYQ) and human TARS1 (PDB ID 4HWT) (
To purify ΔU1AmTARS and ΔU2AmTARS, ΔU1AmTARS bound to pET21a vector and ΔU2AmTARS bound to pET21a vector were separately introduced into E. coli through heat shock, and then cells containing genes were selected using the antibiotic ampicillin and cultured in liquid. The cells were obtained by centrifuging the cultures, and then suspended using a solution of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole. The cells were lysed using sonication treatment, and then centrifuged at high speed to obtain supernatants. The supernatants were passed through Ni-NTA agarose affinity chromatography to purity ΔU1AmTARS-His and ΔU2AmTARS-His proteins. The obtained proteins were intensively dialyzed with a solution of 1×PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2K2HPO4), and then LPS from E. coli was removed using TX-114 to obtain high-purity proteins (
TARS is an aminoacylase involved in protein synthesis, and the domain and site involved in its function are well known. The constructed deletion mutants were confirmed to have deletions of parts irrelevant to the function, but the aminoacylation reaction experiment was conducted to determine whether the deletion mutants performed the intrinsic functions thereof appropriately.
Specifically, a 2× reaction solution (100 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 200 mM NaCl, 200 mM KCl, 2 mM DTT, 500 μM ATP, and 5 mM L-threonine) was prepared, and 25 μL of a reaction solution, 20 μL of distilled water, and 5 μL of protein were added to make a final 50 μL volume, followed by incubation for 30 min. Thereafter, a reaction was conducted using a malachite green phosphate assay kit (Bioassay Systems) for 30 min, and the free monophosphate group (Pi) was measured at a wavelength of 620 nm.
As a result, as shown in
Next, BMDMs were treated with AmTARS, ΔU1AmTARS, and ΔU2AmTARS separately and analyzed by ELISA by way of the same method as in Example 2. As a result, Neither mutant protein was different from the wild-type (WT) AmTARS with respect to its enzymatic activity (
Next, to determine whether the regulation of immune cytokine secretion by AmTARS in an inflammatory environment was induced by the two functional sites, the mouse-derived immune cell Raw 264.7 cells were treated with LPS to induce an immune response by the method as in Example 3, and treated with AmTARS, ΔU1AmTARS, and ΔU2AmTARS for 24 h separately to obtain cell cultures. The obtained cell cultures were analyzed for Immune cytokine secretion by way of the same method as in Example 2.
As a result, as shown in
To determine whether U1 and U2 as deletion sites exhibited an anti-inflammatory effect of AmTARS, peptides were prepared using respective sequences, and the effect thereof was investigated.
A total of four types of fragments (AmTARS, U1 (R1, SEQ ID NO: 3), U2 (R2, SEQ ID NO: 4), Tat-function site 1 (Tat-R1), and Tat-function site 2 (Tat-R2)) were synthesized including U1, U2, and fragments obtained by adding, to the functional sites, Tat tag, which is a tag for favorable delivery into cells.
An inflammatory environment was created through LPS in BMDMs by way of the same method as in Example 3, and then the cells were treated with the four types of fragments, and the immune cytokine secretion was determined by ELISA.
As a result, as shown in
When the concentrations of U2 were 100 μM, 200 μM, 300 μM in the same conditions, the secretion of IL-6 was reduced and the secretion of IL-10 was increased by all of U2(R2) 100 μM and 300 μM and Tat-R2 100 μM, 200 μM, and 300 μM. Of these, the secretion of IL-10 was increased by 2-fold in the Tat-R2 treatment groups compared with AmTARS.
It was therefore confirmed that the AmTARS-specific regulation of immune cytokine secretion was exerted from U1 and U2.
To verify the anti-inflammatory efficacy of specific functional sites of AmTARS, AmTARS, ΔU1AmTARS, and ΔU2AmTARS were separately administered to the inflammatory bowel disease mouse model of Example 5, and then an anti-inflammatory efficacy experiment was performed.
Neither mutant protein promoted recovery of body weight loss, disease activity scores, shortening of the intestine, or epithelial lining disruption and damage scores, in colitis-induced mice (
In addition, as shown in
It was therefore confirmed that U1 and U2 were essential for anti-inflammatory efficacy in AmTARS.
To identify the host target of AmTARS, we performed membrane protein interactome analysis using THP1 cells. Several potential interactors, which are receptors involved in innate immunity, were identified, and their interactions with AmTARS were evaluated using a pull-down assay.
Specifically, THP1 was separated into cytoplasmic and cell membrane components by using Mem-PER™ Plus Membrane Protein Extraction Kit. The separated lysate was incubated with His-tagged AmTARS and then pulled down with His-tagged resin. Proteins bound to the resin were eluted and then loaded on SDS-PAGE gel to identify proteins by CBB staining.
As a result, as shown in
Then, each gel was largely divided into five, which were then subjected to liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis. A part of the gel to be analyzed was placed in a 1.5 mL tube. The gel was de-stained with 25 mM ammonium bicarbonate (ABC)/50% acetonitrile (ACN) until the gel was transparent. Thereafter, 10 mM DL-dithiothreitol (DTT) and 55 mM iodoacetamide (IAA) were added, followed by reduction and alkylation. Upon the end of the reaction, the gel was dried, and then 0.0125 μg/μL trypsin was added with the same volume as the gel, followed by incubation overnight. Thereafter, the sample was collected and then dried with a vacuum pump.
The sample was dissolved in 10 μL of 2% ACN/0.1% formic acid (FA) solution, and 5 μL thereof was injected. The LTQ-Orbitrap Elite and Easy nano-LC 1000 by Thermo were used for analysis, and solution A used for LC was 0.1% FA/water, and solution B was 0.1% FA/ACN, and solution B was allowed to flow at a gradient of 2% to 32% for 120 min, and the flow rate was 0.3 μL/min. The trap column used was pepamap 100 (75 μm×2 cm, C18, 3 μm, 100 Å), and the analytical column was pepmap RSLC (C18 2 μm, 100 Å, 75 μm×15 cm). The analytical column temperature was 50° C., and the full MS resolution was 60,000, and AGC target was 1E5. MS/MS used the HCD method with a resolution of 15,000, and MS/MS was set to top 15, IT 100 ms, and threshold 200,000.
The raw data obtained by LC-MS/MS were analyzed using Maxquant. For the database, uniprot Homo sapiens was used, and modification was set as fixed modification—carbamidomethyl(c), variable modification—oxidation (M), acetyl (protein N-term). Peptide 2 or more were assigned, and LFQ and match between run were applied to perform analyses. “LFQ intensities” were compared by filtration through at least 2 razor+unique peptide.
As a result, as shown in
Then, Strep-tagged AmTARS, AmTARS having deletions of two functional site (AmTARSΔ1Δ2), and FLAG-tagged TLR2 were transduced into human-derived HEK 293T cells through PEI, and thereafter, the cells were lysed using a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1 mM EDTA, 0.25% Na-deoxycholate, and a protease inhibitor cocktail. The cell lysates each were incubated with Strep Tactin beads and washed with a buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 1 mM EDTA, and proteins attached to the beads were eluted and analyzed by immunoblotting.
In addition, His-tagged AmTARS and FLAG-tagged protein were mixed at a ratio of 1:2, incubated with Ni-NTA beads, and then washed with a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, and 1% NP-40, and the proteins attached to the beads were eluted and identified on SDS-PAGE gel.
The results suggested that among the candidates tested, AmTARS interacted only with TLR2 (
The TLR2-dependent immune function of AmTARS was further corroborated using TLR2 knock-out (KO) mice. AmTARS did not induce IL-10 production in BMDMs from TLR2-KO mouse, demonstrating that interaction between AmTARS and TLR2 is indispensable for anti-inflammatory immune activation (
We assessed the role of TLR2 in AmTARS function in vivo. To verify whether the binding between AmTARS and TLR2 actually acted on the anti-inflammatory effect of AmTARS, an inflammatory bowel disease mouse model was prepared from TLR2-deficient mice by way of the same method as in Example 5 and investigated for anti-inflammatory efficacy after the administration of AmTARS. TLR2-KO mice were slightly more susceptible to DSS-induced colitis than WT mice, as reported previously (Rakoff-Nahoum et al., 2004).
Colitis-induced body weight loss and the disease activity score in TLR2-KO mice did not improve after AmTARS treatment (
As shown in
These results strongly suggest that AmTARS is a commensal-derived TLR2 ligand required to maintain host immune homeostasis.
To identify the signaling pathway triggered by AmTARS, we first evaluated kinases and CREB. IL-10 production and M2 polarization were measured in BMDMs treated with inhibitors of ERK (PD98059), p38 (SB203580), MSK1/2 (SB747651A), CREB (666-15), PI3K (Wortmannin) and AKT (AKT1/2) before subsequent stimulation with AmTARS. Inhibition of each of these molecules resulted in significant suppression of IL-10 production (
Subsequently, we conducted immunoblotting assays to measure the time-dependent activation of AmTARS/TLR2-induced signaling molecules in both BMDMs and THP1 cells. AmTARS rapidly activated ERK, p38, and MSK1/2 within 15 min and, subsequently, phosphorylation of CREB at Ser133 (which is essential for IL-10 production) within 30 min in BMDMs (
CREB phosphorylated at Ser133 inhibits NF-
AmTARS activated PI3K and AKT, and subsequently inhibited GSK3β activity via AKT-mediated phosphorylation at Ser9, in BMDMs (
Since TLR2 forms heterodimers with either TLR1 or TLR6 during activation, we assessed which form is targeted by AmTARS. An immunoassay showed that AmTARS-induced TLR2 signaling is shut down by antibodies targeting TLR1, TLR6, and TLR2, indicating that AmTARS targets both the TLR2/1 and TLR2/6 forms to activate anti-inflammatory signaling (
We next performed global gene expression profiling of BMDMs treated with AmTARS or Pam3 for 1 or 3 h (
Next, we validated the anti-inflammatory function of AmTARS using the Tg(NF
Taken together, these data provide further evidence that AmTARS secreted by the commensal bacterium A. muciniphila is a steady-state immune homeostatic mediator that is distinct from typical TLR2 ligands, and which maintains and induces immune homeostasis in response to inflammatory challenges (
To verify whether the administration of AmTARS via an intramuscular injection exhibits an anti-inflammatory efficacy, an inflammatory bowel disease mouse model was prepared in accordance with Example 5. Further, AmTARS was intramuscularly injected into the mouse model to confirm the anti-inflammatory efficacy thereof.
As a result, AmTARS, when intramuscularly injected at 0.4 mg/kg or more, was confirmed to have the efficacy of decreasing the weight and intestinal length of the mouse model caused by the inflammatory bowel disease. Further, AmTARS was confirmed to alleviate the damage to intestinal epithelial cells caused by the same (
The results of the above examples confirmed that Akkermansia muciniphila TARS or a fragment thereof according to the present invention increased the secretion of the anti-inflammatory cytokine IL-10 by promoting the differentiation of M2 macrophages as anti-inflammatory macrophages to attain the proliferation of macrophages, and thus led to the proliferation of B cells to exhibit an anti-inflammatory effect, thereby exhibiting an effect of alleviating not only an inflammatory disease but also an immune disease, an infectious disease, and a metabolic disease, which were accompanied by inflammation, and in particular, had an excellent effect on the prevention or treatment of inflammatory bowel disease or the like.
As set forth above, a person skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics thereof. Therefore, the embodiments described above should be construed as being exemplified and not limiting the present invention. The scope of the present invention is not defined by the detailed description as set forth above but by the accompanying claims of the invention, and it should also be understood that all changes or modifications derived from the definitions and scopes of the claims and their equivalents fall within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0036379 | Mar 2020 | KR | national |
This application is a continuation-in-part of International Application No. PCT/KR2021/003720 filed on Mar. 25, 2021 which is based upon and claims the benefit of priority to Korean Patent Application No. 10-2020-0036379 filed on Mar. 25, 2020, in the Korean Intellectual Property Office, both of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2021/003720 | Mar 2021 | US |
| Child | 17952990 | US |
