JP6445802B2 - Screening method for inflammasome activation regulator - Google Patents

Description

  The present invention relates to a screening method for an inflammasome activation regulator.

  Inflammasomes are protein complexes that are present in the cytoplasm and induce inflammatory responses. Abnormalities or unwanted activation of inflammasome can cause many diseases such as abnormal glucose metabolism such as diabetes (especially type 2 diabetes), abnormal lipid metabolism, stroke, cardiovascular diseases such as myocardial infarction, chronic hepatitis, self It has become clear that it causes various inflammatory diseases such as inflammatory syndrome and gout.

  The inflammasome is mainly formed from a receptor protein that recognizes a danger signal such as a pathogen, an adapter protein ASC (a complex protein having a pilin domain and a caspase recruitment domain), and caspase-1. Known receptor proteins include NLR protein family proteins such as NLRP1, NLRP3, NLRP4, and NLRP6, AIM2, and the like. Normally, these receptor proteins exist in an inactive form. However, when an activation signal (danger signal such as a pathogen) is received, the self-polymerized receptor protein binds to ASC and caspase-1, and the inflammasome To form an active form. In this state, caspase-1 becomes close to each other, and this proximity induces caspase-1's own cleavage from inflammasome, thereby activating caspase-1. The activated caspase-1 processes pro-IL-1β and pro-IL-18, which are precursors of IL-1β and IL-18, to IL-1β and IL-18, respectively. For example, in the case of the NLRP3 inflammasome having NLRP3, which is known to be activated by various endogenous and exogenous danger signals, as a receptor protein, LRRs (leucine that play an essential role in signal responsiveness at the C-terminus Rich repeat), NOD at the center, NLRP3 with pilin domain (PD) at the N-terminus, ASC with PD at the N-terminus, CARD at the C-terminus, CARD at the N-terminus, p20 at the center, p10 at the C-terminus By binding caspase-1, inflammasome is formed and activated (FIG. 1). The pilin domain (PD), one of the constituent domains of the inflammasome, exists in both NLRP3 and ASC, and the binding between these PDs is involved in inflammasome formation. PD has an amino acid sequence rich in lysine (lysine-rich sequence) in part, and it has been reported that this site is involved in the binding between PDs (Non-patent Document 1).

  IL-1β and IL-18 are inflammatory cytokines, and excessive secretion of these cytokines has been reported to be strongly associated with many diseases. For example, cryopyrin-related periodic fever syndrome, a type of autoinflammatory syndrome, is improved by anti-IL-1 drugs (Non-patent Documents 2 and 3), and type 2 diabetes is improved by IL-1 receptor antagonists (Non-Patent Document 4) has been reported. In addition, in studies using mice, caspase-1 inhibitors improve insulin sensitivity and adipocyte metabolic function (Non-patent Document 5), and in studies using ASC-deficient mice, arteriosclerosis-like intima It has also been reported that hypertrophic lesions are suppressed (Non-Patent Document 6). Furthermore, the relationship between various diseases and inflammasome has been reported (Non-patent Document 7).

  As described above, caspase-1 is released when the receptor protein binds to ASC to form an inflammasome, and pro-IL-1β and pro-IL-18 are processed into IL-1β and IL-18. . Inhibition of inflammasome formation (inflammasome activation) leads to suppression of caspase-1 production, suppression of IL-1 production, and creates a state similar to ASC deficiency. Effects similar to those seen with one drug, caspase-1 inhibitor, or ASC deficiency may be obtained. However, the search for substances that inhibit inflammasome formation has not progressed sufficiently. In particular, no inflammasome formation inhibitor has been obtained from low molecular weight compounds that may be useful in drug development.

  A Pyrin-only protein that can act as an inhibitor of inflammasome has been reported (Patent Document 1). Patent Document 2 discloses the use of an inhibitor of NLRP3 inflammasome as a therapeutic agent for gout. Patent Document 3 discloses that a substance that suppresses the release of IL-1β is screened as an inflammasome inhibitor and used as a UV damage recovery agent. However, no effective screening method has been found for substances that control inflammasome activation by inhibiting inflammasome formation, particularly low molecular weight inflammasome activation regulators.

International Publication WO2006 / 110591 International Publication WO2007 / 077261 JP 2012-046448 A

Vajjhala PR, et al., J. Biol. Chem., 287: 41732-41743 (2012) Raphaela Goldbach-Mansky, M.D., et al., N. Engl. J. Med., 355: 581-592 (2006) Helen J. Lachmann, M.D., et al., N. Engl. J. Med., 360: 2416-2425 (2009) Claus M. Larsen, M.D., et al., N. Engl. J. Med., 356: 1517-1526 (2007) Rinke Stienstra, et al., Cell Metab., 12: 593-605 (2009) Noriyuki Yajima, Circulation, 117: 3079-3087 (2008) S. L. Masters, Clinical Immunology (2013) 143, p.223-228

  An object of the present invention is to provide an effective screening method for an inflammasome activation regulator.

  As a result of intensive studies to solve the above problems, the present inventors have found that inflammasomes are obtained by binding various monocarboxylic acids to lysine-rich sequences in the pilin domain (PD) constituting the inflammasomes. The inventors have found that the activation of can be controlled, and have completed the present invention.

That is, the present invention includes the following.
[1] A screening method for an inflammasome activation regulator, comprising contacting a monocarboxylic acid with a polypeptide containing a lysine-rich sequence of a pilin domain and selecting a monocarboxylic acid that binds to the lysine-rich sequence.
In one embodiment of this method, the polypeptide is
i) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or
ii) A polypeptide comprising an amino acid sequence showing 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and retaining a lysine-rich sequence.
[2] A composition for controlling inflammasome activation, comprising a monocarboxylic acid selected by the method according to [1] above.
In one embodiment, the monocarboxylic acid contained in the inflammasome activation control composition is preferably a short-chain fatty acid or alanine. Preferred examples of short chain fatty acids may be selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and enanthic acid.
In a preferred embodiment, the inflammasome activation control composition is for control of inflammatory cytokine production. In that case, the inflammatory cytokines can be IL-1β and IL-18.
[3] A method for controlling inflammasome activation using the inflammasome activation control composition according to [2] above.
[4] A medicinal product or food or drink for controlling inflammasome activation, comprising the composition for controlling inflammasome activation according to [2] above.
[5] A feed additive for inflammasome activation control, comprising the inflammasome activation control composition according to [2].

  According to the present invention, a substance capable of effectively controlling inflammasome activation can be screened.

FIG. 1 is a schematic diagram showing the process of inflammasome formation and its activation. FIG. 2 is a photograph showing inhibition of inflammasome formation by propionic acid or butyric acid. FIG. 3 is a diagram showing an outline of a method for producing propionic acid-immobilized microbeads and butyric acid-immobilized microbeads. FIG. 4 is a schematic diagram showing a tool used for screening for an inflammasome formation control substance. A shows microbeads on which the test substance is immobilized, and B shows ASC proteins of different lengths. Lys Rich indicates a lysine rich sequence. FIG. 5 is a photograph showing the binding of propionic acid to the lysine-rich sequence of ASC. FIG. 6 is a photograph showing the binding of propionic acid to various NLRP family proteins. FIG. 7 is a photograph showing the results of a competitive inhibition test of short chain fatty acids on ASC. FIG. 8 is a diagram showing the effect of suppressing the increase in caspase-1 activity by propionic acid and butyric acid. * P <0.05, ** P <0.01. (-) Is an additive-free control. FIG. 9 is a diagram showing the inhibitory effect on the increase in IL-1β secretion concentration by propionic acid and butyric acid. ** P <0.01. (-) Is an additive-free control. FIG. 10 is a diagram showing the IL-1β production inhibitory effect of various carboxylic acids. For each carboxylic acid, the left bar shows a sample added with 0.5 mM, and the right bar shows a sample added with 1 mM. Ac: Acetic acid, Pro: Propionic acid, But: Butyric acid, C5: Valeric acid, C6: Caproic acid, C7: Enanthate, Lac: Lactic acid, Ala: Alanine, Glu: Glutaric acid, Suc: Succinic acid, Cit: Citric acid . * P <0.05 and ** P <0.01 compared to the LPS / ATP addition group. (-) Is an additive-free control. FIG. 11 is a diagram showing the IL-18 production inhibitory effect of various carboxylic acids. For each carboxylic acid, the left bar shows a sample added with 0.5 mM, and the right bar shows a sample added with 1 mM. Ac: Acetic acid, Pro: Propionic acid, But: Butyric acid, C5: Valeric acid, C6: Caproic acid, C7: Enanthate, Lac: Lactic acid, Ala: Alanine, Glu: Glutaric acid, Suc: Succinic acid, Cit: Citric acid . * P <0.05 and ** P <0.01 compared to the LPS / ATP addition group. (-) Is an additive-free control.

  The lysine-rich sequence in the pilin domain (PD), one of the constituent domains of the inflammasome, is involved in PD binding. This lysine-rich sequence is a very rare sequence that is hardly observed in other proteins. When there is a substance that binds to this lysine-rich sequence, the present inventors specifically inhibit the binding via PD by binding the substance in advance to PD, that is, effectively form inflammasome formation. As a result of searching for substances that bind to lysine-rich sequences, it was found that various monocarboxylic acids could bind to lysine-rich sequences and specifically control inflammasome formation. It was. The present invention is based on this finding. Hereinafter, the present invention will be described in detail.

1. Screening method The present invention examines the test substance binding ability to a lysine-rich sequence in the pilin domain (PD), which is one of the constituent domains of the inflammasome, and detects the test substance bound to the lysine-rich sequence. The present invention relates to a method for screening an inflammasome activation controlling substance by selecting it as a potential candidate substance for the inflammasome activation controlling substance.

  In the present invention, the term “inflammasome” broadly means an intracellular protein complex that activates caspase-1 and thereby induces production of IL-1β, IL-18, etc. that cause an inflammatory response. In the present invention, the “inflammasome” that is the target of activation control preferably has an inflammasome binding between a receptor protein such as an NLR family protein and caspase-1 via a lysine-rich sequence of a pilin domain (PD). Examples of inflammasomes essential for some formation include NLRP1 inflammasome, NLRP3 inflammasome, NLRP4 inflammasome, NLRP6 inflammasome, AIM2 inflammasome, and the like.

  In the present invention, the “lysine rich sequence” in the pilin domain (PD) can be easily recognized by those skilled in the art. Examples of the lysine-rich sequence in the pilin domain (PD) include KKFKLK (SEQ ID NO: 7) in the PD of the adapter protein ASC constituting the inflammasome, KKFKMHI (SEQ ID NO: 8) in the PD of the NLRP3 protein, POP1 Examples include, but are not limited to, KKFKMKI (SEQ ID NO: 9), etc. Specifically, for example, KKFKLK in ASC PD contributing to the formation of NLRP3 inflammasome is 21 of SEQ ID NO: 2. It corresponds to the 26th amino acid.

  In the present invention, the test substance used for screening is preferably a monocarboxylic acid. This is based on the finding found by the present inventors that monocarboxylic acids tend to bind to lysine-rich sequences in the pilin domain, thereby strongly affecting inflammasome formation (activation). . In the present invention, monocarboxylic acid refers to a compound having one carboxyl group, and examples thereof include short-chain fatty acids or amino acids. In the present invention, the short chain fatty acid refers to a fatty acid having 8 or less carbon atoms. Examples of short chain fatty acids include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, enanthic acid, and caprylic acid. It is also preferred that the short chain fatty acid is a straight chain fatty acid. The monocarboxylic acid in the present invention is not limited, but preferably has 8 or less carbon atoms, may have 7 or less carbon atoms, and may have 2 to 7 carbon atoms, for example. The monocarboxylic acid, for example, a short-chain fatty acid or an amino acid in the present invention may preferably be one having no hydroxyl group (excluding those contained in the carboxyl group). Examples of amino acids that are monocarboxylic acids having no hydroxyl group include glycine, alanine, valine, and leucine.

  Typically, in the screening method of the present invention, the test substance is contacted with a polypeptide containing a lysine rich sequence (for example, KKFKLK) of the pilin domain, and a test substance that binds to the lysine rich sequence is selected. A screening method for an inflammasome activation regulator.

  In the present invention, the “polypeptide containing a lysine-rich sequence of a pilin domain” means a protein having a pilin domain containing a lysine-rich sequence (for example, an inflammasome-constituting protein or a pilin protein), or a partial fragment of the protein. Means containing lysine rich sequence.

  In one embodiment of the present invention, a monocarboxylic acid is contacted with a polypeptide comprising a lysine rich sequence (eg, KKFKLK) of a pilin domain derived from an inflammasome-constituting protein to bind the monocarboxylic acid that binds to the lysine rich sequence. By selecting, an inflammasome activation control substance can be screened.

  Here, the inflammasome-constituting protein refers to a receptor protein or adapter protein ASC that is a constituent protein of the inflammasome and includes a pilin domain. “Polypeptide containing a lysine-rich sequence of a pilin domain derived from an inflammasome-constituting protein” is a partial fragment of an inflammasome-constituting protein that includes a lysine-rich sequence within the pilin domain of the protein. .

  More specifically, as a preferred embodiment of the screening method of the present invention, (i) a test substance is a polypeptide containing a lysine-rich sequence of a pilin domain, or a lysine-rich sequence obtained by deleting the lysine-rich sequence therefrom. (Ii) detecting a polypeptide bound to a test substance, and (iii) a polypeptide containing a lysine-rich sequence but not containing a lysine-rich sequence. And a method of selecting a test substance as a substance that binds to the lysine-rich sequence when it is not detected. In this method, the polypeptide containing the lysine-rich sequence of the pilin domain and the polypeptide not containing the lysine-rich sequence may have substantially the same amino acid sequence except that the lysine-rich sequence has been deleted. preferable. Here, “substantially the same” means, for example, that several amino acid residues adjacent to the lysine-rich sequence are deleted together with the lysine-rich sequence in a polypeptide that does not contain the lysine-rich sequence. It means that a difference that does not affect the determination of the binding ability to the sequence may exist in addition to the deletion of the lysine rich sequence.

  Specific examples of the polypeptide containing the lysine-rich sequence of the pilin domain, and the polypeptide without the lysine-rich sequence from which the lysine-rich sequence is deleted include ASC partial fragments containing the lysine-rich sequence KKFKLK, and the lysine-rich sequence KKFKLK ASC partial fragment not containing. As a more specific example, an ASC partial fragment containing a lysine-rich sequence (typically KKFKLK) in which the N-terminal side of human ASC is deleted before the lysine-rich sequence in the pilin domain (PD), An ASC partial fragment that does not contain a lysine-rich sequence (typically KKFKLK) that is deleted from the N-terminal side of human ASC to the end of the lysine-rich sequence or its vicinity. An example schematic can be seen in FIG. 4B. Specific examples of the ASC partial fragment containing the lysine-rich sequence KKFKLK include a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, and 80% or more, preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 4. More preferred is a polypeptide having an amino acid sequence showing 95% or more sequence identity and retaining a lysine rich sequence KKFKLK. Specific examples of the ASC partial fragment not containing the lysine rich sequence KKFKLK include a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6, or 80% or more, preferably 90% or more, more preferably 95% or more of the same sequence. And a polypeptide consisting of an amino acid sequence that exhibits sexiness and does not contain the lysine-rich sequence KKFKLK. These ASC partial fragments can be brought into contact with the test substance, respectively, and a test substance that binds only to the ASC partial fragment containing a lysine-rich sequence can be selected. As a positive control, full-length ASC (FIG. 4B) is preferably tested in the same manner by contacting with a test substance. Instead of ASC partial fragments, partial fragments containing lysine-rich sequences and partial fragments not containing lysine-rich sequences from other constitutive proteins of the inflammasome containing lysine-rich sequences of the pilin domain (eg, NLRP1, NLRP4, NLRP6) Can be prepared and used for screening as well. Since the lysine-rich sequence of the pilin domain is thought to be involved in the formation of inflammasomes in these constituent proteins, screening methods using such partial fragments derived from other inflammasome constituent proteins are similarly infrastructural. It is suitable for obtaining a masome activation controlling substance.

  A polypeptide containing a lysine-rich sequence of a pilin domain, and a polypeptide without a lysine-rich sequence obtained by deleting a lysine-rich sequence from the polypeptide, for example, chemically or enzymatically cleaves a protein having a lysine-rich sequence. By doing so, it can be produced by a conventional method. Alternatively, these polypeptides can be chemically synthesized using peptide synthesis methods well known to those skilled in the art, or can be recombinantly produced or synthesized using genetic engineering techniques.

  Detection of those polypeptides bound to the test substance can be performed using any method. Such detection may include a step of recovering the polypeptide bound to the test substance. In a preferred embodiment, the test substance is preferably immobilized on a solid support in order to facilitate recovery of those polypeptides bound to the test substance. Examples of the solid support include beads (for example, microbeads such as SG beads, magnetic beads, etc.), thin films, microtubes, filters, plates, tubes, chips, and the like, but are not limited thereto. Although the test substance can be immobilized on the solid support by a conventional method, for example, it can be immobilized via any linker. Many solid supports (beads, etc.) having various surface modifications for immobilizing a substance are commercially available, and those skilled in the art can select and use an appropriate one according to the type of test substance. . FIG. 3 shows an example of immobilization of a test substance on beads. For example, a test substance may be bound to a functional group (for example, amino group) at the linker terminal of microbeads such as SG beads and FG beads according to the method described in the bead instruction manual. A schematic diagram of such a bead is shown in FIG. 4A. As an example of immobilization of a test substance using such beads, for example, when the test substance is propionic acid, propionic acid is reacted via the linker by reacting the amino group of the linker with succinic anhydride. Bound beads can be made. Further, when the test substance is butyric acid, beads having butyric acid bonded thereto via the linker can be prepared by reacting glutaric anhydride with the amino group of the linker.

  A test substance immobilized on a solid support is contacted with a polypeptide containing a lysine-rich sequence or a polypeptide not containing a lysine-rich sequence, and then bound to the test substance by using a recovery method using the solid support. The recovered polypeptide can be easily recovered. For example, when the solid support is a free bead, after contacting the bead and polypeptide, the bead is collected by filtration, washed, and then treated with an eluate to elute the polypeptide bound to the bead from the bead. The presence or absence of the binding of the polypeptide to the test substance can be determined by detecting the polypeptide eluted by electrophoresis and silver staining. For example, the polypeptide is applied to a column packed with beads (solid support) on which a test substance is immobilized, the column is washed to wash away unbound polypeptide, and then treated with an eluate to bind the bound polypeptide. By eluting from the beads and detecting the eluted protein, the presence or absence of binding of the polypeptide to the test substance can be determined. When a polypeptide containing a lysine-rich sequence is contacted with a test substance, a polypeptide bound to the test substance is detected, but when a polypeptide that does not contain a lysine-rich sequence is contacted with the same test substance, When the polypeptide bound to the test substance is not detected, the test substance can be determined as a substance that binds to the lysine-rich sequence of the pilin domain.

  The test substance that binds to the lysine-rich sequence thus selected is a promising candidate for an inflammasome activation control substance. In the screening method according to the present invention, it is further preferable to confirm the inflammasome activation controlling action of the test substance preferably in a cell. Confirmation of the intracellular inflammasome activation control action of the test substance may be performed prior to the above-described test for examining the binding ability to the lysine-rich sequence, or may be performed in parallel. It may be performed after confirming the binding ability to the rich sequence.

  Confirmation of the intracellular inflammasome activation control action of the test substance can be performed, for example, in the presence of the test substance by transferring mammalian cells (for example, human cells) to lipopolysaccharide (LPS) and adenosine triphosphate ( ATP) and detection of inflammasome formation in the cell can be performed. Mammalian cells used in this test may be any cells having inflammasome-forming ability. In the case of human cells, for example, U937 cells induced to differentiate with PMA (phorbol myristate acetate), ASC gene introduced And recombinantly expressed 293T cells, THP1 cells, primary macrophages, blood cells, and the like.

  Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) are known to induce intracellular inflammasome formation. Those skilled in the art can appropriately set the addition concentrations of LPS and ATP in a range suitable for inflammasome formation according to the type of cells used and culture conditions.

  Detection of inflammasome formation in cells can be performed by measuring caspase-1 activity in cells after stimulation of inflammasome formation with LPS and ATP in the presence of a test substance. If an increase in caspase-1 activity equal to or greater than that of stimulation with LPS and ATP in the absence of the test substance is observed, it is determined that inflammasome formation (activation) has been detected. can do. On the other hand, inhibition of inflammasome formation (activation) was detected when significant increase in caspase-1 activity was observed compared to stimulation with LPS and ATP in the absence of the test substance It can be judged. If inhibition of inflammasome formation (activation) is detected, it can be confirmed that the test substance has an inflammasome activation inhibitory action. A substance having an effect on inflammasome activation selected as such is referred to as an inflammasome activation control substance in the present invention.

  The detection of inflammasome formation in cells is performed by stimulating inflammasome formation with LPS and ATP from the cells in the presence of the test substance, and then the IL-1β and / or IL-18 concentration (secretion) in the culture medium. It can also be performed by measuring (concentration). Inflammasome formation (activation) if IL-1β and / or IL-18 concentrations are elevated, equivalent to or higher than those stimulated with LPS and ATP in the absence of the test substance Can be determined to be detected. On the other hand, if significant suppression of increase in IL-1β or IL-18 concentration is observed compared to when stimulated with LPS and ATP in the absence of the test substance, inhibition of inflammasome formation (activation) is observed. It can be determined that it has been detected. If inhibition of inflammasome formation (activation) is detected, it can be confirmed that the test substance has an inflammasome activation inhibitory action.

Alternatively, after inducing the inflammasome formation by treating cells that have been recombinantly expressed by introducing DNA encoding FLAG ^(R) -ASC with LPS and ATP in the presence of the test substance , Western blotting analysis was performed on cell lysate samples using anti-FLAG ^(R) antibody and anti-inflammasome receptor protein antibody (eg, anti-NLRP3 antibody), and FLAG ^(R) -ASC The formation (activation) of inflammasome can also be detected by confirming whether it has bound to the receptor protein. If a band of a size that appears when FLAG ^(R) -ASC is bound to an inflammasome receptor protein is observed, it can be determined that formation (activation) of the inflammasome has been detected. On the other hand, when the band of the size disappears, inhibition of inflammasome formation (activation) is detected, and it can be determined that the test substance has an inflammasome activation inhibitory action.

  Any one of these methods may be performed to detect inflammasome formation in the cell, or a combination of two or more methods may be performed. Also, any other method that can detect inflammasome formation may be used.

  In the present invention, by using the screening method as described above, an inflammasome activation controlling substance having an action of controlling inflammasome formation through binding to a lysine-rich sequence can be obtained. According to the method of the present invention, a low molecular compound inflammasome activation controlling substance can be efficiently obtained. The present invention also provides an inflammasome activation regulator obtained by the above screening method.

2. Inflammasome activation regulator and its use The inflammasome activation regulator selected using the screening method as described above binds to a lysine-rich sequence, thereby binding the pilin domain necessary for inflammasome formation. Affects the coupling through For example, an inflammasome activation regulator having an inflammasome activation inhibitory action selected using the screening method of the present invention binds to a lysine-rich sequence, thereby binding via a pilin domain (PD). Can specifically inhibit, inhibit inflammasome activation, suppress the production of inflammatory cytokines IL-1β and IL-18, resulting in inflammatory reaction and inflammasome activity induced by inflammatory cytokines Other biological reactions caused by conversion can be suppressed.

  Therefore, the present invention also provides an inflammasome activation control composition containing the inflammasome activation control substance obtained as described above. In the present invention, “inflammasome activation control” means that inflammasome activation (inflammasome formation) is controlled in vivo, in vitro or ex vivo. Typically, “inflammasome activation control” is inhibition of inflammasome activation, in which case inhibiting the production of inflammatory cytokines IL-1β, IL-18 as a result of such inhibition, and It also includes inhibiting the inflammatory reactions they induce and other biological reactions caused by inflammasome activation. As described above, the inflammasome activation control substance contained in the inflammasome activation control composition according to the present invention is preferably a monocarboxylic acid, more preferably a short-chain fatty acid or an amino acid. It is. The monocarboxylic acid that can be used as the inflammasome activation controlling substance in the present invention is not limited, but preferably has 8 or less carbon atoms, and may have 7 or less carbon atoms. It may be ~ 7. The monocarboxylic acid which is an inflammasome activation controlling substance in the present invention, for example, a short-chain fatty acid or an amino acid, may preferably not have a hydroxyl group (excluding those contained in a carboxyl group). Preferable examples of the monocarboxylic acid that is an inflammasome activation controlling substance include, but are not limited to, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and enanthic acid. Preferable examples of amino acids that are inflammasome activation regulators include alanine and the like, but are not limited thereto.

  The composition for controlling inflammasome activation according to the present invention comprises an inactive carrier (solid or liquid carrier), an excipient, a surfactant, a binder, and a solubilizing agent in addition to the inflammasome activation controlling substance. , A suspending agent, a coating agent, a coloring agent, a preservative, a buffering agent, a pH adjusting agent and the like may be included. The composition for controlling inflammasome activation according to the present invention may be used, for example, for controlling the production of inflammatory cytokines via the activation control of inflammasome. Typically, the inflammasome activation control composition according to the present invention may be used, for example, for inhibiting inflammatory cytokine production through inhibition of inflammasome activation.

  In one embodiment, the inflammasome activation control composition according to the present invention causes inflammasome activation inhibition in a subject who has been administered or ingested, preferably the inflammatory cytokines IL-1β, IL- The production of 18 can be suppressed, and the inflammatory reaction induced by them and other biological reactions caused by inflammasome activation can be suppressed. The inflammasome activation control composition according to the present invention can be administered orally or parenterally, but is preferably administered parenterally.

  The inflammasome activation control composition according to the present invention can be administered to cells or tissues, humans, domestic animals (horses, cows, sheep, goats, pigs, etc.), pet animals (dogs, cats, rabbits). Etc.), experimental (test) animals (mouse, rat, monkey, etc.), etc., can be administered to any mammal (subject). Mammals having diseases (inflammatory reaction or inflammatory diseases or other diseases) with abnormalities such as inflammasome activation or enhancement thereof, mammals having the disease tendency or predisposition, life factors, environmental factors or others Mammals having a high risk of developing such diseases due to factors such as these diseases are preferred administration subjects (subjects). Examples of inflammatory diseases or other diseases accompanied by inflammasome activation include abnormal glucose metabolism such as autoinflammatory syndrome, diabetes (particularly type 2 diabetes), abnormal lipid metabolism, chronic hepatitis, stroke, myocardial infarction, atheroma Examples include, but are not limited to, cardiovascular diseases such as atherosclerosis, gout, obesity, autoimmune diseases, inflammatory bowel diseases, cytopenias, and allergies.

  The dose or intake of the inflammasome activation control composition according to the present invention may be administered as a pharmaceutical or a food or drink, administration route, age of a subject including human, body weight, symptoms It can be set as appropriate in consideration of various factors. The inflammasome activation control composition according to the present invention may be administered once, but may be administered multiple times at intervals of several hours to several months.

  The present invention provides an inflammasome activation control method comprising administering the inflammasome activation control composition according to the present invention or the inflammasome activation control substance according to the present invention to a subject as described above. And methods for controlling the production of inflammatory cytokines (particularly IL-1β and / or IL-18). In a preferred embodiment, the methods are a method for inhibiting inflammasome activation and a method for suppressing inflammatory cytokine (especially IL-1β and / or IL-18) production. The present invention also provides inflammasome activation comprising administering the inflammasome activation control composition according to the present invention or the inflammasome activation control substance according to the present invention to a subject as described above. Also provided are methods for inhibiting accompanying inflammatory responses or other biological responses caused by inflammasome activation, and methods for ameliorating, treating or preventing inflammatory diseases or other diseases associated with inflammasome activation. The present invention also relates to a method for controlling inflammasome activation using the inflammasome activation control composition. The method for controlling the activation of inflammasome may be a method performed in vivo or a method performed in vitro. The method for controlling the activation of inflammasome may be performed on isolated cells or tissues or cultures thereof.

  The composition for controlling inflammasome activation according to the present invention can be suitably used in pharmaceuticals or foods and drinks. Therefore, this invention also provides the pharmaceutical or food-drinks containing the composition for inflammasome activation control which concerns on this invention. The pharmaceutical or the food or drink containing the inflammasome activation control composition according to the present invention contains the above inflammasome activation control substance as an active ingredient.

  The pharmaceutical comprising the inflammasome activation control composition according to the present invention is a pharmaceutically acceptable formulation adjuvant such as an inert carrier (solid or liquid carrier), excipient, surfactant, binder, disintegration. Agents, lubricants, solubilizers, suspending agents, coating agents, coloring agents, flavoring agents, preservatives, buffering agents, pH adjusting agents and the like may be further included. Specifically, water, other aqueous solvents, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, Xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and other artificial cell structures such as liposomes, etc. May be included. The formulation adjuvant can be selected appropriately or in combination depending on the dosage form of the formulation. The pharmaceutical comprising the inflammasome activation control composition according to the present invention may further contain an appropriate amount of vitamins, minerals, sugars, amino acids, peptides, etc., and further contains other active ingredients such as antibiotics. May be included.

  The pharmaceuticals containing the inflammasome activation control composition according to the present invention are solid preparations such as tablets, granules, powders, pills and capsules, liquid preparations such as liquids, suspensions and syrups, and gels. In addition, it may be formulated into any dosage form such as an aerosol. When used as a liquid preparation, the pharmaceutical product containing the inflammasome activation control composition according to the present invention is prepared, for example, in physiological saline immediately before using the inflammasome activation control composition according to the present invention. It can also be formulated as a dry product intended to be reconstituted. The amount of the inflammasome activation controlling substance in the pharmaceutical product according to the present invention varies depending on the dosage form, the formulation adjuvant to be used, the severity of the target disease, etc., and is not particularly limited.

  The food or drink containing the composition for controlling inflammasome activation according to the present invention may contain a formulation adjuvant acceptable as a food. As such formulation adjuvants, the same formulation adjuvants as those described above for the pharmaceutical products can be used. The food / beverage products containing the composition for inflammasome activation control according to the present invention may be any food / beverage products such as processed foods, side dishes, confectionery, seasonings, and beverages. The food or drink according to the present invention may be a functional food. In the present invention, “functional food” means food having a certain functionality with respect to a living body, and includes, for example, food for specified health use (including conditional tokuho [food for specified health use]) and nutritional function food. So-called functional health foods, special-purpose foods, dietary supplements, health supplements, supplement products (for example, tablets, coated tablets, dragees, capsules, liquids, etc.) and beauty foods (for example, diet foods) Includes all health foods. The functional food of the present invention also includes a health food to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied. The functional food according to the present invention may be, for example, a supplement for a human having a disease or symptom having an inflammatory response associated with inflammasome activation or prone to the inflammatory response. The functional food according to the present invention may also be a supplement for humans who have or are prone to other diseases or other symptoms associated with inflammasome activation.

  The food and drink according to the present invention may be in any food form such as solid, liquid, mixture, suspension, paste, gel, powder, granule, capsule and the like. The composition for controlling inflammasome activation or the inflammasome activation controlling substance according to the present invention may be contained in a food or drink by any appropriate method available to those skilled in the art. For example, after preparing the inflammasome activation control composition or inflammasome activation control substance according to the present invention in a liquid, gel, solid, mixture, powder or granule, You may mix | blend with, and after adding directly in the raw material of food-drinks, you may process into food-drinks. The inflammasome activation control composition or inflammasome activation control substance according to the present invention may be encapsulated, or may be encapsulated with an edible film or an edible coating agent. Moreover, after adding an appropriate excipient | filler etc. to the composition for inflammasome activation control which concerns on this invention, or an inflammasome activation control substance, you may shape | mold into arbitrary shapes, such as a tablet. The food and drink according to the present invention include, for example, various foods (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared powdered milk, liquid foods, food for the sick, nutritional foods, frozen foods. , Processed foods and other commercial foods) may be prepared by adding the inflammasome activation control composition or inflammasome activation control substance according to the present invention.

  The food and drink containing the inflammasome activation control composition according to the present invention should also contain water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Can do. Examples of the protein include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, their degradation products; butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. It may contain peptides and amino acids such as casein phosphopeptide, arginine and lysine. Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, tartaric acid, erythorbic acid, and the like. These components can be used in combination of two or more, and each may be a synthetic product or a natural product.

  The medicinal product or food or drink containing the composition for controlling inflammasome activation according to the present invention is more specifically for controlling inflammasome activation or for controlling production of inflammatory cytokines (particularly IL-1β and / or IL-18). Specifically, it may be used for suppression of an inflammatory response (particularly an inflammatory response induced by IL-1β and / or IL-18) associated with inflammasome activation.

  The present invention also provides a feed additive containing the inflammasome activation control composition or inflammasome activation control substance according to the present invention. The feed additive according to the present invention may be in any form such as solid, liquid, mixture, suspension, paste, gel, powder, granule, capsule and the like. The feed additive containing the composition for controlling inflammasome activation according to the present invention may be any adjuvant that can be used for the feed additive, such as an inert carrier (solid or liquid carrier), excipient, interface. Activators, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, coloring agents, flavoring agents, preservatives, buffering agents, pH adjusting agents and the like may further be included. The feed additive may further contain other components such as vitamins, minerals, sugars, amino acids, peptides and the like in appropriate amounts, and may further include other active ingredients such as antibiotics. The feed additive according to the present invention can be appropriately blended in the feed of animals such as domestic animals and pets (preferably mammals). By blending the feed additive according to the present invention into the feed, it becomes easy to ingest animals such as domestic animals and pets with the inflammasome activation control substance.

  The inflammasome activation control composition or inflammasome activation control substance-containing pharmaceutical or food / drink product or feed additive (subject) to be administered or ingested according to the present invention is subject to inflammasome activation. The same as described for the control composition.

  The pharmaceutical, food and drink, and feed additive containing the inflammasome activation inhibiting composition or inflammasome activation controlling substance according to the present invention may be for inflammasome activation control, for example, inflammasome It is preferably used for the control (particularly suppression) of inflammatory reactions or other biological reactions associated with some activation, or the improvement, treatment, or prevention of inflammatory diseases or other illnesses associated with inflammasome activation. it can. Control of the inflammatory response accompanying inflammasome activation is not limited, but for example, the amount of inflammatory cytokines, particularly the amount of IL-1β and IL-18 can be determined as an index.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1]
(1) Preparation of FLAG-ASC expression vector and NLRP3 expression vector
In order to recombinantly express FLAG-ASC and NLRP3 in 293T cells, an animal cell expression vector was prepared as follows.

(A) FLAG-ASC expression vector A DNA fragment containing the full length of human ASC gene (SEQ ID NO: 1; GenBank Accession No. AB023416) was PCRed using ASC cDNA cloned from human leukemia cell line U937 cells as a template and using the following primers: FLAG-ASC expression vector by inserting into the Eco RI-Bgl II site of the expression vector pCMV10-FLAG (Sigma), which can be amplified with the FLAG epitope tag and fused to the 5 'end of the cloned gene did.
Primer (forward): 5'-TTTGAATTCAATGGGGCGCGCGCGCGCGACGCC-3 '(SEQ ID NO: 10)
Primer (reverse): 5'-TTTTAGATCTTCAGCTCCGCTCCAGGTCCTC-3 '(SEQ ID NO: 11)

(B) NLRP3 expression vector
Using NLRP3 cDNA cloned from U937 cells as a template, the following primers are used to amplify a DNA fragment containing the full length of the NLRP3 gene by PCR and insert it into the Bam HI-Xho I site of animal cell expression vector pcDNA3.1 (INVITROGEN) Thus, an NLRP3 expression vector was prepared.
Primer (forward): 5'-TTTTGGATCCATGAAGATGGCAAGCACCCGC-3 '(SEQ ID NO: 12)
Primer (reverse): 5'-TTTTCTCGAGCTACCAAGAAGGCTCAAAGAG-3 '(SEQ ID NO: 13)

(2) Selection of inflammasome formation regulator using the binding of FLAG-ASC and NLRP3 as an indicator 293T cultured in the medium DMEM + 10% FCS in the 10cm dish of the FLAG-ASC expression vector and NLRP3 expression vector prepared above Cells were transfected with Lipofectamin (registered trademark) 2000 (Invitrogen) according to the attached protocol of Invitrogen. After culturing the cells for 24 hours after gene introduction, the cells were cultured for 12 hours in the presence of monocarboxylic acid (1 mM propionic acid, butyric acid, or lactic acid) as a test substance, and added LPS (100 ng / ml) / ATP ( 1 mM) was allowed to react for 1 hour to recover the cells. The collected cells were washed twice with 1 ml of PBS, suspended in 500 μl of lysis buffer (20 mM Hepes (pH 7.9), 100 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1% NP-40) and allowed to stand on ice for 10 minutes. And dissolved. The lysate was centrifuged at 20,000 × g and 4 ° C. for 5 minutes to recover the supernatant, which was reacted with 10 μl of anti-FLAG M1 antibody-agarose gel (Sigma) for 1 hour for immunoprecipitation. After the reaction, after washing three times with 500 μl of lysis buffer, add 20 μl of SDS loading buffer (0.125M Tris-HCl, 2% SDS, 10% glycerin, 0.005% BPB, 5% mercaptoethanol) to the remaining agarose gel. Elute by heating. After subjecting the sample to SDS-PAGE, Western blotting was performed using an anti-FLAG antibody (Sigma) or an anti-NLRP3 antibody (Abcam) to detect FLAG-ASC or NLRP3. As a result, in the detection with the anti-FLAG antibody, a band corresponding to FLAG-ASC recovered with the anti-FLAG antibody agarose and a band of NLRP3 which was combined with this to form an inflammasome were detected. Therefore, whether or not NLRP3 was able to form an inflammasome was determined based on the detection result of the anti-NLRP3 antibody based on the presence or absence of a band corresponding to the latter.

  FIG. 2 shows the results of detection with anti-NLRP3 antibody. The control represents a sample to which no test substance was added, and (−) represents a sample in which FLAG-ASC and NLRP3 were not introduced into the 293T cells. As shown in FIG. 2, in the control, NLRP3 recovered by the anti-FLAG antibody agarose by binding to FLAG-ASC to form an inflammasome was detected as a band. On the other hand, the binding of NLRP3 was significantly attenuated in the presence of propionic acid or butyric acid. From these results, it was shown that propionic acid and butyric acid, which are short-chain fatty acids, inhibit NLRP3 inflammasome formation, respectively.

[Example 2]
(1) Preparation of test substance-immobilized microbeads Affinity beads with propionic acid or butyric acid immobilized thereon were prepared by reacting succinic anhydride or glutaric anhydride with the linker of affinity nanobeads (FG beads). (FIG. 3, FIG. 4A). Specifically, amino group type affinity beads (amino beads; Tamagawa Seiki Co., Ltd.) were mixed with 10 mM glutaric anhydride, reacted at room temperature for 12 hours, then added with 4% acetic anhydride and reacted for 6 hours. By masking the remaining amino groups, affinity nanobeads with butyric acid immobilized thereon were prepared. Similarly, amino group type affinity beads (amino beads; Tamagawa Seiki Co., Ltd.) were mixed with 10 mM succinic anhydride, reacted at room temperature for 12 hours, then added with 4% acetic anhydride and reacted for 6 hours. By masking the group, affinity nanobeads with immobilized propionic acid were produced.

(2) Production of full-length ASC fragments and partial fragments
From the N-terminal side of ASC to the front of the lysine-rich sequence for detection of substances that bind to the amino acid sequence part (KKFKLK; lysine-rich sequence) rich in basic amino acids (lysine) of the pilin domain (PD) of ASC ASC partial fragment from which 18 amino acids were deleted (Δ18 ASC; 177 amino acids long; FIG. 4B) and ASC partial fragment from which 27 amino acids up to the end of the lysine-rich sequence were deleted (Δ27 ASC; 168 amino acids long; FIG. 4B) did.

Specifically, first, the human ASC cDNA (195 amino acid long human ASC full-length amino acid sequence or its N) was inserted into the Eco RI-Sal I site of an E. coli expression vector pGEX6P-1 (GE healthcare) for GST tag fusion protein expression. A vector in which a cDNA encoding an amino acid sequence lacking 18 or 27 amino acids at the terminal was inserted was prepared. Human ASC cDNA was prepared by PCR amplification using the following PCR primers.
Primer (forward; ASC full length): 5'-TTTGAATTCATGGGGCGCGCGCGCGACGCC-3 '(SEQ ID NO: 14)
Primer (forward; ASCΔ18): 5'-TTTGAATTCGCCGAGCTCAAGAAG-3 '(SEQ ID NO: 15)
Primer (forward; ASCΔ27): 5'-TTTGAATTCCTGCTGTCGGTGCCGCTG-3 '(SEQ ID NO: 16)
Primer (reverse): 5'-TTTTGCTAGCTCAGCTCCGCTCCAGGTCCTC-3 '(SEQ ID NO: 17)

  The prepared vector was introduced to transform Escherichia coli strain BL21 (DE), cultured in LB medium, and 1 mM IPTG (Wako Pure Chemical Industries, Ltd.) was added to induce recombinant protein expression. The cells were disrupted by sonication, the recombinant protein was concentrated using glutathione sepharose (GE healthcare), and the GST tag was cleaved with the GST fusion protein cleavage protease Precision protease (GE healthcare) and released. The replacement ASC protein was recovered.

  Among the obtained recombinant ASC proteins, the amino acid sequence of full-length ASC is shown in SEQ ID NO: 2, and the base sequence encoding it is shown in SEQ ID NO: 1. The amino acid sequence of Δ18 ASC is shown in SEQ ID NO: 4, and the base sequence encoding it is shown in SEQ ID NO: 3. Further, the amino acid sequence of Δ27 ASC is shown in SEQ ID NO: 6, and the base sequence encoding it is shown in SEQ ID NO: 5.

(3) Detection of substance binding to lysine-rich sequence of ASC pilin domain 5 μg of the recombinant ASC protein (full-length ASC or ASC partial fragment) prepared above and 0.2 μg of propionate-immobilized beads prepared above were combined with a binding buffer ( binding buffer) (20 mM Hepes, 100 mM NaCl, 10% glycerol, 0.1% NP40) was mixed in 200 μl and allowed to react for 1 hour. The beads were collected by centrifugation, washed 3 times with a binding buffer, and the protein bound to the beads was eluted with an SDS loading buffer. The eluate was run on a 12.5% SDS polyacrylamide gel, and the protein on the gel was detected by silver staining. As a result, as shown in FIG. 5, in the sample reacted with the full-length ASC and the ASC partial fragment (Δ18 ASC) containing the lysine-rich sequence of PD, binding was observed with propionate-immobilized beads. No binding was observed in the sample reacted with the ASC partial fragment (Δ27 ASC) deleted to the lysine-rich sequence. As a result, it was revealed that propionic acid binds to the lysine-rich sequence of PD of ASC. This indicates that propionic acid may inhibit inflammasome formation by binding PD to lysine-rich sequences. Furthermore, the same experiment was conducted for butyric acid, and the binding of butyric acid to lysine-rich sequences was shown.

(4) Detection of substances that bind to NLRP family proteins having a pilin domain
The lysine-rich sequence contained in the ASC pilin domain is also conserved in the pilin domain of the NLRP family proteins that make up the inflammasome. Therefore, in order to examine whether short-chain fatty acids bind to factors having a pilin domain other than ASC, recombinant proteins of NLRP1, NLRP3, NLRP4, and NLRP6 were prepared and the binding activity was verified.

First, human NLRP1, NLRP3, NLRP4 and NLRP6 cDNAs were amplified by PCR using the following primers and inserted into the Sgf I-Pme I site of the in vitro translation expression vector pF3AWG (PROMEGA). The vector which expresses was produced.
Primer (NLRP1 forward): 5'-TTTTCGATCGCCATGGCTGGCGGAGCCTGGGGC-3 '(SEQ ID NO: 18)
Primer (NLRP1 reverse): 5'-TTTTGTTTAAACTCAGCTGCTGAGTGGCAGGAG-3 '(SEQ ID NO: 19)
Primer (NLRP3 forward): 5'-TTTTCGATCGCCATGAAGATGGCAAGCACCCGC-3 '(SEQ ID NO: 20)
Primer (NLRP3 reverse): 5'-TTTTGTTTAAACCTACCAAGAAGGCTCAAAGAC-3 '(SEQ ID NO: 21)
Primer (NLRP4 forward): 5'-TTTTCGATCGCCATGGCAGCCTCTTTCTTCTCTG-3 '(SEQ ID NO: 22)
Primer (NLRP4 reverse): 5'-TTTTGTTTAAACTCAGATCTCTACCCTTGTGAT-3 '(SEQ ID NO: 23)
Primer (NLRP6 forward): 5'-TTTTCGATCGCCATGGACCAGCCAGAGGCCCCC-3 '(SEQ ID NO: 24)
Primer (NLRP6 reverse): 5'-TTTTGTTTAAACTCAGAAGGTCGAGATGAGTTC-3 '(SEQ ID NO: 25)

  3 μg of the expression plasmid prepared above was expressed in vitro using the TNT SP6 in vitro translation kit (PROMEGA), and the expressed protein was fluorescently labeled with FluoroTECT (PROMEGA). These expressed proteins were reacted with 0.2 μg of propionate-immobilized beads for 1 hour, and then purified. Proteins bound to propionic acid beads were subjected to SDS-PAGE and then detected with a fluorescence scanner (Typhoon FLA 9500). As a result, as shown in FIG. 6, binding to propionic acid was detected in all of NLRP1, NLRP3, NLRP4 and NLRP6. From these facts, it was considered that monocarboxylic acids such as propionic acid bind widely to proteins having a pilin domain such as NLRP family as well as ASC.

[Example 3]
Using the recombinant ASC full-length protein prepared in Example 2 (2), a competitive inhibition experiment was conducted to verify the binding ability of the test substance (propionic acid, butyric acid or lactic acid) to the ASC protein. Add an excess amount of propionic acid, butyric acid, or lactic acid (1 mM or 5 mM) to the recombinant ASC protein solution, incubate at room temperature for 30 minutes, add butyric acid-immobilized beads, and react for 1 hour to collect the beads. After the substance bound to the beads was eluted, the eluted substance was subjected to SDS-PAGE, and the protein was detected by silver staining.

  The results are shown in FIG. In the figure, (−) is a sample using control beads to which a test substance is not immobilized, and (+) is a sample using beads to which butyric acid is immobilized. The binding with ASC seen in butyric acid-immobilized beads disappeared with the addition of propionic acid or butyric acid, whereas the addition of lactic acid had no effect on the binding. This indicates that most of the ASC protein was bound to an excessive amount of propionic acid or butyric acid, and was hardly bound to butyric acid-immobilized microbeads, so that the ASC protein could not be recovered by the beads. This confirms that propionic acid and butyric acid surely bind to ASC protein, and that lactic acid does not bind to ASC protein.

[Example 4]
When the inflammasome is formed, caspase-1 is activated and processes pro-IL-1β and pro-IL-18 into IL-1β and IL-18, respectively. Therefore, the inflammasome formation control action by short-chain fatty acids was tested using U937 cells based on changes in caspase-1 activity. U937 cells are deposited under the catalog number JCRB9021 to the Pharmaceutical Research Institute JCRB Cell Bank (7-6-8 Saito Asagi, Ibaraki City, Osaka, Japan).

  Differentiation was induced by adding 100 nM PMA (phorbol myristate acetate) to RPMI 1630 medium (10% FCS) and culturing for 24 hours to U937 cells. After differentiation induction, the medium was replaced with a new medium, and LPS 100 ng / ml was added and cultured for 12 hours. To this, 1 mM butyric acid, propionic acid or lactic acid was added for 1 hour and cultured for 1 hour, and 5 mM ATP was added and cultured for 1 hour to activate inflammasome formation. Thereafter, the cells were washed twice with PBS and collected, and analyzed using a caspase-1 assay kit (abcam). According to the attached protocol of abcam, the caspase-1 assay detects the intensity of fluorescence emitted from YVAD-AFC, a fluorescent substrate cleaved by caspase-1, using a plate reader (Wallac ARVO SX), and activates caspase-1 This was done by measuring the amount of -1.

  The result is shown in FIG. When cultured in the absence of a test substance (butyric acid, propionic acid or lactic acid), the activity of caspase-1 was significantly increased by the addition of LPS and ATP. On the other hand, when cultured in the presence of butyric acid or propionic acid, the increase in caspase-1 activity due to the addition of LPS and ATP was significantly suppressed. This result shows that butyric acid or propionic acid inhibits the formation of inflammasome, so that inflammasome is not formed by stimulation with LPS and ATP, resulting in the release of caspase-1 (activation of caspase-1) ) Has occurred. Since lactic acid did not suppress the increase in caspase-1 activity, it was also confirmed that lactic acid does not inhibit inflammasome formation.

[Example 5]
To further investigate the inflammasome formation control effect of short-chain fatty acids, the effect on the secretion of IL-1β from U937 cells was examined. Specifically, in the same scheme as in Example 4, PMA was added to U937 cells to induce differentiation, butyric acid, propionic acid or lactic acid was added, and LPS and ATP were added to stimulate to form inflammasomes. Activated. The concentration of IL-1β released into the medium was measured using a human IL-1β ELISA kit (R & D). The procedure of this measurement was performed according to the R & D attached document, the absorbance developed by the reaction was measured with a plate reader (BioRad: Benchmark PLUS), and the IL-1β concentration was quantified.

  The result is shown in FIG. When cultured in the absence of a test substance (butyric acid, propionic acid or lactic acid), the addition of LPS and ATP markedly increased the IL-1β concentration (IL-1β secretion concentration) in the medium. On the other hand, when cultured in the presence of butyric acid or propionic acid, the increase in the secretion concentration of IL-1β due to the addition of LPS and ATP was significantly suppressed.

  From this result and the results of Example 4, butyric acid and propionic acid suppress caspase-1 activation through inhibition of inflammasome formation, thereby inhibiting pro-IL-1β processing. Indicated. It was also shown that lactic acid did not suppress the increase in IL-1β secretion concentration and did not have an action to suppress the increase in IL-1β secretion concentration.

[Example 6]
To further investigate cytokine production control based on inflammasome formation control, using human acute leukemia cell line THP1 cells, including short-chain fatty acids for IL-1β and IL-18 production induced by inflammasome stimulation The effects of various monocarboxylic acids were investigated. In the same procedure as in Example 4, PMA was added to THP1 cells at 100 nM in RPMI 1630 medium (10% FCS) and cultured for 24 hours to induce differentiation. After differentiation induction, the medium was replaced with a new medium, and LPS 100 ng / ml was added and cultured for 12 hours.

  Short-chain fatty acids (acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and enanthic acid), branched monocarboxylic acids (lactic acid and alanine), di- or tri-carboxylic acids (glutaric acid, succinic acid, And citric acid) were added for 0.5 hour or 1.0 mM and cultured for 1 hour, respectively, and 5 mM ATP was added and cultured for 1 hour to activate inflammasome formation. The produced IL-1β and IL-18 were measured using ELISA kits from R & D or MBL, respectively.

  As a result, as shown in FIGS. 10 and 11, IL-1β production (FIG. 10) and IL-18 production (FIG. 10) were observed in short chain fatty acids (acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and enanthic acid). 11) The effect of suppressing the increase was observed, but lactic acid, dicarboxylic acid, and tricarboxylic acid did not affect IL-1β and IL-18 production. Alanine tended to suppress the increase in IL-1β and IL-18 production. From these results, it was shown that monocarboxylic acids, particularly short-chain fatty acids and amino acids having no hydroxyl group (such as alanine) have an inflammasome formation control action.

  The inflammasome formation control composition of the present invention, for example, inhibits inflammasome formation, suppresses the release of caspase-1 and the activation of IL-1β and IL-18 by caspase-1, It can be used to specifically suppress an inflammatory reaction involving asome. The present invention is useful for efficiently obtaining a low-molecular-weight and highly safe inflammasome activation control substance.

SEQ ID NOs: 3 to 6: Synthetic constructs SEQ ID NOs: 10 to 25: Primers

Claims (8)

  A method for screening an inflammasome activation regulator, comprising contacting a monocarboxylic acid with a polypeptide containing a lysine-rich sequence of a pilin domain and selecting a monocarboxylic acid that binds to the lysine-rich sequence. The polypeptide is
i) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, or
ii) The method according to claim 1, which is a polypeptide comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and retaining a lysine rich sequence. A composition for inflammasome activation control comprising a monocarboxylic acid selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, and alanine . The composition for controlling inflammasome activation according to claim 3 for controlling inflammatory cytokine production. The inflammasome activation control composition according to claim 4 , wherein the inflammatory cytokines are IL-1β and IL-18. A method for controlling inflammasome activation using the inflammasome activation control composition according to any one of claims 3 to 5 . A medicinal product or food or drink for inflammasome activation control comprising the inflammasome activation control composition according to any one of claims 3 to 5 . The feed additive for inflammasome activation control containing the composition for inflammasome activation control of any one of Claims 3-5 .

Images