JP5821128B2 - Immunoregulatory composition for colonic mucosa tissue - Google Patents

Description

  The present invention relates to a polypeptide that affects the intestinal tract immune system of the colonic mucosal tissue, a method for producing the polypeptide, and an immune control composition for the colonic mucosal tissue.

  Numerous microorganisms live in the mucous membrane that covers the digestive tract and respiratory organs. Many of these microorganisms are eubacteria, and are referred to as the resident bacterial flora, distinguishing them from those that temporarily invade the living body and express pathogenicity. Although the constituent bacterial species of the resident bacterial flora vary greatly depending on the resident site, the intestinal flora is composed of the most bacterial species. The intestinal flora metabolizes the host's undigested dietary components and supplies the host with monosaccharides, oligosaccharides, amino acids, vitamins and short chain fatty acids. Furthermore, it has been found that it has many irreplaceable beneficial physiological functions for host survival as a microbial barrier against intestinal mucosal epithelial proliferation and differentiation, angiogenesis, intestinal plexus differentiation and enteropathogenic bacteria.

  As a steady-state resistance to stress invasion including microbial infection, the intestinal mucosa is stimulated by resident bacterial flora, and an immune response centering on effector T cells is constantly induced and maintained. This is called “physiological inflammation” in the mucosa. When effector T cells are activated, regulatory T cells (inducible Treg (iTreg)) function under physiological inflammation. Regulatory T cells are thought to be induced and maintained under stimulation of the intestinal flora, as in the case of effector T cells. Therefore, the constitutive abnormality of the intestinal microflora or the excessive activation of effector T cells resulting from the constitutive abnormality may cause the onset of various diseases such as inflammatory bowel disease, obesity, cancer, rheumatoid arthritis or type I diabetes. It has become clear that it is closely related to the severity (see Non-Patent Document 1 to Non-Patent Document 3).

  Furthermore, with the advancement of comprehensive analysis methods for intestinal flora genomes, the effects of the intestinal flora on the intestinal tract immune system have been gradually clarified. For example, Non-Patent Document 4 to Non-Patent Document 8 Have been described. In Non-Patent Document 4 and Non-Patent Document 5, segmented filamentous bacteria belonging to the Firmicutes gate develop immune responses related to immunoglobulin A (IgA) and development of intestinal interepithelial cell lymphocytes. It is described that it contributes to. Non-Patent Document 6 describes that the bacteria contribute to cell differentiation of helper T cell Th17 cells. In Non-Patent Document 7 and Non-Patent Document 8, an intestinal bacterium Bacteroides fragilis that produces a capsular polysaccharide called polysaccharide antigen A (PSA) has a regulatory cytokine, interleukin-10 (IL-10). It has been reported that it contributes to the differentiation of regulatory T cells produced.

Turnbaugh PJ, Ley RE, Mahowald MA, Margrini V, Mardis ER, Gordon JI., 2006, An obesity-associated gut microbiome with increased capacity for energy harvest., Nature 444, 1027-1031. Wu HJ, Ivanov II, Darce J, Hattori K, Mathis D, et al., 2010, Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells., Immunity 32, 815-827 Wen L, Ley RE, Yu P, Gordon JI, Chervonsky AV, et al., 2008, Innate immunity and intestinal microbiota in the development of Type 1 diabetes., Nature 455, 1109-1113. Talham GL, Jiang HQ, Bos NA, Cebra JJ., 1999, Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system., Infection and Immunity 67, 1992-2000 Umesaki Y, Okada Y, Matsumoto S, Imaoka A, Setoyama H., 1995, Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM 1 glycolipids on the small intestinal epithelial cells in ex- germ-free mouse., Microbiol. Immunol. 39, 555-562. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T, et al., 2009, Induction of intestinal Th 17 cells by segmented filamentous bacteria., Cell 139, 1-14 Mazmanian SK, Round JL, Kasper DL., 2008, A microbial symbiosis factor prevents intestinal inflammatory disease., Nature 453, 620-625. Round JL, Mazmanian SK., 2010, Inducible Foxp 3+ regulatory T-cell development by a commensal bacterium of the intestinal microbiota., Proc. Natl. Acad. Sci. USA. 107, 12204-12209.

  However, in the above-mentioned Non-Patent Document 4 to Non-Patent Document 8, any of the segment bacteria, probiotics or Bacteroides fragilis described therein is directly taken up by cells in the mucosal immunity. is not. In addition, any cell component or effector molecule produced by these bacteria is initiated by the interaction of any host immune system cell with any biomolecule, ensuring the development, functional differentiation or maturation of the intestinal immune response. It is not clear at all.

  In recent years, regulatory T cells related to the mucosal immune system are important as a regulator of mucosal inflammation, and it is clear that the use of regulatory T cells can prevent and treat the development of mucosal inflammatory diseases. It has become to. Taking these into account, elucidate how the mucosal immune system containing regulatory T cells interacts with the intestinal microflora, especially intestinal resident bacteria that act directly on colonic mucosa resident cells. It is extremely important to elucidate species and bacterial species-derived inducers.

  The present invention has been made in view of the above circumstances, elucidating a resident bacterial species and a bacterial species-derived inducing factor that parasitize colonic mucosa resident cells and have mucosal immune control action, and directly in the intestinal mucosa It is an object of the present invention to provide a polypeptide that regulates the above, a method for producing the polypeptide, and an immunoregulatory composition for large intestine mucosa tissue.

  In order to achieve the above-mentioned object, the present inventors conducted extensive research. As a result, colonic mucosa resident macrophages that initiate immune response activation in the intestinal mucosa have a specific environmental bacterium (especially Proteobacteria). It was found by the 16S ribosome gene method that the environmental bacteria species of the phylum are oligopolistically parasitic. Furthermore, focusing on Stenotrophomonas maltophilia among the environmental bacteria, the result of infecting macrophage cell line RAW264.7 with the standard strain Stenotrophomonas maltophilia ATCC 13636 strain The inventors have found that production of the regulatory cytokine interleukin-10 is enhanced.

Next, the present inventors orally infected a sterile mouse with the same bacterium to prepare a notobiotic mouse. In notobiauto mice, it was found that not only increased production of regulatory cytokine interleukin-10 in colonic mucosa resident macrophages but also induction of Foxp3 ⁺ regulatory T cell responses. Furthermore, Stenotrophomonas maltophilia culture supernatant-enriched fraction containing 25 kDa (kilodalton) protein also has the effect of enhancing the production of regulatory cytokine interleukin-10 in macrophage cell line RAW264.7. As a result of MALDI-TOF MS analysis, it was revealed that the 25 kDa protein is a smlt2713 gene product of unknown function. That is, it was suggested that the 25 kDa smlt2713 protein produced from Stenotrophomonas maltophilia may be a synbiotic factor of colonic mucosa resident macrophages that controls the functional differentiation and maturation of colonic mucosal immunity (see Examples). ).

  Therefore, the polypeptide according to the first aspect of the present invention is the amino acid sequence shown in SEQ ID NO: 1, or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, inserted and / or Alternatively, it has an added amino acid sequence and has an immunoregulatory effect on the mucosa of the large intestine.

Preferably, the immunoregulatory effect in the colonic mucosa tissue by the polypeptide is enhanced production of regulatory cytokine interleukin-10 and / or induction of Foxp3 ⁺ regulatory T cell response.

A method for producing a polypeptide according to the second aspect of the present invention comprises:
A culture process for culturing Stenotrophomonas maltophilia;
An extraction step of extracting the polypeptide according to the first aspect from the cell culture obtained by the culturing step;
It is characterized by including.

  The immunoregulatory composition for large intestine mucosa tissue according to the third aspect of the present invention comprises the polypeptide according to the first aspect, a pharmaceutically acceptable derivative or pharmaceutically acceptable salt of the polypeptide, or It contains stenotrophomonas maltophilia that produces the polypeptide as an active ingredient.

  According to the present invention, a resident bacterial species parasitic on colonic mucosa resident cells and having a mucosal immunomodulatory action and a bacterial species-derived inducing factor have been elucidated, and a polypeptide that directly controls the immune system in the intestinal mucosa, the polypeptide Methods for producing peptides and immunoregulatory compositions for colonic mucosal tissues are provided.

It is a figure which shows the change of the production amount of the regulatory cytokine interleukin-10 by the influence of Stenotrophomonas maltophilia which concerns on Example 2. FIG. It is a figure which shows the ratio of Foxp3 ⁺ T cell measured with the FACSCalibur flow cytometer which concerns on Example 2. FIG. It is a figure which shows the result of SDS-PAGE which concerns on Example 4. FIG. It is a figure which shows the result by the MALDI-TOF MS analysis which concerns on Example 4. FIG. It is a figure which shows the result by Mascot analysis which concerns on Example 4. FIG. It is a figure which shows the change of the production amount of regulatory cytokine interleukin-10 by the influence of 25kDa smlt2713 which concerns on Example 5. FIG.

  Hereinafter, embodiments of the present invention will be described in detail. In this specification, expressions such as “having”, “including”, or “containing” also mean “consisting of” or “consisting of”.

(Polypeptide)
Embodiment 1 of the present invention relates to a mucosal immune regulatory polypeptide having a specific amino acid sequence. Specific examples include mucosal immune regulatory polypeptides having the amino acid sequence set forth in SEQ ID NO: 1 (smlt2713 gene product in Stenotrophomonas maltophilia ATCC 13636). A mucosal immune regulatory polypeptide having an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 1, and which provides an immunoregulatory action in the colonic mucosal tissue It doesn't matter. In the present specification, the “immunoregulatory effect” specifically means mainly the production of regulatory cytokine interleukin-10 and the induction of Foxp3 ⁺ regulatory T cell response.

  The polypeptide according to the first embodiment may be produced by a method using Stenotrophomonas maltophilia described in detail in the second embodiment, but a polypeptide synthesis method, a genetic engineering method, etc. May be produced by a conventional method in the art.

  For example, in the case of a polypeptide synthesis method, a liquid phase method or a solid phase method can be exemplified. The liquid phase method is a method in which a reaction is carried out in a solution state, a product is isolated and purified from a reaction mixture, and this product is used as an intermediate for the next peptide extension reaction. On the other hand, the solid phase method is a method in which an amino acid is bound to a solid phase carrier that is insoluble in a reaction solvent, a condensation reaction is sequentially performed on the amino acid, and a peptide chain is elongated.

  Specifically, in polypeptide synthesis, first, an amino acid protected with an amino group is dehydrated and condensed with an amino acid protected with a carboxyl group to form a peptide bond. Next, after removing the amino protecting group, the next amino group-protected amino acid is sequentially extended to the free amino group one by one from the C-terminal to the N-terminal. In the dehydration condensation reaction, the carboxyl group is activated and reacted with an amino group to be bonded. Examples of activation include dicyclohexylcarbodiimide (DCC) method, active ester method, acid anhydride method or azide method, etc., selected appropriately in consideration of high reactivity, racemization and other side reactions. do it. In order to prevent side reactions during the condensation reaction, a protective group is introduced into the amino group, carboxyl group and / or side chain functional group of the amino acid. These protecting groups are preferably those which are stable under the conditions in the condensation reaction and can be removed quickly when necessary. Further, it is preferable that the amino protecting group and the carboxyl protecting group can be selectively removed from each other.

  Examples of amino-protecting groups include benzyloxycarbonyl (Bz), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and the like. it can. Examples of the protecting group for the carboxy group include groups capable of forming an alkyl ester or a benzyl ester.

  However, in the case of the solid phase method, the C-terminal carboxyl group is bonded to a carrier such as chlorotrityl resin, chloromethyl resin, oxymethyl resin or p-alkoxybenzyl alcohol resin, so the condensation reaction is not possible with a condensing agent such as carbodiimide. It is preferably carried out in the presence or with N-protected amino acid active esters or peptide active esters. After completion of the condensation reaction, the protecting group is removed. In the solid phase method, the bond between the C-terminus of the peptide and the resin is also cleaved. Thereafter, the chemically synthesized peptide is purified by, for example, ion exchange chromatography, high performance liquid chromatography (HPLC), reverse phase chromatography, affinity chromatography, Edman degradation method or gas chromatography mass spectrometry (GC-MS). It is possible to analyze.

  In the genetic engineering method, the smlt2713 gene encoding the polypeptide according to the first embodiment is used. The smlt2713 gene can be obtained by various methods of Stenotrophomonas maltophilia (including the aforementioned standard strain Stenotrophomonas maltophilia ATCC 13636 or K279a) using a conventional method by those skilled in the art. DNA can be separated and purified. Further, for example, DNA synthesis may be performed artificially using a DNA synthesis kit or the like.

  Thereafter, the synthesized smlt2713 gene and various regulatory elements (promoter, ribosome binding site, terminator, enhancer and / or various cis elements that control the expression level) for expressing the gene in the host cell are used. A recombinant vector having a gene construct for expression is constructed according to the host cell. The recombinant vector thus constructed is introduced into, for example, a predetermined host cell and cultured under predetermined conditions. Thereby, expression, production and extraction of the polypeptide according to Embodiment 1 in the host cell can be performed.

  The mucosal immune regulatory polypeptide according to the first embodiment produced by such a peptide synthesis method or a method using a genetic engineering method includes a pharmaceutically acceptable derivative of the polypeptide, etc. It can be used as an active ingredient of the immunoregulatory composition for large intestine mucosa tissue according to Form 3.

(Polypeptide production method)
Embodiment 2 of the present invention relates to a method for producing a mucosal immune regulatory polypeptide according to Embodiment 1 described above. Specifically, from a culturing step for culturing Stenotrophomonas maltophilia, and a cell culture obtained by the culturing step (preferably fractionating and concentrating the culture supernatant (see Examples)) An extraction step of extracting the mucosal immune regulatory polypeptide according to Form 1.

  The culture medium and culture method are not particularly limited as long as the Stenotrophomonas maltophilia grows well, such as stationary culture, neutralized culture with a constant pH, rotary culture, or continuous culture. For examples of culture conditions for Stenotrophomonas maltophilia, see Examples. In the culturing step, a polypeptide (smlt2713 gene) produced by culturing a large number of strains of Stenotrophomonas maltophilia species and using a conventional method for protein extraction (eg, cell disruption method etc.) in the art. Product) can be easily obtained. Moreover, you may obtain a smlt2713 gene product using the method described in detail in an Example.

(Immunoregulatory composition for colonic mucosa tissue)
Embodiment 3 of the present invention relates to a composition using the mucosal immune regulatory polypeptide according to Embodiment 1 described above. Specifically, the mucosal immune regulatory polypeptide according to Embodiment 1, the pharmaceutically acceptable derivative or pharmaceutically acceptable salt of the polypeptide, or the Stenotrophomonas that produces the polypeptide An immunoregulatory composition for colonic mucosal tissue, containing maltoophilia (Stenotrophomonas maltophilia) as an active ingredient.

In the present specification, “immunoregulatory composition of large intestine mucosal tissue” specifically refers to, for example, controlling and regulating immunity of large intestine mucosal tissue (mainly increased production of regulatory cytokine interleukin-10) And Foxp3 ⁺ regulatory T cell response can be induced).

  More specifically, in the case of foods, for example, health foods, supplements, foods for specific health, and the like based on the concept of prevention, improvement, and treatment of inflammatory bowel disease, which is an immune system disease of the large intestine tissue, can be mentioned. . In the case of pharmaceuticals, for example, liquids, tablets, granules, fine granules, powders, tablets or capsules can be mentioned, and an oral administration form is preferred.

  In the immunoregulatory composition for large intestine mucosal tissue according to Embodiment 3, in order to prepare various foods or pharmaceuticals, other pharmaceutically acceptable compositions may be combined as appropriate. The peptide and the composition may be used as a derivative or salt form. Examples of the derivatives include polypeptide derivatives such as partially substituted products or addition compounds of polypeptides. More specifically, for example, derivatives in which a carboxyl group is amidated or acylated can be exemplified. As salt forms, inorganic acid salts such as hydrochloride, nitrate or hydrobromide, or organic acid salts such as p-toluenesulfonate, metasulfonate, fumarate, succinate or lactate Etc.

  In these foods or pharmaceuticals, the content of the polypeptide according to Embodiment 1 as an active ingredient and the daily dose can be appropriately adjusted depending on the type of the composition and the like. In addition to direct use for pharmaceuticals, etc., by utilizing the mechanism of action of the synbiotic factor in the immune control of the mucosal tissue of the large intestine, it is possible to live against the synbiotic factor using functional nanomagnetic fine particles (solder beads). This suggests that it is useful for the search for target molecules in the body, the development of a mucosal immunity enhancement method using a synbiosis factor, and the research and development of a mucosal inflammatory disease prevention, amelioration and treatment method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, an Example does not limit this invention.

Example 1
In Example 1, an example relating to the identification of bacterial species that inhabit colonic mucosal tissue resident macrophages will be described.

  In order to prepare colonic mucosa resident macrophages, first, female 8-week-old BALB / c mice (Claire Japan) were bred for 4 weeks and acclimated. The acclimated mice were euthanized by carbon dioxide inhalation, and then laparotomized with sterile ophthalmic scissors in a clean bench, and the large intestine tissue was aseptically removed. Subsequently, the mesentery and adipose tissue adhering to the large intestine tissue were removed as carefully as possible by placing on a sterile Kim towel (registered trademark, Crecia) moistened with sterile RPMI 1640 medium (Nacalai). Further, a longitudinal incision was made along the mesentery adhering portion, followed by washing with cold sterilized PBS to remove as much as possible the intestinal lumen contents (mainly feces and mucus) adhering to the incised large intestine.

  Next, the aforementioned large intestine tissue was transferred into a sterile Erlenmeyer flask to which 20 ml of sterile RPMI 1640 medium containing 5 mM EDTA (pH 8.0) was added, and stirred with a stirrer at a reaction temperature of 37 ° C. for 20 minutes. Sexual mucus and epithelial layers were removed as much as possible. Further, in a sterile Erlenmeyer flask containing only 20 ml of sterile RPMI 1640 medium (Nacalai), the same stirring operation was performed for 10 minutes to remove EDTA remaining in the large intestine tissue. Thereafter, in order to increase the efficiency of the enzyme treatment, in a sterile scintillation vial to which 10 ml of sterile RPMI 1640 medium containing 1 mg / ml collagenase S-1 (Nitta gelatin) and 2% fetal bovine serum (2% FBS) was added, The aforementioned large intestine tissue was transferred and finely chopped to about 5 mm using sterile ophthalmic scissors.

  The vial was gently stirred with a stirrer at 37 ° C. Thirty minutes after the start of stirring, the supernatant in the vial was carefully aspirated with a sterile Komagome pipette and collected in a sterile 50 ml centrifuge tube (BD Falcon) as the fraction of cells that migrated from the lamina propria of the large intestine. Left to stand. Further, the above-described collagenase treatment was repeated again, and the supernatant was combined with the first sample.

The centrifuge tube containing the cell suspension collected in this manner was centrifuged at 1300 rpm for 10 minutes at 4 ° C., the supernatant was discarded, and the cell sediment remaining on the bottom of the centrifuge tube was carefully loosened. To this, 30 ml of ice-cold RPMI 1640 medium was added, and the cell sediment was evenly stirred, followed by repeated centrifugation at 1300 rpm for 10 minutes at 4 ° C. to obtain a cell washing operation. The colon mononuclear cell fraction was aseptically collected by sterilized Percoll (GE Healthcare) 75% / 40% discontinuous density gradient centrifugation (2000 rpm, 20 minutes, room temperature). The collected large intestine mononuclear cell fraction was washed twice with a cold 2% FBS-containing RPMI 1640 medium, and the number of viable cells was calculated by trypan blue method. A 100-fold diluted Fc-block antibody (purified anti-mouse CD16 / 32 monoclonal antibody (BD Bioscience)) was added to the colon mononuclear cell fraction suspended in sterile 2% FBS / PBS to a concentration of 10 ⁷ / ml. PE-labeled anti-mouse CD11b monoclonal antibody (BD Bioscience) diluted 40-fold with sterile 2% FBS / PBS containing, APC-labeled anti-mouse CD11c monoclonal antibody (BD Bioscience), and Pacific Blue-labeled anti-mouse CD45 monoclonal antibody (BD Bioscience) ) Were added to a final concentration of 1 μg and reacted aseptically for 30 minutes under ice cooling and light shielding. The reaction was carried out in a sterile polystyrene test tube (FALCON 2058). After the reaction, the cells are washed with sterilized 2% FBS / PBS, aseptically subjected to FACSAria (BD Bioscience), a mononuclear cell fraction by forward scatter / side scatter, and CD11b ^high CD11c ^medium CD45 ^high . The indicated fractions were isolated aseptically.

  Next, genomic DNA purification in colonic mucosa resident macrophage parasitic bacteria was performed.

First, the aforementioned CD11b ^high CD11c ^medium CD45 ^high cells separated aseptically (10 ⁵ cells) by FACSAria, use and assembly suction filtering device (Millipore), sterile polycarbonate membrane filter (pore size 0.2 [mu] m, Millipore) Adsorbed on top. Next, this membrane was suction-washed with about 50 ml of sterilized water. By these operations, CD11b ^high CD11c ^medium CD45 ^high cells on the membrane are almost completely crushed, and only bacteria that are parasitic in the cells are retained on the membrane. The membrane was transferred to a sterilized Eppendorf tube containing 500 μl of sterilized water and chopped as much as possible using sterilized scissors. The tube was sufficiently stirred with a vortex mixer, then immersed in liquid nitrogen and allowed to stand at room temperature, and the sample containing the shredded membrane was frozen and thawed three times.

Thereafter, an equal amount of a phenol / chloroform / isoamyl alcohol (PCI) mixture (Nippon Gene) was added, and the mixture was stirred well using a vortex mixer, and then centrifuged at 11000 × g at room temperature for 5 minutes. After centrifugation, the upper layer (aqueous layer) was collected and PCI extraction was repeated. The aqueous layer was collected again, 1/10 volume of 3M sodium acetate (Nippon Gene) and 2.5 volumes of 100% ethanol (Nacalai) were added, and the mixture was allowed to stand at −20 ° C. for 16 hours. The next day, after centrifugation at 15000 rpm and 4 ° C. for 45 minutes, the sediment was washed with 70% ethanol. The precipitate was vacuum dried and then dissolved in 20 μ1 of T ₁₀ E ₁ buffer (pH 8.0, Nippon Gene). Subsequently, in order to remove contaminants such as bacterial cell components typified by polysaccharide antigens, which are inhibitors of the subsequent PCR (Polymerase chain reaction) reaction, the bacteria are used using Wizard SV Genomic DNA Purification Kit (Promega). The genomic DNA was purified.

  Next, PCR was carried out targeting the 16S ribosomal RNA gene for elucidation of environmental bacteria parasitic on colon resident macrophages.

  In this PCR, Rudi et al. Used a universal primer designed for eubacterial 16S ribosomal RNA gene analysis (Rudi K, et al., 1997, Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8, Appl. Environ. Microbiol. 63, 2593-2599). The base sequence of the primer is shown as SEQ ID NO: 2 for forward (Bac334F) and SEQ ID NO: 3 for reverse (Bac939R). These primers were commissioned by Greiner Japan. The PCR reaction was performed according to the method described in the aforementioned Rudi et al. Great care was taken to avoid contamination of the bacterial genome via reagents and laboratory environment during the PCR reaction. The PCR product was purified using Nucleospin (Macherey-Nagel). For PCR reaction, Applied Biosystems thermal cycler GeneAmp PCR System 9700 and Takara's ExTaq Hot Start Version were used, and reaction conditions were basically 94 ° C./60° C./72° C. and 32 cycles.

  Next, cloning of the PCR product and in vitro amplification of the recombinant plasmid were performed to determine the DNA base sequence. Cloning of the PCR product obtained in the above step was performed using pGEM-T Easy Vector System manufactured by Promega. Competent cells (High efficiency competent E. coli HB101) used for transformation were obtained from Toyobo. As a result of transformation, 96 to 192 clones were randomly selected from the obtained recombinant Escherichia coli, and a recombinant plasmid was obtained using a DNA chain extension reaction by phi29 DNA polymerase (New England Biolabs) according to the method of Maruyama et al. (See Maruyama et al., 2009, Cost effective DNA sequencing and template preparation from bacterial colonies and plasmids., Journal of Bioscience and Bioengineering 107, 471-473). The amplified recombinant plasmid DNA was quantified and then subjected to a DNA chain elongation reaction for base sequencing based on Sanger's dideoxy method using ABI Prism BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems). In this sequencing reaction, two types of primers, M13M4 (forward) and M13R (reverse), were used. The base sequence of the reaction product was determined by requesting the commissioned sequence service provided by Hokkaido System Science.

  When the determined base sequence was investigated by Blast search using Ribosomal Datebase Project Virsion 10, it was found that environmental bacteria species of Proteobacteria were oligopolistic. Among these detected environmental bacterial species, the present inventors have focused on Stenotrophomonas maltophilia, which is often detected.

(Example 2)
In Example 2, preparation of Notobiauto mice, and measurement of the concentration of the regulatory cytokine interleukin-10 and control by a flow cytometer when the Notobiauto mice were inoculated with Stenotrophomonas maltophilia The analysis of sex T cells will be described in detail.

Female biweek-old Germ-Free IQI / Jic mice were used as notobiotic mice. In order to prepare Stenotrophomonas maltophilia intestinal colonization nototobioauto mouse, standard strain Stenotrophomonas maltofilia ATCC 13636 was enriched by shaking culture at 30 ° C. for 16 hours in Luria Bertani liquid medium. Germ-Free IQI / Jic mice were inoculated with 1 × 10 ⁸ CFU of the same bacterium, which was enriched by oral intragastric intubation using a sterile sonde, and 10 ⁹ CFU / ml of the bacterial solution was mixed on the floor. Things were given for 4 consecutive days.

Four weeks after the start of the experiment, the animals were euthanized by carbon dioxide inhalation, and then the large intestine tissue was aseptically removed in a safety cabinet. According to the method of Example 1 described above, colonic mucosa resident mononuclear cells were isolated from the isolated large intestine tissue. Next, a cell fraction rich in macrophages was isolated from the mononuclear cell fraction using the adhesion to the bottom of a 12-well cell culture plate (Falcon). The cell fraction (5 × 10 ⁵ cells) was cultured in 10% FBS / RPMI1640 medium containing penicillin and streptomycin for 24 hours under conditions of 37 ° C. and 5% CO ₂ , and the culture supernatant was recovered. The regulatory cytokine interleukin-10 contained therein was measured using DuoSet mouse Cytokine ELISA Kit (R & D Systems).

  FIG. 1 is a graph showing changes in the production amount of regulatory cytokine interleukin-10 due to the influence of Stenotrophomonas maltophilia according to Example 2. FIG. As shown in FIG. 1, a sterile mouse (GF-SM) inoculated with Stenotrophomonas maltophilia is more potent than the leftmost sterile mouse (GF) and the rightmost general mouse (SPF). The production amount of leukin-10 (IL-10) was greatly increased.

In addition, the APC-labeled anti-diluted 40-fold with 2% FBS / PBS containing Fc-block antibody (purified anti-mouse CD16 / 32 monoclonal antibody (BD Bioscience)) diluted 100-fold with respect to the above-mentioned colon mononuclear cell fraction. Mouse CD3 monoclonal antibody (BD Bioscience) was added to a final concentration of 1 μg and allowed to react for 30 minutes under ice cooling and light shielding. After completion of the reaction, antibody-stained cells were washed with 2% FBS / PBS. Subsequently, antibody staining for Foxp3 in cells was performed using Foxp3 Staining Buffer Set (eBioscience) and FITC-labeled anti-Foxp3 monoclonal antibody (clone FJK-16s). The percentage of Foxp3 ⁺ T cells was measured using a FACSCalibur flow cytometer (BD Bioscience).

FIG. 2 is a graph showing the ratio of Foxp3 ⁺ T cells measured by a FACSCalibur flow cytometer according to Example 2. As shown in FIG. 2, as a result of measuring the ratio of Foxp3 ⁺ T cells with a flow cytometer, it was found that the mice inoculated with S. maltophilia rather than sterile mice (sterile animals, Germfree). The ratio of Foxp3 ⁺ T cells increased.

(Example 3)
In this Example 3, an example relating to the preparation of 25 kDa protein and SDS-PAGE, which is suggested to be a synbiosis factor in Stenotrophomonas maltophilia, will be described in detail.

  First, Stenotrophomonas maltophilia culture supernatant was prepared and concentrated. The culture supernatant of Stenotrophomonas maltophilia ATCC 13636 was prepared using the synthetic medium RPMI1640. From the Luria Bertani agar medium smeared with the same strain, an independent colony was inoculated into 2 ml of RPMI 1640 medium (Nacalai) and subjected to shaking culture at 30 ° C. and 170 rpm for 48 hours. The total amount of this culture solution was added to 13 ml of RPMI 1640 medium dispensed into a 50 ml conical tube (BD Falcon), and shaking culture was further performed at 30 ° C. and 150 rpm for 70 hours with the lid loosened. After incubation, the mixture was centrifuged at 4 ° C. and 3000 rpm for 10 minutes, and the supernatant was sterilized by filtration using a 0.22 μm filter (Advantech). Fractionation, concentration, and PBS replacement of the Stenotrophomonas maltophilia culture supernatant were performed using Centriprep YM-10 (Millipore). In addition, it is possible to concentrate a fraction having a molecular weight of 10 kDa or more by using Centriprep YM-10. Furthermore, it concentrated by centrifuging sequentially at 4 degreeC and 2330 * g for 60 minutes, 15 minutes, and 8 minutes. Next, 3 ml of PBS was added, and centrifugation was sequentially performed at 4 ° C. and 2330 × g for 15 minutes and 8 minutes. Further, 3 ml of PBS was added again and centrifuged in the same manner to replace the PMI1640 medium with PBS.

The Smlt2713 homologous protein of Stenotrophomonas maltophilia culture supernatant concentrated by Centriprep YM-10 was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). MODEL AE6530 (Ato) was used for the electrophoresis layer, and e-PAGEL (Ato) and Ez Run C ⁺ (Ato) were used for the polyacrylamide gel and the electrophoresis buffer, respectively. Electrophoresis was performed at a constant current of 20 mA for 1 hour. For Coomassie Brilliant Blue (CBB) staining, Ez stain AQua (Ato) was used, staining was performed at room temperature for 1 hour, and decolorization was performed using ultrapure water.

  FIG. 3 is a diagram showing the results of SDS-PAGE according to Example 3. As shown in FIG. 3, in the results of SDS-PAGE, both Lane 1 (4 μl), 2 (6 μl) and 3 (8 μl) contained within the fractional concentration of the culture supernatant of Stenotrophomonas maltophilia. Detected a protein of about 25 kDa. That is, it means that the 25 kDa protein has a high possibility of functioning as a synbiotic factor in the environmental bacterium Stenotrophomonas maltophilia.

Example 4
In this Example 4, an example relating to MALDI-TOF MS analysis in the aforementioned 25 kDa protein will be described.

  After separation by SDS-PAGE, the Coomassie-stained target protein band was cut out, and the gel piece was cut into a 1 to 2 mm square dice and transferred to an Eppendorf tube. Next, 100 μl of decolorizing solution (50% acetonitrile and 25 mM ammonium bicarbonate) was added per tube and shaken at room temperature for 10 minutes. Subsequently, the decoloring solution was removed so as not to suck the gel piece, and this operation was repeated until the blue color was completely removed from the gel piece. Further, using a Speed Vac, vacuum was applied to dry the gel.

  After dehydration, 100 μl of reducing solution (10 mM dithiothreitol and 25 mM ammonium bicarbonate) was added per tube, shaken at 56 ° C. for 1 hour, then returned to room temperature and discarded. Add 100 μl of washing buffer (25 mM ammonium bicarbonate) per tube and shake for 10 minutes. Add 100 μl of alkylating agent (55 mM iodoacetamide and 25 mM ammonium bicarbonate) per tube and shield with aluminum foil or the like. And shaken at room temperature for 45 minutes. Thereafter, the liquid was removed and 100 μl of washing buffer (25 mM ammonium bicarbonate) was added per tube and shaken for 10 minutes.

  200 μl of dehydrated liquid (50% acetonitrile and 25 mM ammonium bicarbonate) per tube was added to the tube that had been reduced and alkylated by the above-described operation, and shaken at room temperature for 10 minutes to remove the liquid. Such an operation was repeated to dehydrate the gel. Furthermore, the gel was dried by applying a vacuum using Speed Vac. Next, 10 μg / ml trypsin solution (modified trypsin (containing 10 μg trypsin in 50 mM ammonium bicarbonate solution), Promega) and Promega) is placed on ice for 30 minutes to allow the trypsin solution to soak into the dried gel piece. Removed with pipette. In this state, the reaction was allowed to proceed overnight at 37 ° C. Thereafter, 50 μl of extraction liquid (50% acetonitrile and 5% trifluoroacetic acid) was added per tube, and the liquid was collected after shaking at room temperature for 30 minutes. Further, 25 μl of the extract was added and shaken at room temperature for 30 minutes, and then the solution was collected, and the two extracts were combined and concentrated with a Speed Vac to about 5 to 10 μl. In this way, in-gel digestion was performed.

  Next, the sample was desalted. 1/10 amount of 1% trifluoroacetic acid was added to the sample concentrated in the aforementioned in-gel digestion to make the final concentration of trifluoroacetic acid 0.1%. Next, a desalting chip (ZipTip C18, Millipore) was swollen with acetonitrile, and then replaced with a 0.1% trifluoroacetic acid solution and equilibrated. Further, the substituted and equilibrated ZipTip C18 was attached to the micropipette of P20, and the concentrated sample was slowly pipetted several times and attached to the resin. Thereafter, 0.1% trifluoroacetic acid was sucked up and discarded in a waste liquid tube, and this operation was repeated several times to wash away those that were not adsorbed on a resin such as salt. The trypsin digest was then eluted with a small amount of eluent (around 2 μl of 50% acetonitrile and 0.1% trifluoroacetic acid). Thereafter, a saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroacetic acid was used as a matrix solution at a ratio of 1: 1 with the eluted sample solution (sample stage). ), Dried and crystallized. In this way, the sample was desalted.

  Next, measurement by MALDI-TOF MS and identification of a 25 kDa protein were performed. MALDI-TOF MS was performed using a Biflex IV (Bruker) apparatus. The target plate subjected to the above-described operation was inserted into a mass spectrometer, and the sample was measured while changing the laser intensity and irradiation position to obtain a spectrum. In addition, a peak list was created using data analysis software (biotools). Furthermore, protein identification was performed using the protein identification software Mascot based on the peptide mass fingerprinting method.

  FIG. 4 is a diagram showing the results of MALDI-TOF MS analysis according to Example 4. FIG. 5 is a diagram illustrating a result of Mascot analysis according to the fourth embodiment. From the spectrum in FIG. 4 and the analysis result in the software in FIG. 5, the protein is homologous to smtl2713 having 99% homology with the amino acid sequence of the smlt2713 gene product whose function is unknown in Stenotrophomonas maltophilia strain K279a. It was found to be a protein.

(Example 5)
In this Example 5, an example relating to the immune response of the macrophage cell line RAW264.7 to Stenotrophomonas maltophilia and smlt2713 homologous protein in vitro will be described.

First, the macrophage cell line RAW264.7 purified as in Example 1 described above was prepared using 10% FBS (JIH, lot.4L0299), 2-mercaptoethanol (GIBCO), 100 μg / ml streptomycin and 100 units / ml penicillin (Meiji). Using RPMI1640 medium (Nacalai) containing confectionery), culture was performed at 37 ° C. under 5% CO ₂ conditions.

Thereafter, macrophage cells RAW264.7 were seeded at 1.5 × 10 ⁵ cells / well in a 12-well plate (BD Falcon) and pre-cultured for 18 hours. Further, the 12-well plate was washed with RPMI1640 medium (containing 10% FBS, 2-mercaptoethanol and penicillin), and the medium was changed. After adding 24 ml of smt2713 homologous protein added to a final concentration of 0.1 μM. I was stimulated.

  Next, the concentration of the regulatory cytokine interleukin-10 in the RAW264.7 cell culture supernatant was measured. Concentration was measured using R & D Systems' DuoSet mouse Cytokine ELISA Kit, followed by solid phase immobilization, antibody reaction and streptavidin-biotin reaction according to the methods recommended by R & D Systems, and TMB 1-Component Microwell Peroxidase Substrate , SureBlue (Funakoshi) and 2N sulfuric acid (Nacalai) were used for color development. The absorbance was measured using a microplate reader Model 550 (Bio-Rad), and measuring the absorbance at 450 nm and correcting the absorbance at 570 nm.

  6 is a graph showing changes in the production amount of regulatory cytokine interleukin-10 due to the influence of 25 kDa smlt2713 according to Example 5. FIG. As shown in FIG. 6, it was found that the production amount of the regulatory cytokine interleukin-10 was increased by adding the smlt2713 homologous protein (smlt2713) as compared to the case of not adding (None). That is, a method for preventing, improving, and treating various diseases such as inflammatory bowel disease, obesity, cancer, rheumatoid arthritis, or type I diabetes, which is a constitutive abnormality of the intestinal bacterial flora or an immune system abnormality caused by the constitutive abnormality This suggests that the study of Stenotrophomonas maltophilia and smlt2713 protein is extremely important for the development, and the smlt2713 protein itself may be an active pharmaceutical ingredient for inflammatory bowel disease and the like. It suggests that there is.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

  The contents of the papers and published patent gazettes specified in this specification are incorporated by reference in their entirety.

The present inventors have found that specific environmental bacteria are predominately parasitic on colonic mucosa resident macrophages that initiate immune response activation in the intestinal mucosa. Of the environmental bacteria, focusing on Stenotrophomonas maltophilia, and as a result of infection with the macrophage cell line RAW264.7, it was found that the production of regulatory cytokine interleukin-10 is enhanced. Furthermore, as a result of oral infection of the same bacteria in sterile mice, it was found that even a Foxp3 ⁺ regulatory T cell response was induced. Furthermore, it was discovered that Stenotrophomonas maltophilia culture supernatant concentrated fraction containing 25 kDa protein also has the effect of enhancing the production of regulatory cytokine interleukin-10 in macrophage cell line RAW264.7. As a result of MALDI-TOF MS analysis, it was revealed that the 25 kDa protein was a smlt2713 gene product of unknown function. That is, it was suggested that smlt2713 produced from Stenotrophomonas maltophilia may be a symbiotic factor responsible for functional differentiation and maturation of intestinal mucosal immunity.

  Therefore, according to the present invention, a resident bacterial species parasitic on colonic mucosa resident cells and having a mucosal immunomodulatory action and a bacterial species-derived inducing factor have been elucidated, and a polypeptide that directly controls the immune system in the intestinal mucosa, A method for producing the polypeptide and an immunoregulatory composition for colonic mucosal tissue are provided.

Claims (2)

It has the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids are substituted, deleted, inserted and / or added, and A polypeptide that provides an immunoregulatory action , a pharmaceutically acceptable derivative or pharmaceutically acceptable salt of the polypeptide, or Stenotrophomonas maltophilia that produces the polypeptide as an active ingredient An immunoregulatory composition for large intestine mucosal tissue, comprising 2. The immunoregulatory action in the colonic mucosa tissue by the polypeptide is enhanced production of regulatory cytokine interleukin-10 and / or induction of Foxp3 ⁺ regulatory T cell response. An immunoregulatory composition for large intestine mucosa tissue according to claim 1 .