KR101494275B1 - Expanding Bone Marrow-Derived Immune Regulatory Cells and Immune Regulatory B cells by Activating GPCR19 Pathway In vivo or In vitro - Google Patents

Abstract

The present invention provides a method for screening a therapeutic agent for GPCR19-related diseases. The present invention also provides a method for analyzing the efficacy of GPCR19 agonists. The present invention also provides a Myeloid-derived suppressor cell (MDSC) which is formed by in vivo physiological activity regulator and GPCR19 agonist treatment and has Gr-1 ⁺ CD11b ⁺ surface trait and has anti-inflammatory activity. In addition, the present invention provides immune regulatory B cells (regulatory B cells) formed by treatment with physiological activity in vivo and GPCR19 agonists or by H-MDSC treatment and having B220 ⁺ surface expression type and having anti-inflammatory activity do. The present invention also provides an anti-inflammatory pharmaceutical composition comprising H-MDSC or B _HM cells as an active ingredient. The present invention also provides an anticancer pharmaceutical composition comprising H-MDSC, 48H B _H or B _HM cells as an active ingredient. The present invention also provides an anti-inflammatory pharmaceutical composition comprising a GPCR19 agonist for the purpose of in vivo amplification of H-MDSC or 48H B _H cells. According to the present invention, administration of sodium taurodeoxycholate (HY-2191) in mice in which inflammation was induced by the administration of lipopolysaccharide (LPS) increased the number of H-MDSC cells and increased H-MDSC Has a significant sepsis treatment effect. According to the present invention, administration of sodium taurodeoxycholate (HY2191) in mice in which inflammation is induced by the administration of lipopolysaccharide (LPS) increases the number of immunoregulatory B cells (48h B _H cells) 48h B _H cells show significant sepsis treatment effect. According to the present invention, administration of H-MDSC in mice in which inflammation was induced by administration of lipopolysaccharide (LPS) increased the number of B _HM cells and increased B _HM Cells show significant sepsis treatment effect.

Description

In vivo amplification of bone marrow-derived immunoregulatory cells and immunoregulatory B lymphocytes by activation of the GPCR19 pathway {Expanding Bone Marrow-Derived Immune Regulatory Cells and Immune Regulatory B cells by Activating GPCR19 Pathway In vivo or In vitro}

The present invention relates to a method of increasing the number of myeloid-derived suppressor cells (MDSC) or immunoregulatory B cells and the anti-inflammatory phenotype of these cells by treating GPCR19 agonists in vitro or in vivo . The present invention relates to a method for screening GPCR19 agonists for the treatment of diseases (hereinafter referred to as GPCR19-related diseases) in which the activation of the signal transduction system via the GPCR19 receptor has a significant influence on the progression and development of disease. The present invention also relates to a method for analyzing the efficacy of a GPCR19 agonist as a therapeutic agent for GPCR19-related diseases.

Sepsis has been associated with systemic inflammatory response syndrome (SIRS) due to simultaneous multiple activation of complement system, blood coagulation system and innate immune cell system after microbial infection. Is a disease that causes death. Therefore, it is easy to presume that these sepsis will not be inhibited through the blocking of one or two TLR4 pathways (complement and coagulation pathway). In most patients, There is a lot of established sepsis. It is estimated that the reason why the therapeutic agent developed for the purpose of blocking one inflammatory pathway is not effective in clinical trials. Lilly's Xygris, which is the only drug worldwide marketed under the FDA's approval, also has a 28% survival rate that increases only in 6% of patients because its active component is activated protein C (APC), which regulates blood coagulation.

After ligation of the connective area of the mouse appendix and puncture of the appendix (CLP, cecal ligation and puncture), septicemia is induced, leading to the formation of CD11b ⁺ Gr-1 ⁺ bone marrow-derived immunosuppressive cells -derived suppressor cell, MDSC) is significantly increased. These cells inhibit the activation of CD4 ⁺ T cells and inhibit the IFN gamma secretion of CD8 ⁺ T cells [Delano MJ , et al., (2007) J Exp Med 204 (6): 1463-1474]. This report supports the prediction that MDSCs play an important role in the induction of immunosuppressed state in sepsis patients, which would facilitate secondary infection and increase patient mortality (Delano MJ , et al., (2007) J Exp Med 204 (6): 1463-1474]. Contrary to this expectation, however, adoptive transfer of 5 × 10 ⁶ MDSCs intravenously 1 hour before and 24 hours after induction of CLP suppresses systemic inflammation due to sepsis (MDSC) in patients with septicemia may increase the mortality rate by increasing the incidence of secondary infection by immunosuppression in patients with septicemia [Sander, LE, et al., (2010) J Exp Med 207 (7): 1453-64] There is controversy as to whether to increase the survival rate of patients or to suppress the systemic inflammatory response.

MDSC has been reported to increase in numbers not only in sepsis but also in several autoimmune disease mouse models, but there is no definitive conclusion as to whether it will aggravate or inhibit disease progression. In the case of systemic lupus erythematosus, MDSC is increased in the kidney and blood of MRL-fas ^lpr mice with symptom-active state, and ex Vivo experiments have been shown to inhibit the proliferation of CD4 ⁺ T cells [Cripps, JG and JD Gorham, (2011) Int Immunopharmacol; Iwata Y., et al., (2010) Clin Exp Nephrol 14: 411417].

B cells (B cells) have traditionally been known to be involved in the humoral immune response by producing antibodies. Recently, B10 immunoregulatory B cells, CD5 ⁺ B1a cells, CD1d ⁺ marginal zone B cells and T2-MZP (Transitional-2-marginal zone precursor) B cells with the function of regulating cellular immune response and inflammatory response (Breg) have been reported. The specific characteristics of these cells have not yet been elucidated, as immunomodulatory B cell specific surface molecules and intracellular target molecules have not been identified. However, immunoregulatory B cells are mostly derived from B2 B cells (with the exception of CD5 ⁺ B1a cells) and secrete IL-10. Among the immunoregulatory B cells ex Immunoregulatory B cells that have been shown to be immunosuppressed through vivo and in vivo experiments are B10 immunoregulatory B cells and T2-MZP immunoregulatory B cells [Mauri, C. and PA Blair, (2010) Nat Rev Rheumatol, 6 11): 636-43.

T2-MZP immunoregulatory B cells (CD19 ⁺ , CD21high, CD23 ⁺ , CD24 high, CD93 ⁺ , CD1d ⁺ , IL-10 ⁺ ) were detected in mesenteric lymph nodes of the mouse model of chronic colitis (TCRα - / - MLN, Mesenteric lymph node), and it has been reported that colitis is suppressed when the cells isolated from the spleen are injected by intravenous injection (Mizoguchi, A., et al., Immunity 16, 219 -230, (2002). T2-MZP immunoregulatory B cells were found to be increased in the spleen of the mouse model of rheumatoid arthritis (CIA, collagen induced arthritis), particularly in the spleen of symptom-improved mice, and the inhibition of arthritis was demonstrated by the quantum transfer method. IL-10 and CD1d knockout mouse experiments demonstrated that increased expression of IL-10 and CD1d plays an important role in inhibiting inflammation by inhibiting IFN-y secretion T _H 1 cell proliferation [Evans, JG et al., J Immunol 178, 7884-7878 (2007).

B10 immunoregulatory B cells (CD5 ⁺ , CD1d ⁺ , IL-10 ⁺ , B220 ⁺ ) have been reported to be produced in the spleen and lymph nodes of mouse models of inflammatory diseases and autoimmune diseases [DiLillo, DJ, T. Matsushita, and TF Tedder, (2010) Ann NY Acad Sci, 1183: 38-57.]. The inflammation-inhibiting effect of B10 immunoregulatory B cells was demonstrated by the adoptive transfer of ex vivo generated B10 immunoregulatory B cells to chronic collagen-induced arthritis (CCIA), a mouse model of rheumatoid arthritis. Quantitation of B10 immunoregulatory B cells reduced the arthritis index by more than 50% and disease severity by more than 90% [Mauri, C., et al., (2003) J Exp Med, 197 (4): 489-501).

However, despite the continued reports of anti-inflammatory effects of MDSC and immunoregulatory B cells, there has been no report of artificial amplification of these cell populations using drugs in vitro or in vivo. The present invention relates to a method of using a GPCR19 agonist to artificially amplify these MDSCs and immunoregulatory B cells in vitro or ex vivo, and also relates to screening of a GPCR19 agonist and a method for measuring the efficacy of the same, (HY2191, Sodiumtaurodeoxycholate) and derivatives thereof, which are different from those known in the art, in terms of gene expression and protein expression in vitro and ex vivo.

A number of patent documents and articles are referenced and cited throughout this specification. The disclosures of the cited patent documents and papers are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a technique for artificially amplifying MDSCs and immunoregulatory B cells with enhanced antiinflammatory activity in vitro and ex vivo. The present inventors also sought to develop a method for screening GPCR19-related diseases, screening methods thereof, and methods for analyzing the efficacy of GPCR19 agonists as GPCR19-related diseases. As a result, the present inventors have developed a technique for amplifying MDSC (Myeloid-derived suppressor cell) and immunoregulatory B cells by administering or treating GPCR19 agonists to a mammalian or bone marrow-derived cell population, and the GPCR19 agonist as an active ingredient Of the present invention has antiinflammatory activity in vivo by mediating MDSC (Myeloid-derived suppressor cell) and immunoregulatory B cells, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a method for numerically increasing a novel phenotype of MDSC or immunoregulatory B cells, wherein the GPCR19 agonist and the derivative thereof are administered in vivo to improve the inflammation-suppressing effect.

It is an object of the present invention to provide a method for increasing the number of new phenotypic MDSCs or immunoregulatory B cells whose GPCR19 agonist is treated in vitro in bone marrow cells or peripheral blood to improve inflammation inhibitory effect.

It is an object of the present invention to provide a screening method for a GPCR19-related disease therapeutic agent.

It is another object of the present invention to provide a method for analyzing the efficacy of GPCR19 agonists.

Another object of the present invention relates to MDSCs having anti-inflammatory activity.

Another object of the present invention relates to immunomodulatory B cells having anti-inflammatory activity.

Another object of the present invention relates to an anti-inflammatory pharmaceutical composition comprising MDSC or immunoregulatory B cells as an active ingredient.

A further object of the present invention is to provide a method of increasing the number of new phenotypic MDSCs or immunoregulatory B cells with improved inflammation inhibitory effect by administering in vivo or treating bone marrow cells with sodium taurodeoxycholate (HY2191, Sodiumtaurodeoxycholate) ≪ / RTI >

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention there is provided a method of in vivo screening for a GPCR19-associated disease therapeutic agent comprising the steps of:

(a) administering an in vivo physiological activity modulating substance to a mammal other than a human;

(b) administering to said mammal a GPCR19 (G-protein coupled receptor 19) agonist test substance; And

(c) In the mammal, (i) Gr-1 ⁺ CD11b ⁺ MDSC (Myeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf, Klra17, Slc40a1, Fcer2a, Hspa1a, Il8rb, Lyz2, Pi16, Dfna5h, Expresses high expression of Mtus1, 4732429D16Rik, Myadm, P2ry6, Fosb, Cd177, Sort1, Gcnt2, Chac1, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcl1 gene, or Il1a, Edn1, Cxcl10, Adora2b, Cxcl9, Serpina3f , Cxcl1, Ptx3, Ifng, Slc7a11, Batf2, Gbp5, Cfl1, Mefv, Bcl2111, Gbp2, Sphk1, Cd274, Icam1, Il10, Ahq, Gbp3, Cirbp, Gpr84, Bcl2a1c, Tgtp, Cd86, Hif1a, Smpd3b, B930041F14Rik, D330014H01Rik , Tnfaip2, Pex11a, Arrdc4, St6galnac4, Irgm1, Pde4d, Marcksl1, Tgm2, Ccl3, Il12a, Tap1, Mt1, Rbak, Rundc3b, Sdc4, Clec4e, Klhl15, Dnase113 or Mx1 (Iii) CD23 ^hi immunoregulatory B cells (CD23 ^hi Regulatory B cell or CD23 ^hi B _H cell) or (iv) Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40 , Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, C1qbp, Id3, Il4i1, Gbp1, Cd83, Mybl2, Ift140, Snx11, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7 , Hbb-bh1, Hspa1a, Srpk3, Hhex, Insig1, Cbfa2t3h, Taf15, Ddx17, Cnn2, Brwd1, Cr2, Arggap4, Pira3, Flna, Cdk5rap1, Cugbp1, Ankrd37, Cd44, Mrps7, Mapk11, Pycard, Cap1, , Cd180, Cfp, Cmah, LOC100046855, Srebf1, Ciita, Ryr1, E2f2, Dap3, Wdr9, Ccr6, Pcbd2, Lmnb1, Pira4, Blvrb or Actb genes, or 1190002H23Rik, Bhlhb8, Mist1, Fkbp11, Cacna1h, Rsph1, Rbm47, Slpi, Ly6c1, Hist1h1c, Tg, Klra7, Creb3l2, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgals1, 2010001M09Rik, Ctsw, Cd160, C Rc1, Rc1, Rc1, Rc1, Rc2, Rc1, Rc2, Rc2, Rc2, Rc2, Rc2, Rc2, Rc2, Rc2, Lc667370, Irf7, Ifit2, Slfn1, Rf1, Rf1, Rf1, Rf1, Rf1, Rf1, Rf1, Rf1, Rf1, Rf2, Rf3, Analyzing whether or not immunoregulatory B cells that express low levels of the Apoe, Lat, Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes are analyzed; When the degree of production of the cells of (i) to (iv) is increased in the mammal to which the test substance is injected as compared with the control group to which the test substance is not injected, the test substance is determined to be a GPCR19-related disease therapeutic agent.

The present inventors sought to develop a therapeutic agent for GPCR19-related diseases, a screening method thereof, and a method for analyzing the efficacy of GPCR19 agonists as a therapeutic agent for GPCR19-related diseases. As a result, the present inventors have developed MDSC (Myeloid-derived suppressor cell) and immunoregulatory B cells formed by administering GPCR19 agonist to a mammalian or bone marrow-derived cell population and found that a composition comprising the same as an active ingredient has anti- Respectively.

The present invention provides a method for screening a therapeutic agent for GPCR19-related diseases. The diseases which can be prevented or treated by the composition of the present invention are GPC19-related diseases, preferably sepsis, autoimmune diseases, inflammatory diseases, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease, ulcerative colitis, type 1 diabetes, , Psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, Castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia areata and systemic lupus erythematosus, most preferably Is sepsis.

The in vivo screening method of the GPCR19-related disease therapeutic agent of the present invention will be described in detail in each step as follows:

Step (a): administration of a physiological activity regulator in vivo

In the method of the present invention, in order to screen for a therapeutic agent for GPCR19-related diseases, in vivo physiological activity regulating substances are first administered to mammals other than humans to induce physiological responses in vivo.

The in vivo physiological activity controlling substance used in the present invention means a substance which causes a physiological reaction in vivo. For example, the in vivo physiological activity modulating substance binds to a receptor of a cell (for example, TLR (toll-like receptor)) to regulate a signal transduction reaction or various components of a general immune system such as vascular endothelial cells, , T cells and B cells) to regulate the signal transduction reaction.

Preferably, the in vivo physiological activity modulating substance interacts with various humoral factors or cells of the immune system to secrete a specific cytokine or chemokine, increase surface molecule expression of the biological cell, or form an antibody Or more specifically a molecule that is composed of a synthetic organic chemical, a protein, a lipid protein, a glycoprotein, a peptide, an RNA, a DNA or a polysaccharide, or a cell structure Or fungi) or part of a virus, but is not limited thereto.

The in vivo physiological activity controlling substance used in the present invention is a substance derived from various kinds of substances and is preferably a bacterial-derived substance, a fungal-derived substance , a virus-derived substance , a self-derived substance or an allergen, Derived material or a fungal-derived material, more preferably a bacterial-derived molecule, and most preferably a lipopolysaccharide (LPS) or a peptidoglycan.

Bacteria-derived material as the in vivo physiological activity regulatory substance used in the method of the present invention can be selected from the group of bacteria is infected with a mammal it causes a number of diseases and, preferably, Salmonella typhimurium (Salmonella typhimurium), Salmonella emban Dhaka (Salmonella mbandaka ), Salmonella entereraitidis ( Salmonella enteritidis), Streptococcus pneumoniae (Streptococcus pneumoniae , Staphylococcus aureus , Staphlylococcus epidermidis , Neisseria < RTI ID = 0.0 > meningitidis , Neisseria gonorrheae), Haemophilus influenza claim (Haemophilus influenzae , Esherichia coli , Klebsiella pneumoniae), L. monocytogenes cytokines Regensburg (Listeria monocytogenes), Vibrio Collet LES (Vibrio cholerae), Clostridium perfringens (Clostridium perfringens), Clostridium botulinum (Clostridium botulinum , Pseudomonas aerations , Borrelia burgdorferi , Shigella flexneri , Shigella boydii , Shigella dysentriae , Alloiococcus otitidis) and the group B Streptococcus, and Koshihikari (streptococci), most preferably, Salmonella typhimurium (Salmonella typhimurium), Salmonella emban Dhaka (Salmonella mbandaka) and Salmonella entera itty display (Salmonella enteritidis ), but is not limited thereto.

As the in vivo physiological activity controlling substance used in the method of the present invention, the fungal-derived substance is a substance derived from a fungus which is infected with a mammal and causes various diseases, for example, superficial mycosis in the skin and mouth of a mammal It is a substance derived from fungus that causes systemic mycosis such as intestine, brain, etc. The fungal-derived material can be selected from a variety of fungi and is preferably selected from the group consisting of Candida , Aspergillus , Cryptococcus , Mucor , Rhizopus , Absidia , Nocardia , Histoplasma , Blastomyces , Coccidioides , Trichophyton , Microsporum , Epidermophyton , ( Epidermophyton ), Sporotrix ( Sporothrix ), Black fungus ( Dematiaceous fungi , Malassezia , Pneumocystis , Penicillium , Alternaria , Cladosporium , Botrytis , aerobacidium , But are not limited to, those selected from the group consisting of Aureobasidium , Fusarium , Trichoderma , Helminthosporium , Neurospora , Wallemia and Rhodotorula , It is not limited.

As the in vivo physiological activity regulating substance used in the method of the present invention, the virus-derived substance can be selected from viruses that are infected with mammals and cause various diseases, and preferably selected from the group consisting of adenovirus, alphavirus, calicivirus, corona (For example, Hep B, hepatitis delta substance, Hep E or F virus), hepatitis A virus (for example, hepatitis B virus, hepatitis B virus or hepatitis B virus), viruses, CMV, distemper virus, Ebola virus, enterovirus, EBV, , VP1), GBV-C, a herpes virus (e.g., a herpes simplex virus protein such as Type I glycoprotein G, gpD, CP27, varicella zoster virus glycoprotein (e.g., IE62, gp1 or coat protein) Defective viruses such as HIV (e. G., Coat or protease), infectious peritonitis virus, influenza viruses (E. G., HPV capsid protein), parainfluenza virus (e. G., HPV < / RTI > (Eg, hemagglutinin / neuraminidase), paramyxovirus [eg RSV (eg F or G protein)], parvovirus, pestivirus, picornavirus [eg, poliovirus capsid polypeptide VP2 or VP3, or Hep A antigen), poxvirus (e.g., vaccinia virus polypeptides such as a coat protein), rabies virus (e.g., rabies virus glycoprotein G), reovirus, retrovirus, Human rhinovirus capsid), lubelavirus (e.g., capsid protein), and rotavirus, but are limited thereto No.

As the in vivo physiological activity controlling substance used in the method of the present invention The autogenous material may cause various signal transduction reactions in the same individual in the normal constituent of the body [e.g., a protein (cytokine, hormone, interleukin or a mixture thereof), a nucleic acid, a mixture thereof or a fragment thereof] It is a substance that causes an autoimmune disease by stimulating the reaction to form an autoantibody. Self-generated substances that cause an autoimmune response are arranged in a configuration different from that of naturally occurring self-derived substances. (The basal membrane protein, thyroid protein PM-1 or glutamate dicarboxylase (64K)), which is an autologous agent that causes an autoimmune response, preferably insulin, myelin basic protein, rh factor, acetylcholine receptor, And carboxypeptidase H, but are not limited thereto. Autoimmune diseases caused by autologous-derived substances are preferably selected from the group consisting of rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), lupus nephritis, skin lupus erythematosus (CLE), autoimmune hepatitis, Systemic sclerosis, systemic sclerosis, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome, ARDS, meningitis, encephalitis, uveitis, chronic obstructive pulmonary disease , Glomerulonephritis, pemphigus, macrophage activation syndrome, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, diabetes mellitus, type I diabetes mellitus, insulin dependent diabetes mellitus, allergic rhinitis, autoimmune reactions associated with organ transplantation, Syndrome, autoimmune thyroiditis, Hashimoto thyroiditis, allergic encephalomyelitis, Sjogren's syndrome, juvenile onset diabetes, cytokines And diseases associated with acute and delayed hypersensitivity mediated by T-lymphocytes, tuberculosis, sarcoidosis, multiple myelitis, granulomatosis, vasculitis, malignant anemia (Addison's disease), leukocyte leukemia, central nervous system CNS) inflammatory disorders, multiple organ damage syndrome, hemolytic anemia, cold anemia, Coombs positive anemia, severe muscle weakness, antigen-antibody complex mediated disease, anti-glomerular basement membrane disease, anti-phospholipid syndrome, IgA nephropathy, IgM neuralgia, IgA nephropathy, neuropathic pain syndrome, neuropathic pain syndrome, autoimmune multiple endocrine neuropathy, lupus erythematosus, muscle stiffness syndrome, Myeloproliferative disorder, autoimmune thrombocytopenia, autoimmune thrombocytopenic purpura (ITP), and autoimmune thrombocytopenia.

As an in vivo physiological activity modulating substance used in the method of the present invention, allergen may be an allergen known in the art, i.e., all naturally occurring substances known to cause IgE mediated reactions upon repeated exposure to an individual, ≪ / RTI > As a naturally occurring allergen inducing substance, preferably, a pollen allergen (for example, a pollen allergen of a tree, a weed, an herb or a grass), a tick allergen (such as a house dust mite or a storage mite) (Allergen-inducing substances derived from saliva, hair and dandruff of dogs, cats, rats and mice), fungal allergens and food allergens But are not limited to, mixtures thereof. These allergen forms include, but are not limited to, allergen extracts, purified allergens, modified allergens, recombinant allergens, recombinant allergens, allergen fragments of 30 amino acids or more, or combinations thereof.

In the method of the present invention, the in vivo physiological activity modulating substance can be administered to a mammal other than a human, and is preferably a mammal, more preferably a companion animal (e.g., a dog, a cat), a farm animal Mouse, or guinea pig), more preferably a mouse, a rat, a dog, a monkey, a goat, a sheep, a rabbit, and most preferably a mouse.

The administration of the physiological activity modulating substance in vivo to the mammal can be carried out through various methods known in the art, and it can be administered orally or parenterally, and in the case of parenteral administration, intravenous infusion, subcutaneous Intraperitoneal injection, intramuscular injection, intraperitoneal injection, intraperitoneal injection, intravenous injection, intraperitoneal injection, intravenous injection, intraperitoneal injection, intradermal injection, , And most preferably intraperitoneally.

On the other hand, the dosage of a physiological activity regulator in vivo can be determined routinely in a laboratory. For example, when lipopolysaccharide (LPS) is used as a physiological activity regulator in a mouse, Preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.1 mg / kg to 50 mg / kg, most preferably 20 mg / kg.

Step (b): GPCR19 Administration of agonist test substance

The GPCR19 agonist test substance used in the method of the present invention is a substance or medicament which interacts with the GPCR19 receptor to cause an effect, preferably has the same (or similar) action as the action of the substance, Height means the molecule. The GPCR19 agonists of the present invention act as agonists or partial agonists of the GPCR19 receptor. The term "agonist" as used herein generally refers to a compound that enhances the activity of another molecule or receptor site. In the classical definition, an agonist has the property of causing a biological action by binding to a receptor and altering its receptor status, whether an orthosteric agonist, an allosteric agonist, an inverse agonist or a co-agonist, Respectively. In conclusion, efficacy is defined as the ability of an agonist or ligand to produce a biological action. More specifically, GPCR19 agonists are receptor ligands or compounds that increase the concentration of cyclic adenosine monophosphate (cAMP) by more than 3% in cells that bind to GPCR19 and express the receptor.

According to a preferred embodiment of the present invention, the GPCR19 agonist used in the present invention comprises a compound represented by the following formula (1), a salt thereof or a hydrate thereof, more preferably sodium taurodeoxycholate (HY2191).

The dose of sodium taurodeoxycholate is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.1 mg / kg to 10 mg / kg, most preferably 0.5 to 8 mg / kg use.

Formula 1

Wherein R < ₁ > is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; R ₂ is hydrogen or? -Hydroxy; R ₃ is selected from the group consisting of hydroxy, NH (CH ₂ ) _m SO ₃ H where m is an integer 0, 1, 2, 3, 4 or 5 or NH (CH ₂ ) _n CO ₂ H , 3, 4 or 5); R ₄ is hydrogen, alkyl or halogen; R < ₅ > is hydrogen, substituted or unsubstituted alkyl or aryl; R ₆ is hydrogen or R ₅ and R ₆ are attached together with the carbon 3, 4, 5, or ring formed of 6 atoms in size and; R ₇ is hydrogen, hydroxy, substituted or unsubstituted alkyl; R ₈ is hydrogen or substituted or unsubstituted alkyl; R ₉ is hydrogen or substituted or unsubstituted alkyl; R ₁₀ is hydrogen or substituted or unsubstituted alkyl; R < ₁₁ > is hydrogen or substituted or unsubstituted alkyl.

Since the method of administering the GPCR19 agonist test substance used in the method of the present invention to a mammal other than a human subject to be administered is the same as that described above in step (a), the contents common to both of them are In order to avoid excessive complexity of the specification, the description thereof is omitted.

As used herein, the term "alkyl" includes saturated aliphatic groups such as straight chain alkyl groups (eg, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), branched chain alkyl groups cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term " cycloalkyl " In certain embodiments, straight chain or branched chain alkyl has 6 or fewer carbon atoms in the backbone [e.g., C ₁ -C ₆ (straight chain), C ₃ -C ₆ (branched chain)]. In some examples, straight chain or branched chain alkyl has 4 or fewer carbon atoms in the backbone. Also, cycloalkyl has 3 to 8 carbon atoms in the ring structure.

The term "substituted alkyl" as used herein refers to an alkyl moiety in which one or more hydrogen atoms on at least one carbon of the hydrocarbon backbone are replaced with a substituent. Such substituents include for example alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl (Alkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, dialkylamino, (Including alkylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, aryl Thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, Or it may include a heteroaromatic moiety.

As used herein, the term "aryl" includes aromatic groups, such as 5- and 6-membered "unbonded" or single-ring aromatic groups which may contain from 0 to 4 heteroatoms, "Conjugated" or multi-cyclic systems. Examples of aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine and pyrimidine And the like. The term "aryl" as used herein also includes a multicyclic aryl group such as tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxy Phenyl, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, deazapurine or indolizine. The aryl group having a hetero atom in the ring structure may also be referred to as "aryl heterocycle", "heterocycle", "heteroaryl" or "heteroaromatic". The aromatic ring may be substituted at one or more ring positions with a substituent as described above, such as halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl , Alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate (Including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, Amido, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfone Saturday and also sulfamoyl, sulfonamido, which may be substituted with nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Furthermore, the aryl group may be fused or bridged with an alicyclic or heterocyclic ring, and they are not aromatic, and thus form a multicyclic system (e.g., tetralin or methylenedioxyphenyl). Unless the number of carbons is otherwise specified, "lower alkyl" includes alkyl groups as defined above having 1 to 10, such as 1 to 6, carbon atoms in the backbone structure.

As used herein, the term " hydroxy "or" hydroxyl "includes groups having -OH or -O-.

The term "halogen" as used herein includes fluorine, bromine, chlorine or iodine.

Step (c): GPCR19 as a GPCR19 -associated disease therapeutic agent In vivo validation of agonist test substances

In the method of the present invention, when the degree of production of the cells of (i) to (iv) is increased in a mammal after administering the GPCR19 agonist test substance to a mammal other than human, the GPCR19 agonist test substance is a GPCR19- It is judged to be a therapeutic agent for the disease.

(i) Gr-1 ⁺ CD11b ⁺ Myeloid-derived suppressor cell (MDSC) cells

The results of quantum transfer of MDSC (Gr-1 ⁺ CD11b ⁺ MDSC) cells with increased expression of the surface trait markers Gr-1 ⁺ and CD11b ⁺ in sepsis-induced mice have been reported to reduce mortality in sepsis-induced mice.

The present inventors have GPCR19 agonist test substance administered were confirmed to Gr-1 ⁺ CD11b ⁺ increase in the MDSC cells of the spleen, these Gr-1 ⁺ CD11b ⁺ MDSC cells are the known Gr-1 ⁺ CD11b on sepsis induced mouse ⁺ MDSC cells with different phenotypic phenotypes, and were able to treat sepsis by quantum transfer. The present inventors have found that GPCR19 is involved in the up-signaling pathway that regulates the increase of MDSC cells. GPCR19 agonists are then treated to induce GPCR19 activation and increase Gr-1 ⁺ CD11b ⁺ MDSC cells, Suggesting that inflammation can be controlled through signal transduction pathways.

Therefore, when an increase in the number of Gr-1 ⁺ CD11b ⁺ MDSC cells is observed as compared with a control group to which no test substance is injected as a result of administration of the test substance, it is determined to be a therapeutic agent for a GPCR19-related disease. More specifically, when the number of Gr-1 ⁺ CD11b ⁺ MDSC cells is increased by 2% or more, preferably 1% or more, more preferably 0.5% or more as compared with the control group to which the test substance is not injected as a result of administration of the test substance The substance is judged to be a therapeutic agent for GPCR19-associated diseases. For example, the number of Gr-1 ⁺ CD11b ⁺ MDSC cells can be analyzed by FACS.

(ii) Analysis of the GPCR19 agonist test substance can be carried out by measuring the number of Gr-1 ⁺ CD11b ⁺ MDSC cells that highly express or under-express the specific gene described in (ii) above.

According to a preferred embodiment of the present invention, high expression or low expression of the specific gene described in (ii) above in MDSC cells is expressed 1.5 times or more and 1.5 times or less, respectively, as compared with the control group (the test substance is not treated) .

The degree of expression of the gene may be determined by a gene expression analysis method commonly used in the art such as a real-time RT-PCR method or a microarray method (see Sambrook, J. et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)).

MDSC cells expressing or under-expressing the above-mentioned genes were transformed and in vivo amplified by administration with GPCR19 agonist, and the transformed and in vivo amplified MDSC cells were administered to sepsis mice to confirm the therapeutic effect of sepsis This is shown in more detail in the first embodiment. Sodium taurodeoxycholate (HY-2191) was preferably used as the GPCR19 agonist test substance, and the MDSC cell produced by the administration of the test substance was named "H-MDSC" cell. The sepsis treatment effect of H-MDSC cells used for the determination of GPCR19-associated disease in sepsis mice was significantly higher than that of the other control groups by 80%. These results suggest that the H-MDSC cells generated as a result of the administration of the GPCR19 agonist test substance can be used as a therapeutic agent for sepsis or as a therapeutic agent for GPCR19-related diseases.

Therefore, as a result of administration of the test substance, an increase in the number of Gr-1 ⁺ CD11b ⁺ MDSC cells showing a high expression or a low expression level of the genes described in (ii) above as compared with the control group in which the test substance was not injected indicates that the GPCR19- Of the patients. More specifically, the number of MDSC cells exhibiting high expression or low expression of the genes described in (ii) above is 2% or more, preferably 1% or more, as compared with the control group to which the test substance is not injected as a result of administration of the test substance. More preferably by 0.5% or more, the test substance is judged to be a therapeutic agent for a GPCR19-related disease. For example, the number of MDSC cells can be analyzed by FACS.

Since the list of genes that are highly expressed or underexpressed in (ii) above is the same as described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification in accordance with repetitive descriptions.

(iii) CD23 ^hi Immunoregulatory B cells (CD23 ^hi Regulatory B cell)

B cells are known to be involved in the humoral immune response by producing antibodies in the immune response. Some immunoregulatory B cells are known to regulate the inflammatory response.

Therefore, the present inventors confirmed the increase of CD23 ^hi immunoregulatory B cells in the spleen by administering GPCR19 agonist test substance to sepsis-induced mice, and confirmed the effect of treating CD23 ^hi immunoregulatory B cells with sepsis. We found that CD23 ^hi We have shown that GPCR19 is involved in the signal transduction pathway that regulates the increase of immune regulatory B cells. GPCR19 agonist treatment induces GPCR19 activation and increases CD23 ^hi immunoregulatory B cells, Suggesting that it is possible to control inflammation.

Therefore, compared to the control non-injection administration of the test substance the result of the test substance CD23 ^hi When an increase in immunoregulatory B cells appears, it is determined to be a therapeutic agent for a GPCR19-associated disease. More specifically, by comparison, the administration and the control group not injected with the test substance the result of the test substance CD23 ^hi If the number of immunoregulatory B cells is increased by 2% or more, preferably 1% or more, more preferably 0.5% or more, the test substance is determined to be a therapeutic agent for a GPCR19-related disease. For example, CD23 ^hi The number of immunoregulatory B cells can be analyzed by FACS.

(iv) Analysis of the GPCR19 agonist test substance showed that the CD23 ^hi Lt; RTI ID = 0.0 > B cells. ≪ / RTI >

According to a preferred embodiment of the invention, CD23 ^hi The high expression or low expression of the specific gene described in (iv) above in the immunoregulatory B cells is lower than 1.5-fold and 1.5-fold lower, respectively, as compared with the control group (the test substance is not treated).

The degree of expression of the gene may be determined by a gene expression analysis method commonly used in the art such as a real-time RT-PCR method or a microarray method (see Sambrook, J. et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)).

Highly expressed or underexpressed CD23 ^hi Immunoregulatory B cells were transformed and in vivo amplified by the administration of GPCR19 agonists, and transfected and in vivo amplified CD23 ^hi Immunoregulatory < / RTI > B cells were administered to sepsis mice to confirm the therapeutic effect of sepsis, which is described in more detail in Example 15. < Desc / CD23 ^hi immunoregulatory B cells and in vivo amplification are preferably performed by the GPCR19 agonist test substance, preferably the H-MDSC cells or the GPCR19 agonist test substance produced in (i) or (ii) Deoxycholate (HY2191) was used. The cells produced as a result of administration of H-MDSC cells were named as "B _HM " cells, and 48 hours after administration of sodium taurodeoxycholate, cells generated in vivo were named "48h B _H " cells . The sepsis treatment effect of B _HM cells used for the determination of GPCR19-related disease in sepsis mice was significantly higher than that of the other control groups (Example 13), and the therapeutic effect of 48h B _H cells was also 80 % Survival rate (Example 14). This result was obtained by administering B _HM cells or sodium taurodeoxycholate produced by administration of H-MDSC cells produced in (i) or (ii) preferably by the GPCR19 agonist test substance, (48 _h B _H ) produced after a given time can be used as a therapeutic agent for sepsis or as a therapeutic agent for GPCR19-associated diseases.

Therefore, when the test substance shows an increase in immunoregulatory B cells showing high expression or low expression of the genes described above in (iv) as compared with the control group in which the test substance is not injected, it is judged to be a therapeutic agent for GPCR19-related diseases do. More specifically, the number of immunoregulatory B cells exhibiting high expression or low expression of the genes described in (iv) above is 2% or more, preferably 1% or more, as compared with the control group in which the test substance is not injected as a result of administration of the test substance. Or more, more preferably by 0.5% or more, the test substance is determined as a therapeutic agent for a GPCR19-related disease. For example, the number of immunoregulatory B cells can be analyzed by FACS.

Since the list of genes that are highly expressed or underexpressed in the MDSC or immunoregulatory B cells of (iv) is the same as described above, the common content between the two is that, in order to avoid the excessive complexity of the present specification, The description thereof is omitted.

As a result, in the method of the present invention, in the group (i) to (iv) in the group to which the GPCR19 agonist test substance is administered as compared with the control group not treated with the GPCR19 agonist test substance as described above in the step (c) Of the cells is increased by 2% or more, preferably 1% or more, more preferably 0.5% or more, the test substance is determined to be a therapeutic agent for a GPCR19-related disease.

According to another aspect of the present invention, the present invention provides a method for screening in vitro of a therapeutic agent for a GPCR19-associated disease comprising the steps of:

(a) treating in vivo physiological activity regulators and GPCR19 agonist test substances in a bone marrow cell population; And

(i) Gr-1 ⁺ CD11b ⁺ MDSC (Myeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf, Klra17, Slc40a1, Fcer2a, Hspa1a, Il8rb, Lyz2, Pi16, Dfna5h , Mtus1, 4732429D16Rik, Myadm, P2ry6, Fosb, Cd177, Sortl, Gcnt2, Chacl, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcl1 gene, or Il1a, Edn1, Cxcl10, Adora2b, Cxcl9, Gpp3, Cirpp, Gpr84, Bcl2a1c, Tgtp, Cd86, Hif1a, Smpdl3b, B930041F14Rq, Cpcl3, Cxc1, Ptx3, Ifng, Slc7a11, Batf2, Gbp5, Cfl1, Mefv, Bcl211, Gbp2, Sphk1, Cd274, Icam1, Il10, MDSCs that under-express D330014H01Rik, Tnfaip2, Pex11a, Arrdc4, St6galnac4, Irgm1, Pde4d, Marcksl1, Tgm2, Ccl3, Il12a, Tap1, Mt1, Rbak, Rundc3b, Sdc4, Clec4e, Klhl15, Dnase1l3 or Mx1 genes, ^hi Immunoregulatory B cells or (iv) Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40, Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, C1qbp, Id3, Il4i1, Gbp1, Cd83, Mybl2, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Brdd2, Brdd1, Brdd1, Brdd1, Cr2, Arrgap4, Pira3, Flna, Cdk5rap1, Cugbp1, Ankrd37, Cd44, Mrs7, Mapk11, Pycard, Cap1, Lta, Rbm4, Cd180, Cfp, Cmah, LOC100046855, Srebf1, Ciita, Ryr1, E2f2, Dap3, Wdr9, Ccr6, Lmnb1, Pira4, Blvrb, or Actb genes, or high expression of the Trk gene, or a mutant of Trk6, , Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgalsl, 2010001M09Rik, Ctsw, Cd160, Ccl5, Sel1l, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inp1, Ssr2 , Reln, Tubb2b, Akp2, F2r, H13, Cd8b1, I Hf-a1, Lgal3bp, Ifitm3, Cfl1, Oas2, Serpina3f, Emb2, P2ry14, Cysl2, Cys1, Cysl2, Cys1, Cysl2, Cirpp, Dhx58, Fcer1g, Ifng, AA467197, Gpr114, Trafd1, Cd3d, Igtp, Rilpl1, Apoe, Lat, Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, D14Ertd668e, Mlkl, Itk, Serpinb6b Analyzing the generation of immunoregulatory B cells that under-express the Cd69 gene; When the degree of production of cells of (i) to (iv) is increased in comparison with the control group in which the test substance is not treated as compared with the control group in the bone marrow cell population treated with the test substance, It is judged as a therapeutic agent.

Step (a): Cellular physiological activity regulator and GPCR19 Treatment of agonist test material with in vitro bone marrow cells

In the method of the present invention, in step (a), the cell physiological activity modulating substance and the GPCR19 agonist test substance are treated in a bone marrow cell population.

Since the cell-physiological activity modulating substance used in the method of the present invention is the same as the in-vivo physiologically active temperate substance and the GPCR19 agonist test substance is also the same as described above, The description thereof will be omitted.

The bone marrow cell population treated with the cell physiological activity modulating substance and the GPCR19 agonist test substance is a population of cells derived from the bone marrow of the femur and tibia and is preferably a hematopoietic or myeloid cell cells, and more preferably all cells of the hematopoietic lineage including B cells, T cells, NK cells, lymphocyte dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes or red blood cells (CMP) or common lymphoid progenitor cells (CLP), which can differentiate into myeloid derived suppressor cells (MDSC) precursor), and most preferably CD23 ^hi capable of forming a plasma expressing surface markers B cells or CD11b ⁺ Gr ⁺ surface transfected A cell capable of forming a MDSC cells expressing large.

In the method of the present invention, treating the in vivo physiological activity modulating substance to a population of bone marrow cells can be carried out in a culture medium of cultured bone marrow cells, and lipopolysaccharide (LPS), which is used as one of in vivo physiological activity regulating substances, Preferably from 0.0001 mg / ml to 10 mg / ml, more preferably from 0.0005 mg / ml to 1 mg / ml, still more preferably from 0.001 mg / ml to 0.1 mg / Can be treated with 0.001 mg / ml. In addition, treatment of GPCR19 agonist test substance to a population of bone marrow cells can be performed on cultures of cultured bone marrow cells, and is preferably administered at a concentration of sodium taurodeoxycoleide (HY2191) used as one of GPCR19 agonist test substances Is more preferably from 0.001 mg / ml to 10 mg / ml, more preferably from 0.01 mg / ml to 1 mg / ml, and most preferably from 0.05 mg / ml to 100 mg / Ml is added to the culture solution.

Step (b): GPCR19 as a GPCR19 -related disease therapeutic agent Agonist test substance determination

(I) to (iv) in the population of bone marrow cells treated with GPCR19 agonist test substance and in vivo physiological activity control substance as compared to the control group in which the test substance was not treated as a result of treatment with the GPCR19 agonist test substance Is increased by 2% or more, preferably 1% or more, more preferably 0.5% or more, the test substance is judged to be a therapeutic agent for a GPCR19-related disease.

The list of genes that are highly expressed or underexpressed in the MDSCs and immunoregulatory B cells of (i) to (iv) produced by in vivo physiological activity regulators and the GPCR19 agonist test substance treatment and the method of treatment of the test substances are described in detail Since the content is the same as the one described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification in accordance with the repetitive description.

According to another aspect of the invention, the present invention provides a method for in vivo potency assay of a GPCR19 agonist comprising the steps of:

(a) administering an in vivo physiological activity modulating substance to a mammal other than a human;

(b) administering to said mammal a GPCR19 agonist; And

(c) In the mammal, (i) Gr-1 ⁺ CD11b ⁺ MDSC (Myeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf, Klra17, Slc40a1, Fcer2a, Hspa1a, Il8rb, Lyz2, Pi16, Dfna5h, Expresses high expression of Mtus1, 4732429D16Rik, Myadm, P2ry6, Fosb, Cd177, Sort1, Gcnt2, Chac1, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcl1 gene, or Il1a, Edn1, Cxcl10, Adora2b, Cxcl9, Serpina3f , Cxcl1, Ptx3, Ifng, Slc7a11, Batf2, Gbp5, Cfl1, Mefv, Bcl2111, Gbp2, Sphk1, Cd274, Icam1, Il10, Ahq, Gbp3, Cirbp, Gpr84, Bcl2a1c, Tgtp, Cd86, Hif1a, Smpd3b, B930041F14Rik, D330014H01Rik , Tnfaip2, Pex11a, Arrdc4, St6galnac4 , Irgm1, Pde4d, Marcksl1, Tgm2, Ccl3, Il12a, Tap1, Mt1, Rbak, Rundc3b, Sdc4, Clec4e, Klhl15, MDSC to low expressing Dnase1l3 or Mx1 gene, (iii) CD23 ^hi Immunoregulatory B cells or (iv) Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40, Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, C1qbp, Id3, Il4i1, Gbp1, Cd83, Mybl2, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Brdd2, Brdd1, Brdd1, Brdd1, Cr2, Arrgap4, Pira3, Flna, Cdk5rap1, Cugbp1, Ankrd37, Cd44, Mrs7, Mapk11, Pycard, Cap1, Lta, Rbm4, Cd180, Cfp, Cmah, LOC100046855, Srebf1, Ciita, Ryr1, E2f2, Dap3, Wdr9, Ccr6, Lmnb1, Pira4, Blvrb, or Actb genes, or high expression of the Trk gene, or a mutant of Trk6, , Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgalsl, 2010001M09Rik, Ctsw, Cd160, Ccl5, Sel1l, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inp1, Ssr2 , Reln, Tubb2b, Akp2, F2r, H13, Cd8b1, I Hf-a1, Lgal3bp, Ifitm3, Cfl1, Oas2, Serpina3f, Emb2, P2ry14, Cysl2, Cys1, Cysl2, Cys1, Cysl2, Cirpp, Dhx58, Fcer1g, Ifng, AA467197, Gpr114, Trafd1, Cd3d, Igtp, Rilpl1, Apoe, Lat, Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, D14Ertd668e, Mlkl, Itk, Serpinb6b Analyzing the generation of immunoregulatory B cells that under-express the Cd69 gene; When the degree of production of the cells of (i) to (iv) is increased as compared with the control group in the mammal injected with the GPCR19 agonist as compared to the control group not injected with the GPCR19 agonist, the GPCR19 agonist is effective .

The method for analyzing the in vivo efficacy of the GPCR19 agonist used in the method of the present invention will be described in detail in each step as follows:

Step (a): administration of a physiological activity regulator in vivo

In the method of the present invention, in order to analyze the efficacy of GPCR19 agonists, in vivo physiological activity regulators are first administered to mammals other than humans to induce physiological responses in vivo. Since the in vivo physiological activity regulator used in step (a), the method of administration thereof, and the mammal are the same as those described above, the common content between the two is to avoid excessive complexity of the present specification , The description thereof is omitted.

Step (b): GPCR19 Agonist  administration

In the method of the present invention, the GPCR19 agonist is administered to the mammal to which the in vivo physiological activity regulator has been administered by the method of step (b). Since the GPCR19 agonist, the method of administering it and the mammal in step (b) are the same as described above, the contents common to both are omitted in order to avoid the excessive complexity of the present specification in accordance with the repetitive description do.

Step (c): GPCR19 Agonist In vivo  Efficacy determination

As a result of the administration of the GPCR19 agonist, the degree of cell formation of the above (i) to (iv) was 2% or more in the mammal to which the in vivo physiological activity regulator and the GPCR19 agonist were administered compared with the control group in which the GPCR19 agonist was not administered , Preferably 1% or more, more preferably 0.5% or more, the GPCR19 agonist is judged to be effective.

A list of genes that are highly expressed or underexpressed in the MDSC and immunoregulatory B cells of the above (i) to (iv) produced by in vivo physiological activity regulators and administration of GPCR19 agonists, and in vivo physiological activity regulators and GPCR19 agonists Are the same as those described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification in accordance with the repetitive description.

According to another aspect of the present invention, the present invention provides a method for in vitro potency assay of a GPCR19 agonist comprising the steps of:

(a) treating cell physiological activity regulators and GPCR19 agonists in a bone marrow cell population; And

(i) Gr-1 ⁺ CD11b ⁺ MDSC (Myeloid-derived suppressor cell), (ii) Prok2, Osm, Ltf, Klra17, Slc40a1, Fcer2a, Hspa1a, Il8rb, Lyz2, Pi16, Dfna5h , Mtus1, 4732429D16Rik, Myadm, P2ry6, Fosb, Cd177, Sortl, Gcnt2, Chacl, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcl1 gene, or Il1a, Edn1, Cxcl10, Adora2b, Cxcl9, Gpp3, Cirpp, Gpr84, Bcl2a1c, Tgtp, Cd86, Hif1a, Smpdl3b, B930041F14Rq, Cpcl3, Cxc1, Ptx3, Ifng, Slc7a11, Batf2, Gbp5, Cfl1, Mefv, Bcl211, Gbp2, Sphk1, Cd274, Icam1, Il10, MDSCs that under-express D330014H01Rik, Tnfaip2, Pex11a, Arrdc4, St6galnac4, Irgm1, Pde4d, Marcksl1, Tgm2, Ccl3, Il12a, Tap1, Mt1, Rbak, Rundc3b, Sdc4, Clec4e, Klhl15, Dnase1l3 or Mx1 genes, ^hi Immunoregulatory B cells or (iv) Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40, Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, C1qbp, Id3, Il4i1, Gbp1, Cd83, Mybl2, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Cr2, Brdd1, Brdd2, Brdd1, Brdd1, Brdd1, Cr2, Arrgap4, Pira3, Flna, Cdk5rap1, Cugbp1, Ankrd37, Cd44, Mrs7, Mapk11, Pycard, Cap1, Lta, Rbm4, Cd180, Cfp, Cmah, LOC100046855, Srebf1, Ciita, Ryr1, E2f2, Dap3, Wdr9, Ccr6, Lmnb1, Pira4, Blvrb, or Actb genes, or high expression of the Trk gene, or a mutant of Trk6, , Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgalsl, 2010001M09Rik, Ctsw, Cd160, Ccl5, Sel1l, Dusp4, Socs2, Ccl4, Cd9, Mcoln2, Trp53inp1, Ssr2 , Reln, Tubb2b, Akp2, F2r, H13, Cd8b1, I Hf-a1, Lgal3bp, Ifitm3, Cfl1, Oas2, Serpina3f, Emb2, P2ry14, Cysl2, Cys1, Cysl2, Cys1, Cysl2, Cirpp, Dhx58, Fcer1g, Ifng, AA467197, Gpr114, Trafd1, Cd3d, Igtp, Rilpl1, Apoe, Lat, Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, D14Ertd668e, Mlkl, Itk, Serpinb6b Analyzing the generation of immunoregulatory B cells that under-express the Cd69 gene; When the degree of production of the cells of (i) to (iv) was increased in the population of bone marrow cells treated with the GPCR19 agonist as compared with the control group not treated with the GPCR19 agonist as compared with the control group, the GPCR19 agonist had an efficacy .

Step (a): Cellular physiological activity regulator and GPCR19 Treatment of agonists

In the method of the present invention, in order to analyze the efficacy of the GPCR19 agonist, a physiological activity regulator is firstly administered to a bone marrow cell population to induce physiological changes of bone marrow cells. Since the in vivo physiological activity regulator and the GPCR19 agonist used in step (a), the method of treating the same, and the population of bone marrow cells are the same as those described above, the contents common to both are the same as those of the present specification In order to avoid excessive complexity, the description is omitted.

Step (b): GPCR19 Agonist In vitro potency determination

As a result of treatment with the GPCR19 agonist test substance, the degree of production of the cells (i) to (iv) in the bone marrow cell population treated with the cell physiological activity regulator and the GPCR19 agonist test substance as compared with the control group without the test substance An increase of 2% or more, preferably 1% or more, more preferably 0.5% or more, judges that the GPCR19 agonist is effective. The test substance is judged to be a therapeutic agent for GPCR19-related diseases.

The list of genes that are highly expressed or underexpressed in the MDSC and immunoregulatory B cells of the above (i) to (iv) and the method of treatment of the test substance produced by the treatment of the cell physiological activity modulating substance and the GPCR19 agonist test substance are as described above Since the contents are the same, the description common to both is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

According to a preferred embodiment of the present invention, the mammal is a mouse, a rat, a dog, a monkey, a goat, a sheep, or a rabbit. The mammal that can be used in the method of the present invention can be administered to a mammal other than a human, preferably a mammal, more preferably a companion animal (e.g., a dog, a cat or a bird), a farm animal Mouse, guinea pig or bird), more preferably a mouse, rat, dog, monkey, goat, sheep, rabbit (for example, And most preferably a mouse.

According to a preferred embodiment of the present invention, the in vivo physiological activity regulating substance or the cell physiological activity regulating substance used in the method of the present invention may be a bacterium-derived fungal-derived substance, a viral-derived substance, an autogenous substance or an allergen . ≪ / RTI > According to a preferred embodiment of the present invention, the in vivo physiological activity regulating substance or the cell physiological activity regulating substance is a lipopolysaccharide (LPS) or a peptidoglycan as a bacteria-derived substance.

According to a preferred embodiment of the present invention, the in vivo physiological activity modulating substance or the cell physiological activity modulating substance used in the method of the present invention acts on receptors of cells other than GPCR19 to promote the inflammatory action of target cells . Since these are the same as those described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

According to a preferred embodiment of the present invention, the GPCR19 agonist used in the method of the present invention is a compound of the formula 1, a salt or a hydrate thereof:

Wherein R < ₁ > is hydrogen, hydroxy, substituted or unsubstituted alkyl or halogen; R ₂ is hydrogen or? -Hydroxy; R ₃ is selected from the group consisting of hydroxy, NH (CH ₂ ) _m SO ₃ H where m is an integer 0, 1, 2, 3, 4 or 5 or NH (CH ₂ ) _n CO ₂ H , 3, 4 or 5); R ₄ is hydrogen, alkyl or halogen; R < ₅ > is hydrogen, substituted or unsubstituted alkyl or aryl; R ₆ is hydrogen or R ₅ and R ₆ are attached together with the carbon 3, 4, 5, or ring formed of 6 atoms in size and; R ₇ is hydrogen, hydroxy, substituted or unsubstituted alkyl; R ₈ is hydrogen or substituted or unsubstituted alkyl; R ₉ is hydrogen or substituted or unsubstituted alkyl; R ₁₀ is hydrogen or substituted or unsubstituted alkyl; R < ₁₁ > is hydrogen or substituted or unsubstituted alkyl.

Since the compound of formula (1) is the same as described above, the content common to both is used in accordance with the method of the present invention in accordance with the preferred embodiment of the present invention, in order to avoid the excessive complexity of the present specification, The genes which are highly expressed or underexpressed in the GPCR19 agonist test substance, the in vivo physiological activity regulator, the cell physiological activity regulator and the MDSC produced by administration or treatment of the GPCR19 agonist used in the present invention are the same as those described above Therefore, in order to avoid the excessive complexity of the present specification according to the repetitive description, the description common to the two is omitted.

According to a preferred embodiment of the present invention, the GPCR19 agonist test substance used in the method of the present invention, the in vivo physiological activity regulator, the cell physiological activity regulator and the immunoregulatory B cell produced by administration or treatment of the GPCR19 agonist Since the genes that are highly expressed or underexpressed in the present invention are the same as those described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

According to another aspect of the present invention, there is provided an in vivo physiological activity modulating substance or a cell physiological activity modulating substance, and MDSC which is formed by GPCR19 agonist treatment and has Gr-1 ⁺ CD11b ⁺ surface trait and has anti- (Myeloid-derived suppressor cell).

The in vivo physiological activity modulating substance or the cell physiological activity modulating substance used in the present invention is the same as the above-mentioned method. The physiological activity modulating substance or the cell physiological activity modulating substance such as lipopolysaccharide (LPS) Is preferably 0.001 mg / kg to 100 mg / kg, more preferably 0.1 mg / kg to 50 mg / kg, and most preferably 20 mg / kg, when administered to mammals other than humans Lt; / RTI >

When the lipopolysaccharide (LPS) is treated in a bone marrow cell population, the concentration is preferably 0.0001 mg / ml to 10 mg / ml, more preferably 0.0005 mg / ml to 1 mg / ml, Preferably 0.001 mg / ml to 0.1 mg / ml, and most preferably 0.001 mg / ml.

On the other hand, when the GPCR19 agonist or the test substance thereof, for example, sodium taurodeoxycholate is administered to a mammal other than a human, it is preferably 0.01 mg / kg to 100 mg / kg, more preferably 0.1 mg / kg to 10 mg / kg, and most preferably 0.5 to 8 mg / kg. When the sodium taurodeoxycholate is treated in a population of bone marrow cells, it is preferably 0.0001 mg / ml to 100 mg / ml, more preferably 0.001 mg / ml to 10 mg / ml, 0.01 mg / ml to 1 mg / ml, and most preferably 0.05 mg / ml.

The time to treat lipopolysaccharide (LPS), for example, as an in vivo physiological activity regulator is preferably 6 hours to 48 hours, most preferably 18 hours. On the other hand, the time for treating the sodium taurodeoxycholate as the GPCR19 agonist is preferably 6 hours to 96 hours, more preferably 12 hours or 72 hours, and most preferably 48 hours.

After treatment of in vivo physiological activity and GPCR19 agonists, MDSC (Myeloid-derived suppressor cell) differentiates into cells that express the Gr-1 ⁺ CD11b ⁺ surface type. Differentiated MDSC cells express macrophage lineage cell marker CD11b and marker Gr1 for granulocytes. Gr1 ⁺ CD11b ⁺ induces an immune response using an antibody against Gr1 ⁺ or CD11b ⁺ and can be screened using a flow cytometer. Administration of MDSC cells with Gr-1 ⁺ CD11b ⁺ surface traits to sepsis mice showed significantly higher survival rates as compared to the control group, suggesting that the MDSC cells exhibit anti-inflammatory activity.

According to a preferred embodiment of the present invention, the MDSC having the Gr1 ⁺ CD11b ⁺ surface expression type is selected from the group consisting of Prok2, Osm, Ltf, Klra17, Slc40a1, Fcer2a, Hspa1a, Il8rb, Lyz2, Pi16, Dfna5h, Mtus1, 4732429D16Rik, Myadm, P2ry6, Expression of Fosb, Cd177, Sort1, Gcnt2, Chacl, Reck, Lyzs, Plekhg3, Csf3r, Aatk, Dgkg, Tacstd2 or Xcl1 gene or Il1a, Edn1, Cxcl10, Adora2b, Cxcl9, Serpina3f, Cxcl1, Ptx3, Ifng, Slc7a11 , Batf2, Gbp5, Cfl1, Mefv, Bcl2111, Gbp2, Sphk1, Cd274, Icam1, Il10, Ahnak, Gbp3, Cirbp, Gpr84, Bcl2a1c, Tgtp, Cd86, Hif1a, Smpd3b, B930041F14Rik, D330014H01Rik, Tnfaip2, Pex11a, Arrdc4, St6galnac4 , Irgm1, Pde4d, Marcksl1, Tgm2, Ccl3, Il12a, Tap1, Mt1, Rbak, Rundc3b, Sdc4, Clec4e, Klhl15, Dnase1l3 or Mx1 genes.

According to a preferred embodiment of the present invention, the in vivo physiological activity controlling substance used in the present invention is characterized by being a bacterial-derived substance, a fungal-derived substance, a virus-derived substance, an autogenous substance, an allergen or a cancer cell- The MDSC is generated by

According to a preferred embodiment of the present invention, the in vivo physiological activity regulating substance is a lipopolysaccharide (LPS) or a peptidoglycan as a bacteria-derived substance. According to a preferred embodiment of the invention, the in vivo physiological activity regulator is an antigen. According to a preferred embodiment of the present invention, the GPCR19 agonist used to make said MDSC in the method of the present invention is a compound of formula (I), a salt thereof or a hydrate thereof.

In accordance with another aspect of the invention, the present invention is formed by the in vivo physiological activity regulatory substance and GPCR19 agonists are processed to form, or in vivo biological activity regulatory substance and GPCR19 agonist MDSC treatment formed by agonist treatment CD23 ^hi Immunoregulatory B cells with surface trait and anti-inflammatory activity are provided.

Since the in vivo physiological activity regulator and the method of agonizing GPCR19 are the same as those described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification according to the repetitive description.

When the in vivo physiological activity regulator and the MDSC prepared by GPCR19 agonist treatment are administered to (H-MDSC) mammals, the number of cells to be administered is preferably 1 x 10 ³ to 1 x 10 ⁸ cells, more preferably 1 x 10 < ⁵ > to 1 x 10 < ⁷ > cells, and most preferably 1 x 10 < ⁶ > cells. Immunoregulatory B cells expressing the CD23 ^hi surface expression marker are generated (for example, B _HM cells) due to administration of H-MDSC, and the cells were administered to sepsis-treated mice, and the survival rate was significantly higher than that of the control , Suggesting that the immunoregulatory B cells (e.g., B _HM cells) exhibit anti-inflammatory activity.

The in vivo physiological activity regulator and the GPCR19 agonist are treated in the same manner as described above, and the administration of CD23 ^hi Immunoregulatory B cells expressing surface trait markers are generated (e. G., 48H B _H cells). The resulting 48H B _H cells were administered to sepsis mice and showed significantly higher survival rates as compared with the control group, suggesting that the immunoregulatory B cells (for example, 48H B _H cells) exhibit anti-inflammatory activity.

Treatment of a GPCR19 agonist or a test substance thereof, such as sodium taurodeoxycholate, to a population of bone marrow cells can be treated with a culture medium of the bone marrow cells to be cultured, preferably from 0.0001 mg / ml to 100 mg / ml, Preferably 0.001 mg / ml to 10 mg / ml, more preferably 0.01 mg / ml to 1 mg / ml, and most preferably 0.05 mg / ml.

CD23 ^hi , a phenotypic trait marker expressed in immunoregulatory B cells resulting from the treatment of GPCR19 agonists or test substances thereof, appears during the maturation of the precursor B cell surface, the early marker of the immune regulatory B cell lineage, It is a marker that remains while B cells are alive. CD23 ^hi immunoregulatory B cells can be screened using a flow cytometer after staining with CD23 ⁺ antibody.

According to a preferred embodiment of the present invention, the immunoregulatory B cells expressing the CD23 ^hi surface expression marker are selected from the group consisting of Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40, Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, Hbb-bh1, Hspa1a, Srpk3, Hhex, Hspa1a, Srpk3, Hpx, Cdp1, Cpbp1, Ankrd37, Cd44, Mrps7, Mapk11, Pycard, Cap1, Lta, Rbm4, Cd180, Cfp, Cmah, LOC100046855, Srebf1, Cbp2t3h, Tbp2t3h, Taf15, Ddx17, Cnn2, Brwd1, Cr2, Arhgap4, Pira3, Flna, The present invention provides a method of expressing high expression of a gene encoding Ci1a, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b1, Ci2b2, , Creb3l2, Prg2, Creld2, Nkg7, Anxa2, Txndc5, Tmem66, Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgals1, 2010001M09Rik, Ctsw, Cd160, , Du Cysl2, Trp53inp1, Ssr2, Reln, Tubb2b, Akp2, F2r, H13, Cd8b1, Ifit3, Rsad2, LOC667370, Irf7, Ifit2, Slfn1, Cxcl10, Ctla4, Rgs1, Oasl2, Apo, Lat, Lt1, Cd3d, Igtp, Rilpl1, Apo, Lat, Cpd, Mx1, LOC100048346, Mx2, Tyki, Hba-a1, Lgal3bp, Ifitm3, Cfl1, Oas2, Serpina3f, Emb, P2ry14, Cirbp, Dhx58, Fcer1g, Ifng, AA467197, Gpr114, Expresses low levels of Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, D14Ertd668e, Mlkl, Itk, Serpinb6b or Cd69 genes.

According to a preferred embodiment of the invention, immunoregulatory B cells expressing the CD23 ^hi surface trait marker express CD21 and CD23.

CD23 (FCεRII) expressed in the immunoregulatory B cells is a type II molecule of the C-lectin family including lymphocyte tracer receptor (MEL-14) and endothelial leukocyte adhesion molecule-1 (ELAM-1). CD23 (FCεRII) is a receptor with low affinity for IgE. A variety of hematopoietic cell types, including dendritic cells, B cells, T cells and macrophages of mammals, express CD23 on the cell surface. CD23 molecules may also be found in dissolved form in biological fluids. Soluble CD23 (sCD23) molecules are produced by proteolytic cleavage of transmembrane receptors. On the other hand, CD21 is a marker expressed in B cells, which forms a complex with CD19 and TAPA-1 and binds with complement component 3Cd, which is a negative stimulus component of B cell activation and proliferation.

According to a preferred embodiment of the present invention, the in vivo physiological activity regulating substance or cell activity regulating substance used in the present invention is characterized by being a bacterial-derived substance, a fungal-derived substance, a virus-derived substance, an autogenous substance or an allergen Lt; RTI ID = 0.0 > B < / RTI > cells.

According to a preferred embodiment of the present invention, the in vivo physiological activity regulating substance or cell activity regulating substance is a lipopolysaccharide (LPS) or a peptidoglycan as a bacteria-derived substance. According to a preferred embodiment of the present invention, the GPCR19 agonist used to make the MDSC or immunoregulatory B cells (48H B _H cells) or more specifically CD21 ⁺ CD23 ⁺ B cells in the method of the present invention is represented by the formula A salt thereof, or a hydrate thereof.

In accordance with another aspect of the present invention, the present invention provides a method for the treatment and / or prevention of diseases caused by in vivo physiologic activity regulating substances and Gr-1 ⁺ CD11b ⁺ MDSC (H-MDSC) formed by GPCR19 agonist treatment, an in vivo physiological activity regulator and GPCR19 agonist (B _HM cells) formed by treatment with Gr-1 ⁺ CD11b ⁺ MDSC formed, more specifically, by Gr-1 ⁺ CD11b ⁺ MDSC treatment formed by GPOR19 agonist treatment and in vivo physiological activity regulator CD21 ⁺ CD23 ⁺ B cells, or in vivo physiological activity regulators and immunoregulatory B cells (48H B _H cells) formed by GPCR19 agonist treatment or more specifically, in vivo physiological activity regulators and CD21 ⁺ CD23 ⁺ B cells as an active ingredient.

The active ingredient of the composition of the present invention is preferably Gr-1 ⁺ CD11b ⁺ MDSC or CD23 ^hi (48H B _H or B _HM cells) or B220 ⁺ CD21 ⁺ CD23 ⁺ B cells.

According to a preferred embodiment of the present invention, the diseases which can be prevented or treated with the anti-inflammatory pharmaceutical composition produced by the method of the present invention are preferably selected from sepsis, autoimmune diseases, inflammatory diseases, rheumatoid arthritis, atopic dermatitis, , Ulcerative colitis, type 1 diabetes, Crohn's disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, Castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia areata and systemic lupus erythematosus But is not limited to, and is most preferably sepsis.

When the composition of the present invention is prepared with an anti-inflammatory pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, and a preservative in addition to the above components. Suitable pharmaceutically acceptable carriers and preparations are described in Remington ' s Pharmaceutical Sciences (19th ed. 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the pharmaceutical composition can be administered by intravenous infusion, subcutaneous injection, intramuscular injection, intra-articular injection, or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Exemplary dosages of the pharmaceutical compositions of the present invention are 0.001-1000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) According to the present invention, administration of sodium taurodeoxycholate (HY2191) increases the number of H-MDSC cells in an inflammation-induced mouse due to the administration of lipopolysaccharide (LPS) Has a significant sepsis treatment effect.

(b) According to the present invention, administration of sodium taurodeoxycholate (HY2191) in mice in which inflammation was induced by the administration of lipopolysaccharide (LPS) increased the number of immunoregulatory B cells (48h B _H cells) , And the increased 48h B _H cell cell shows significant sepsis treatment effect.

(c) According to the present invention, administration of sodium taurodeoxycholate (HY2191) increases the number of B220 ⁺ CD21 ⁺ CD23 ⁺ B cells in mice in which inflammation is induced by the administration of lipopolysaccharide (LPS) Increased B220 ⁺ CD21 ⁺ CD23 ⁺ B cells show significant sepsis treatment.

(d) According to the present invention, administration of H-MDSC in mice in which inflammation is induced by administration of lipopolysaccharide (LPS) increases the number of B _HM cells and increases B _HM Cells show significant sepsis treatment effect.

(e) According to the present invention, administration of H-MDSC in mice in which inflammation was induced by administration of lipopolysaccharide (LPS) increased the number of B220 ⁺ CD21 ⁺ CD23 ⁺ B cells and increased B220 ⁺ CD21 ⁺ CD23 ⁺ B Cells show significant sepsis treatment effect.

(f) The method of the present invention provides a method for screening a therapeutic agent for a GPCR19-related disease or a method for analyzing the efficacy of a GPCR19 agonist, thereby providing an anti-inflammatory pharmaceutical composition.

FIGS. 1A-1C are graphs showing the survival rate after injecting sodium taurodeoxycholate (HY2191) into various kinds of sepsis-induced mice in order to examine the therapeutic effect of sodium taurodeoxycholate (HY2191) on sepsis. An image of the experimental method is attached to the upper part of the graph. 1A is a graph showing the survival rate when HY2191 was injected into LPS-induced sepsis mice 30 minutes and 24 hours after LPS treatment. 1B is a graph showing the survival rate when HY2191 was injected into CLP induced sepsis mice 2 hours after induction of CLP. FIG. 1C is a graph showing that HY2191 does not show a sepsis treatment effect in mice lacking B cells and T cells (Rag2 KO).
Figures 2A-2B are graphs depicting inhibition of inflammatory cytokine production in vivo by HY2191. Figure 2A is a graph showing that HY2191 reduces the serum inflammatory cytokine concentration induced by LPS. FIG. 2B is a graph showing that HY2191 reduces CLP-induced blood inflammatory cytokine levels.
Figures 3A-3B are graphs illustrating mitigation of hypotension due to sepsis after treatment with HY2191. 3A is a graph showing that a blood pressure lowering induced by LPS is normalized by HY2191, and the average blood pressure (mmHg) is increased. FIG. 3B is a graph showing that LPS-induced renal failure and hepatic dysfunction are alleviated by HY2191, respectively, with blood BUN and AST reduction.
4 is a graph showing absolute numbers of spleen cells, particularly CD11b ⁺ cells and B220 ⁺ B cells, in LPS-induced sepsis mice.
FIG. 5 is a graph showing absolute numbers of spleen CD4 ⁺ Foxp3 ⁺ T cells (regulatory T cells, Treg) in LPS-induced sepsis mice.
FIG. 6 is a graph showing that activation of splenocytes by LPS is inhibited by HY2191. FIG. In FIG. 6A, the activation of spleen CD11C ⁺ cells was expressed as mean fluorescence intensity (MFI) of the CD86 activation marker. In Figure 6B, the activation of spleen B220 ⁺ B cells was expressed as mean fluorescence intensity (MFI) of the CD86 activation marker. Figure 6C is a FACS plot showing the relative percentage (%) of B220 ⁺ CD86 ⁺ B cells overexpressing CD86, a cell activation marker in spleen B220 ⁺ B cells.
FIG. 7 is a graph showing that Gr-1 ⁺ CD11b ⁺ cells (H-MDSC) increase in an in vivo LPS-induced sepsis mouse treated with HY2191 and that they exhibit a sepsis treatment effect. FIG. 7A is a graph showing that the number of Gr-1 ⁺ CD11b ⁺ MDSC cells in the spleen of LPS-induced septic mice was significantly increased after 24 hours and 48 hours in the HY2191 treatment group. Also, it is a graph showing that HY2191 does not change the number of Gr-1 ⁺ CD11b ⁺ MDSC cells in a mouse deficient in B cells and T cells (Rag2 KO). Figure 7B is a FACS plot showing the relative percentage (%) of H-MDSC in splenocytes. FIG. 7C is a FACS histogram showing the immaturity of H-MDSC as a percentage of CD31 ⁺ cells as bone marrow immature markers. Figure 7C shows that H-MDSC is Ly6G ⁺ Ly6C ^inter F4 / 80 ^low Is a FACS plot. FIG. 7D is a heat-map comparing the expression genes of H-MDSC and L-MDSC. Red indicates the gene overexpressed in H-MDSC and green indicates the low expression gene compared with L-MDSC (Gr-1 ⁺ CD11b ⁺ MDSC harvested from LPS-induced septicemia mice not treated with HY2191).
FIG. 8 is a graph showing the survival rate after adoptive transfer of various kinds of cells into sepsis-induced mice in order to examine the therapeutic effect of H-MDSC on sepsis. An image of the overall experimental method for analyzing the therapeutic effects of H-MDSC harvesting and administration in LPS-induced sepsis mice was attached to the top of the graph.
FIG. 9 is a graph showing changes in immune cells in vivo induced by H-MDSC transduced into LPS-induced sepsis-induced mice as absolute numbers and relative ratios (%). FIG. 9A is a FACS plot image (top) and a repeating experiment graph (bottom) showing the relative and absolute number increase of splenic B cells 24 hours after H-MDSC proton transfer. 9B is the spleen and bone marrow ^{CD11B + Gr-1 + F4 /} 80 after the H-MDSC both transfected 24 ^hours a FACS plot image (top) and a graph (bottom) of the repeated experiments showing the relative and the increase in the absolute number of cells . FIG. 9C is a FACS plot image (top) and a graph (bottom) showing that the relative and absolute numbers of spleen T cells (CD4 ⁺ and CD8 ⁺ T cells) do not change after 24 hours of H-MDSC proton transfer. Figure 9D is a graph showing no significant change in the absolute number of spleen CD11C ⁺ dendritic cells after 24 hours of H-MDSC proton transfer.
FIG. 10 shows the effect of splenocyte-induced B cells on splenic B-cells (B _HM cells) proliferated by H-MDSCs transfected with LPS into sepsis transfer) and graph the survival rate.
FIG. 11 is a graph showing the relationship between the quantum B _HM FIG. 5 is a graph showing that the cells show the therapeutic effect of sepsis in the presence of H-MDSC in B cells and T cells deficient mice (Rag2 KO).
12A-C are graphs showing changes in in vivo spleen immunity cells induced by 48h B _H cells transfected into LPS-induced sepsis mice. FIG. 12A is a FACS plot image illustrating the activation of splenic B cells by LPS 24 h after 48 _h B _H cells proton transfer. Figure 12B is a FACS plot image illustrating the activation of spleen T cells (CD4 ⁺ and CD8 ⁺ T cells) by LPS 24 h after 48 _h B _H cells proton transfer. FIG. 12C is a FACS plot image illustrating the relative percentage (%) of CD11b ⁺ Ly6G ⁺ Ly6C ^inter cells increased in the spleen 24 hours after transfection of 48h B _H cells.
Figure 13 is a FACS plot showing that the phenotype of 48h B _H cells is different from previously known immunostimulatory B cells. 13A is 48h B _H Lt; / RTI > is a FACS plot in which cells express expression of CD1d and CD5, which are B1a B cell characteristic markers. 13B is 48h B _H Lt; RTI ID = 0.0 > CD3 < / RTI > expression, which is an immunoregulatory T2-MZP B cell characteristic marker. FIG. 13C is a FACS plot in which 48h B _H cells are illustrative of IgM expression, which is a characteristic marker of mature B cells, as compared to B cells observed in mice that induced sepsis in LPS.
FIG. 14A is a graph showing the effect of Gr-1 ⁺ CD11b ⁺ MDSC harvested from LPS-induced septic mice treated with H-MDSC (HY2191 treated Gr-1 ⁺ CD11b ⁺ MDSC) ) And the relative percentage (%) of spleen B220 ⁺ CD23 ^hi CD21 ^int cells increased 24 hours after transfection. 14B is a graph showing the absolute number of B220 ⁺ CD23 ⁺ cell numbers increase in the spleen 24 hours after transfection with L-MDSC or H-MDSC in mice induced to induce sepsis by LPS.
Figure 15A shows the effect of B220 ⁺ CD23 ^hi CD21 ^hi cells (CD23 ^hi B _H Cell) is a FACS plot showing CD23 expression 48 h after HY2191 injection in mice induced to sepsis by LPS. FIG. 15B shows the effect of B220 ⁺ CD23 ^hi CD21 ^int cells (CD23 ^hi B _H Cell) in order to examine the effect of sepsis on the survival rate of mice after adoptive transfer to sepsis-induced mice. The overall experimental method for analyzing the time of harvesting CD23 ^hi B _H cells from the mice induced by LPS and the treatment effect thereof after administration was schematically shown on the graph.
16 is a graph showing that CD23 ^hi B _H cells adoptively transferred to LPS-induced sepsis induce B cell and CD11b ⁺ cell proliferation in vivo in mice. B220 ⁺ B cells and CD11b ⁺ Gr-1 ⁺ absolute numbers of spleen at 24 and 48 hours after transfection of CD23 ^hi B _H cells into mice were compared with control (PBS) and CD23 ^lo B _H cells.
FIG. 17A is a graph illustrating the increase in the absolute number of Gr-1 ⁺ CD11b ⁺ cells after in vitro treatment of mouse bone marrow cells with LPS (1 mg / ml) and HY2191 (50 mg / ml) for 24 h. LysMDSC (Gr-1 ⁺ CD11b ⁺ cells generated after LPS alone treatment without HY2191 in mouse bone marrow cells in vitro) or HexMDSC (generated after simultaneous treatment of HY2191 and LPS in mouse bone marrow cells in vitro) 0.0 ^& gt ^; Gr-1 ^{+ &} lt ^; / RTI ^& gt ^; CD11b ⁺ cells). FIG. 17B is a graph illustrating the survival rate of mice induced to sepsis by LPS surviving after quantum transfer in order to examine the effect of treating HexMDSC, LexMDSC, and cellgro (negative control) in FIG. 17A.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  One. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191 ) Effect

C57BL / 6 mice (SLC, Japan) were used for all experiments to establish the sepsis model induced by LPS (Sigma, USA). (PBS, WelGENE, Korea) or sodium taurodeoxycholate (Sigma, USA) as a tail vein 30 minutes and 24 hours after intraperitoneal administration of Salmonella LPS (Sigma, USA) 20 mg / HY2191, sodium taurodeoxycholate; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand) was intradermally injected for 15 minutes using Medfusion 2001 (Medex, USA) (Medfusion 2001, Medex, USA). The survival rate of the experimental group and the control group over time was examined, and the results are shown in FIG. 1A. As can be seen from FIG. 1A, the survival rate of the mice treated with sodium taurodeoxycholate was 4 times higher than that of the control group, and 5 times higher when the groups were administered after 24 hours.

Example  2. Cecal - ligation and Puncture  ( CLP ) In the sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191 ) Effect

The pharmacological effect of sodium taurodeoxycholate was tested using a CLP model in which mice were artificially ligation and perforation of the cecum to induce peritonitis, followed by sepsis in the course of time. Twenty C57BL / 6 mice (SLC, Japan) were anesthetized by diluting 8 times diluted stock with PBS (15.5 ml of 2,2,2-Tribromoethanol 25 g + 2-Methyl-2-butanol) The right abdomen was incised to a length of 0.5 cm and the cecum was exposed. The lower portion of the ileocecal valve (0.9 cm) was ligated to the cecum. Three holes were made in the cecum with a 23-gauge needle. Suture caused septicemia. Two hours after suturing, 10 C57BL / 6 mice were treated with sodium taurodeoxycholate (HY2191; HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was administered dropwise to the tail vein for 20 minutes (experimental group), and the remaining 10 C57BL / 6 mice were administered with PBS in the same manner (control group). As a negative control group, mice that did not induce sepsis by abdominal skin and peritoneum were sutured after artificially incision of abdominal skin and peritoneum and then operated without surgery for CLP, were used as sham operation group. Survival rates of C57BL / 6 mice in the experimental group and the control group over time were examined. The results are shown in Fig. 1B. HY2191 was injected into CLP induced septic mice two hours later, showing a 7-fold increase in survival compared to the control group.

Example  3. LPS Of B cells and T cells in sepsis-induced sepsis model

The same LPS as in Example 1 was used to induce sepsis. Sodium taurodeoxycholate (HY2191, sodium taurodeoxycholate) was prepared by using B cells and T cell deficient mice (Rag2 KO). HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand)] was tested in the same manner as in Example 1. As shown in FIG. 1C, all the subjects died within 48 hours, and the survival rate was not changed in the experimental group and the control group. This suggests that HY2191 requires T cells or B cells for the treatment of sepsis.

Example  4. LPS In a mouse model that induced sepsis, sodium taurodeoxycholate ( HY2191 , sodium  taurodeoxycholate; HY2191 ) Inhibited the inflammatory cytokine production

Control material (PBS, WelGENE, Korea) or sodium taurodeoxycholate (HY2191) was used as a tail vein 30 minutes after the intraperitoneal injection of 20 mg / kg of LPS into C57BL / 6 mice as in Example 1. HY2191 (New Zealand Pharmaceuticals Ltd, New Zealand). After 6 hours of LPS administration, blood was drawn from the tail of the mouse and centrifuged (2000 × g, 20 minutes) to obtain serum. Changes in cytokine expression were confirmed using a cytometric bead array kit (BD, USA) .

TNF-a and IFN-g, MCP-1 and IL-6 showed a significant decrease by administration of HY2191 after 6 hours of LPS induction (FIG. 2A).

Example  5. CLP  Sodium taurodeoxycholate in inflammatory cytokine changes in sepsis model HY2191 , sodium taurodeoxycholate ; HY2191 ) Effect

The change in inflammatory cytokine during CLP induction as in Example 2 was confirmed by the same method as in Example 4. [ Blood was collected at 4, 24, and 48 hours after CLP, and the serum was separated, and the change of cytokine expression was confirmed using a CBA (cytometric bead array) kit.

TNF-a and IL-1b, and MCP-1 showed a significant decrease by administration of HY2191 at 24h after CLP induction (Fig. 2B).

Example  6. Sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191 ) Sepsis After treatment  Normalized blood pressure lowering effect

Blood pressure was measured using a non-invasive blood pressure (NIBP, CODA, USA) at 1, 2, 4 and 6 hours after induction of LPS in the tail of C57BL / 6 mice treated with the same method as in Example 1 Respectively. From 1 hour after the administration of LPS, the mean blood pressure began to decrease in both groups, and the mean blood pressure of the group treated with HY2191 increased significantly from 4 hours after induction of LPS compared with the blood pressure of the control group treated with PBS (FIG. 3A) .

Example  7. In the sepsis model, sodium taurodeoxycholate ( HY2191 , sodium  taurodeoxycholate; HY2191 ) To protect kidney and liver function

(BUN) and aspartate aminotransferase (AST) concentrations in the blood were measured by measuring the changes in the long-term damage after administration of HY2191 (Fig. 3B). After 30 minutes of LPS induction, HY2191 or PBS was administered dropwise to the tail, and blood was collected at 24 hours and 48 hours to separate the serum. BUN was measured in the separated serum and the degree of damage of the renal function was observed. AST was measured to observe the degree of damage of liver function. HY2191 was found to significantly reduce the BUN elevation that is indicative of renal damage at 48 hours of LPS induction (Fig. 3B, left). In addition, HY2191 significantly inhibited the increase of AST concentration due to sepsis in all of the patients observed for 24 hours and 48 hours. This suggests that HY2191 is effective in treating liver damage caused by sepsis. (Fig. 3B, right).

Example  8. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; By HY2191) Increased Splenocyte  Confirm

As in Example 1, changes in splenocytes were observed at 6 hours, 24 hours, and 48 hours after treatment with HY2191 and PBS in the mouse model of sepsis induced by LPS. The number of total splenocytes was significantly increased in the HY2191-treated group compared with the decrease in the PBS control group after 48 hours of LPS induction. Using flow cytometry, it was found that the absolute number of CD11b ⁺ cells and B220 ⁺ cells was markedly increased in all splenocytes (FIG. 4).

Example  9. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; By HY2191) Increased  Identify T regulatory cells

The experiment was carried out in the same manner as in Example 8, and it was confirmed that the absolute number of CD4 ⁺ Foxp3 ⁺ T cells as T regulatory cells in the spleen cells was significantly increased after 48 hours of LPS induction by HY2191 (FIG. 5).

Example  10. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191) Dendritic cell  Activation inhibition effect

In the same manner as in Example 8, the activity of CD11c ⁺ dendritic cells (CD86 expression) was confirmed in the splenocytes after LPS induction. As shown in FIG. 6A, only CD11c (+) / 7AAD (-) cells were selected for analysis of living dendritic cells, and CD86 expression was analyzed in dendritic cell co-stimulatory molecules. CD86 expression was expressed by mean fluorescence intensity (MFI), and expression was inhibited by HY2191 from 24 hours after LPS induction.

Example  11. LPS In a mouse model that induced sepsis, sodium taurodeoxycholate ( HY2191 , sodium  taurodeoxycholate; HY2191 ) Inhibited B-cell activation

In the same manner as in Example 8, the activity (CD86 expression) of B220 ⁺ B cells in the spleen cells after LPS induction was confirmed. To analyze only live B cells, only B220 (+) / 7AAD (-) cells were selected and CD86 expression was analyzed in B cell co-stimulatory molecules. CD86 expression was expressed by mean fluorescence intensity (MFI), and it was confirmed that HY2191 significantly inhibited CD8 expression at 6 hours and 24 hours after induction of LPS (FIG. 6B, FIG. 6C).

Example  12. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191) Increased  H- MDSC Of sepsis treatment effect

Twenty-four hours after administration of sodium taurodeoxycholate to the mice induced by LPS as in Example 1, mice were sacrificed and spleens were harvested to express CD11b in spleen cells, and Gr-1 Highly expressed cells (bone marrow-derived inhibitory cells, MDSC) were analyzed (Fig. 7A, Fig. 7B). Mice that received LPS alone without sodium taurodeoxycholate were sacrificed and spleens were harvested to induce CD11b expression in splenocytes and to induce a higher level of Gr-1 expression (hereinafter, referred to as L-MDSC) After 24 hours of administration of sodium taurodeoxycholate, the numbers of H-MDSC cells expressing CD11b and simultaneously expressing high levels of Gr-1 in the spleen cells obtained by sacrificing each mouse were significantly (Figs. 7A and 7B). The immature status of L-MDSC and H-MDSC cells was confirmed using CD31, a bone marrow cell immature marker (Fig. 7C, top). There was no significant difference in the ratio of CD31 ⁺ expressing cells in both cells. L-MDSC and H-MDSC showed F4 / 80 ^lo Gr-1 ⁺ and Ly6g ⁺ Ly6c ⁺ phenotype (FIG. MDSCs with these phenotypes showed a significant increase in sepsis mice treated with sodium taurodeoxycholate.

Total RNA was extracted from MDSC using QIAGEN RNeasy Mini KIT (Qiagen, USA). Remove selected without processing the HY2191 in mice that lead to sepsis Gr1 ⁺ CD11b ⁺ MDSC Gr1 ⁺ CD11b ⁺ MDSC (H-MDSC) a process to separate screening HY2191 in mice that lead to sepsis as compared to the (L-MDSC) (Fig. 8B, Table 1, Table 2). The differences in expression of the genes were compared using the Illumina Sentrix MouseRef-8 (v1.1) BeadChip arrays (Illumina, USA) and genes with p-value less than 0.05 and fold change greater than or equal to 2 or less than -2 were selected. Tables 1 to 2 show:

A gene highly expressed in H-MDSC compared to L-MDSC Reference sequence ID sign H-MDSC /
L-MDSC ratio NM_015768.1 Prok2 3.74 NM_001013365.1 Osm 3.33 NM_015768.1 Prok2 3.15 NM_008522.3 Ltf 3.02 NM_133203.1 Klra17 3.01 NM_016917.2 Slc40a1 2.96 NM_008522.2 Ltf 2.91 NM_013517.1 Fcer2a 2.83 NM_010479.2 Hspa1a 2.81 NM_009909.3 Il8rb 2.63 NM_017372.3 Lyz2 2.60 NM_023734.3 Pi16 2.41 NM_018769.3 Dfna5h 2.39 NM_001005864.2 Mtus1 2.38 NM_145437.2 4732429D16Rik 2.29 NM_016969.1 Myadm 2.29 NM_183168.1 P2ry6 2.24 Osm 2.22 NM_001005865.2 Mtus1 2.21 NM_008036.2 Fosb 2.17 NM_026862.3 Cd177 2.16 NM_019972.2 Sort1 2.12 NM_133219.1 Gcnt2 2.12 NM_026929.3 Chac1 2.12 NM_016678.1 Reck 2.11 NM_019972.2 Sort1 2.11 NM_026862.3 Cd177 2.11 NM_017372.2 Lyzs 2.10 NM_153804.3 Plekhg3 2.06 NM_007782.1 Csf3r 2.05 NM_007377.3 Aatk 2.05 NM_138650.1 Dgkg 2.03 NM_020047.3 Tacstd2 2.03 NM_008510.1 Xcl1 2.02 NM_007782.1 Csf3r 2.02

Compared to L-MDSC, genes that are low in H-MDSC Reference sequence ID sign H-MDSC /
L-MDSC ratio Il1a -3.05 NM_010104.2 Edn1 -2.86 NM_021274.1 Cxcl10 -2.56 NM_007413.4 Adora2b -2.35 NM_008599.3 Cxcl9 -2.23 NM_001033335.2 Serpina3f -2.21 NM_008176.1 Cxcl1 -2.19 NM_008987.2 Ptx3 -2.03 NM_008337.3 Ifng -2.00 NM_011990.2 Slc7a11 -2.00 NM_028967.1 Batf2 -1.89 NM_153564.2 Gbp5 -1.88 NM_007687.2 Cfl1 -1.86 NM_008337.1 Ifng -1.83 NM_019453.1 Mefv -1.77 NM_207680.2 Bcl2l11 -1.76 NM_010260.1 Gbp2 -1.74 NM_011451.2 Sphk1 -1.71 NM_021893.2 Cd274 -1.71 NM_010493.2 Icam1 -1.71 NM_010548.1 Il10 -1.71 NM_001039959.1 Ahnak -1.70 NM_018734.2 Gbp3 -1.69 NM_007705.2 Cirbp -1.67 NM_030720.1 Gpr84 -1.67 NM_007535.2 Bcl2a1c -1.67 NM_011579.2 Tgtp -1.66 NM_019388.2 Cd86 -1.65 NM_010431.1 Hif1a -1.65 NM_133888.2 Smpdl3b -1.65 NM_030720.1 Gpr84 -1.65 NM_178699.3 B930041F14Rik -1.64 NM_177617.2 D330014H01Rik -1.63 NM_018734.3 Gbp3 -1.63 NM_019388.2 Cd86 -1.63 NM_009396.1 Tnfaip2 -1.63 NM_011068.1 Pex11a -1.62 NM_001042592.2 Arrdc4 -1.62 NM_011373.2 St6galnac4 -1.62 NM_008326.1 Irgm1 -1.62 NM_011056.2 Pde4d -1.61 NM_010807.3 Marcksl1 -1.61 NM_009373.3 Tgm2 -1.60 NM_011337.2 Ccl3 -1.60 NM_008351.1 Il12a -1.58 NM_013683.1 Tap1 -1.58 NM_013602.2 Mt1 -1.58 NM_021326.2 Rbak -1.58 NM_198620.1 Rundc3b -1.58 NM_011521.2 Sdc4 -1.58 NM_011068.1 Pex11a -1.57 NM_019948.2 Clec4e -1.57 NM_153165.2 Klhl15 -1.57 NM_007870.2 Dnase1l3 -1.56 NM_010846.1 Mx1 -1.55 NM_146004.1 BC024997 -1.55 NM_016674.2 Cldn1 -1.55 NM_008330.1 Ifi47 -1.54 NM_145209.3 Oasl1 -1.54 NM_019948.1 Clecsf9 -1.53 NM_001013370.1 Sesn1 -1.53 NM_008793.1 Pcsk4 -1.53 NM_013928.2 Schip1 -1.53 NM_008332.2 Ifit2 -1.52 NM_010501.2 Ifit3 -1.52 NM_009396.1 Tnfaip2 -1.52 NM_130447.2 Dusp16 -1.52 NM_027261.1 Josd3 -1.52 NM_009251.1 Serpina3g -1.52 NM_009754.2 Bcl2l11 -1.51 NM_025745.2 4933407N01Rik -1.51 NM_007707.2 Socs3 -1.51 NM_010474.1 Hs3st1 -1.51 NM_009895.3 Cish -1.51 NM_019758.2 Mtch2 -1.50 NM_175352.4 Unc119b -1.50 NM_008607.1 Mmp13 -1.50 NM_018738.3 Igtp -1.50 NM_010260.1 Gbp2 -1.50 NM_175687.1 A230050P20Rik -1.50

The cells were divided into three groups according to the degree of expression of CD11b and Gr-1, and they were sorted by FACS sorter (Fig. 8). The number of cells in the separated cells was measured with a hemocytometer, and the number of living cells was adjusted to 1 x 10 < ⁵ > by mixing with PBS to inject into the tail vein into sepsis-induced mice (Fig. 8). At this time, mice were intraperitoneally administered LPS with lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before the cells were injected. Each mouse was used for five rats in the PBS group and the L-MDSC, H-MDSC group, in the group injected with Gr-1 ^int cell 3, Gr-1 ^- in the group injected with the cells were used quadruplets. In the H-MDSC-treated mice, significantly increased survival (80% survival) was seen compared to L-MDSC (40% survival) and other surface phenotype cells. In order to confirm the mechanism of action of the therapeutic effect by H-MDSC, splenocyte changes of cells injected mice were observed (FIG. 9). H-MDSC and L-MDSC were quantitatively transferred to mice 24 hours after administration of LPS. Splenocytes from mice were harvested 24 hours after cell proliferation. As shown in FIG. 9A (above: representative FACS plot, below: repeated experiment graph), it was confirmed that B220 ⁺ cells were significantly increased when H-MDSC was quantitated. In addition, CD11b ⁺ Gr-1 ⁺ cells increase in bone marrow and spleen compared with L-MDSC when H-MDSC is quantitated as shown in Figure 9B (Above: representative FACS plot, Respectively. However, as can be seen in Figures 9C and 9D, there was no change in CD4 ⁺ T cells, CD8 ⁺ T cells and CD11c ⁺ DC counts.

Example  13. LPS In the sepsis-induced sepsis model, sodium To taurodeoxycholate  By Increased  48h B _H  Effect of treatment of sepsis in cells

The same C57BL / 6 mouse as in Example 1 was used. Five mice were used in PBS group, normal mouse B cell administration group, 48h B _L cell administration group, 24h B _H cell administration group, 48h B _H cell administration group and B _HM cell administration group, respectively. To obtain B _HM cells, 24 hours after administration of LPS and sodium taurodeoxycholate, mice were sacrificed and spleens were harvested. MDSCs expressing high levels of CD11b and Gr-1 molecules in the spleen cells were treated with FACS sorter (Fig. 10, left). Live (7AAD ^-) to suspend the CD11b ⁺ Gr-1 ⁺ cells 1 × 10 ⁵ reviews PBS were injected into the tail vein in a B6 mouse sepsis was induced 24 hours. To obtain B _HM cells from these mice, spleens were sacrificed after 24 hours of administration of CD11b ⁺ Gr-1 ⁺ cells, and B cells highly expressing B220 molecules in the splenic cells were collected by FACS sorter (B _HM cells, left in FIG. 10, left). Twenty-four hours or 48 hours after administration of LPS and sodium taurodeoxycholate, the mice were sacrificed to obtain 24h or 48h B _H cells, and spleens were harvested. B cells expressing B220 molecules Cells were sorted by FACS sorter (24h B _H cells or 48h B _H cells, also in the top 10). 48 h B _L cells were harvested 48 hours after the administration of LPS, and the spleens were sacrificed. B cells expressing high B220 molecules in the spleen cells were sorted by FACS sorter (48 h B _L Cell, top 10 right). 1 × 10 ⁶ viable (7AAD ^- ) cells were suspended in PBS and these cells were injected into the tail vein of B6 mice induced sepsis. Mice receiving adoptive cells at this time induced sepsis by intraperitoneal administration of LPS (20 mg / kg, 12,000 EU / g) 24 hours before the injection of B cells. Compared with the group injected with B cells of normal mice (0% survival) and 24h B _H cells injected group (20% survival) and 48h B _L cells (survival of 40%) (80% survival) in mice treated with 48h B _H cells. B _HM cells, the survival rate (100% survival) was the best.

Example  14. LPS In the sepsis-induced sepsis model, sodium To taurodeoxycholate  By Increased  48h B _H  Identifying the importance of cells and H-MDSC

As in Examples 12 and 13, H-MDSC and B _HM cells generated in C57BL / 6 mice were quantitatively transferred to Rag2KO mice without B cells and T cells 24 hours after induction of LPS, and the effect on the sepsis model was confirmed. PBS group, B _HM cells (1 × 10 ⁶ , 5 × 10 ⁶ ), and B _HM cells (1 × 10 ⁶ , 5 × 10 ⁶ ) and 48 h H-MDSC (1 × 10 ⁵ ) . When LPS (20 mg / kg, 12,000 EU / g) was intraperitoneally administered to Rag2KO mice as in Example 3, 24 hours after the injection, 1 × 10 ⁶ of PBS or B _HM cells were quantitatively transferred into the tail vein. It was confirmed that there was no change in the survival rate in the experimental group and the control group. B _HM cells 1 × 10 ⁶ dogs were all objects mortality for 48h H-MDSC 72 hours in case of both transfection, all the objects within 96 hours in case of both transfected to 5 × 10 ⁶ gae B _HM cells mortality. Unlike the other groups, 5 × 10 ⁶ B _HM cells and 1 × 10 ⁵ H-MDSCs of 48 h survived for more than 120 hours only in the quantum transfer group. B _HM cells proliferated more prolonged than PBS group regardless of the number of cells.

Example  15. LPS In the sepsis-induced sepsis model, sodium taurodeoxycholate ( HY2191 , sodium taurodeoxycholate ; HY2191) Increased B _H  After cell proton transfer In splenocytes  Confirm change

48h B _H cells were prepared in the same manner as in Example 13, and 1 x 10 ⁶ of 48h B _H cells were quantitatively injected at 24 hours after induction of LPS. After 24 hours of mice were sacrificed, B220 ⁺ B cells (CD86 expression) of the cells. To analyze only live B cells, only B220 (+) / 7AAD (-) cells were selected and CD86 expression was analyzed in B cell co-stimulatory molecules. Compared with the PBS-only control group at 24 hours, CD86 expression was decreased when B _H cells were transfected (FIG. 12A). In FIG. 12B, it was confirmed that the CD69 ^hi CD62L ^lo T cells were reduced in the CD4 ⁺ T cells or CD8 ⁺ T cells in the spleen cells when LPS was administered. Figure 12C was analyzed after gating only the CD11b ⁺ cells 48h _H B cells by FACS plots image urbanization the relative proportion (%) of both the transfected CD11b ⁺ Ly6G ⁺ Ly6C ^inter cells increased in the spleen after 24 hours. Compared with the PBS group, the relative proportion of CD11b ⁺ Ly6G ⁺ Ly6C ^inter cells increased from 58% to 82% when 48h B _H cells were transfected.

Example  16. B _HM  cell, B _L  And B _H  Cell phenotype analysis

48h B _H cells, which have characteristics similar to those of B _HM cells and can be produced more easily and efficiently than B _HM cells, were compared and analyzed. The normal C57BL / 6 mouse spleen from 8 to 12 weeks was ground with two frosted slides into a single cell culture supernatant, and then blocked with blocking buffer, purified 2.4 G2 (rat IgG2b against FcγRII , eBioscience, USA) and phosphate buffered saline (PBS) containing 10% of inactivated mouse, rat and goat serum were mixed and incubated at 4 ° C for 30 minutes. Mouse spleen cells were treated with anti-mouse CD11b (M1 / 70), anti-mouse B220 (RA3-6B2), anti-mouse Gr-1 (RB6-8C5), anti- mouse F4 / 80 (CI: A3-1) Mouse CD23 (B3B4), anti-mouse CD21 (7G6) and anti-mouse CD93 (AA4.1) and all other antibodies for 15 minutes. Washing conditions After staining, 7-amino actinomycin D (7AAD, 7-amino actinomycin D) was stained for 10 minutes so that only living cells could be identified. Then, the cells were measured using FACS Canto II (BD, USA). Data were analyzed using Flowjo software (Tree Star, Inc., USA). B _HM cells and 48h B _H cells express CD21 and CD23 high (FIG. 14 and FIG. 15A) similarly to conventional T2-MZP regulated B cells, but CD5, CD1d and AA4 (CD93) -B). The expression of IgM was 48h B _L 48 _h B _H The cells were evenly elevated, indicating that they had characteristics of mature B cells (Fig. 13C).

Example  17. LPS In the sepsis-induced sepsis model, sodium Taurodeoxycholate After treatment Increased CD23 ^hi B _H  Effect of treatment of sepsis in cells

Forty-eight hours after administration of LPS and sodium taurodeoxycholate, mice were sacrificed and spleens were harvested. In splenocytes, B220 ⁺ CD23 ^hi CD21 ^hi Gr-1 ^- CD11b ^- CD23 ^hi and selected separation of _H B cells exhibiting a phenotype of a FACS sorter. At the same time ^{B220 + CD23 lo CD21 hi Gr-} 1 - CD11b - CD23 lo B H represents the phenotypic Cells were also selected and used as controls in the same manner. 1 × 10 ⁵ and 0.5 × 10 ⁵ living (7AAD ^- ) cells were suspended in PBS and injected into the tail vein of mice. At this time, mice were intraperitoneally administered LPS at a lethal dose (20 mg / kg, 12,000 EU / g) 24 hours before the cells were injected. 1 × 10 ⁵ and 0.5 × 10 ⁵ of CD23 ^hi B _H (100% and 75% survival) in CD23 ^lo B _H Cell-treated sepsis mice (each 75% and 25% survival), respectively. PBS-only injected mice showed 0% survival. (Fig. 15B). PBS treated group, CD23 ^lo B _H cell treated group and CD23 ^hi B _H (1 × 10 ⁵ and 0.5 × 10 ⁵ ) for the two dose groups in the cell- Four of each were used. Total RNA was extracted from B cells using QIAGEN RNeasy Mini KIT (Qiagen, USA). Sepsis-induced CD23 ^lo CD21 ⁺ B220 ⁺ B cells (CD23 ^lo ) isolated from the B220 ⁺ B cells (48h B _L cell) or sepsis mice sorted and isolated 48 hours after treatment with HY2191 B compared to the _H cell) in the septic mice HY2191 treatment 48 hours after separation screening the B220 ⁺ B cells (expressed specifically in 48h B _H cell) or CD23 ^hi CD21 ⁺ B220 ⁺ B cells (CD23 ^hi B _H cell) (Illumina, USA), and genes with a p-value of less than 0.05 and a fold change of more than or less than -2 were selected. The expression levels of the increased or decreased genes were compared using the Illumina Sentrix MouseRef-8 (v1.1) BeadChip arrays , The list is shown in Tables 3 to 8 below.

24h B _H cells and 48h B _H cells Reference sequence ID sign 48h B _H /
24h B _H ratio NM_001082545.1 Stfa2 34.73 NM_013650.2 S100a8 26.68 NM_181596.3 Retnlg 21.78 NM_009114.1 S100a9 21.15 NM_008491.1 Lcn2 17.54 NM_001082547.1 EG433016 15.16 Il1b 11.76 NM_029796.2 Lrg1 10.60 NM_017370.1 Hp 9.96 NM_008039.2 Fpr2 8.65 NM_007695.2 Chi3l1 8.18 NM_013590.2 Lyz 8.16 NM_008522.3 Ltf 7.98 NM_007585.3 Anxa2 7.54 NM_008230.4 Hdc 7.15 NM_010185.2 Fcer1g 7.12 NM_001082543.1 Stfa1 6.06 NM_133198.1 Pygl 5.91 NM_011313.2 S100a6 5.70 NM_009369.1 Tgfbi 5.63 Chi3l3 5.30 NM_011315.3 Saa3 5.10 NM_026862.3 Cd177 5.00 NM_009663.1 Alox5ap 4.84 NM_009841.3 Cd14 4.83 NM_009477.1 Upp1 4.77 NM_013517.1 Fcer2a 4.65 NM_026414.2 Asprv1 4.64 NM_010058.1 Dmwd 4.61 NM_016917.2 Slc40a1 4.56 NM_133219.1 Gcnt2 4.51 NM_015768.1 Prok2 4.34 NM_021609.3 Ccbp2 4.17 NM_030694.1 Ifitm2 4.14 NM_009402.1 Pglyrp1 4.02 NM_011414.2 Slpi 4.00 NM_172453.1 Pif1 3.88 NM_008694.1 Ngp 3.86 NM_145158.2 Emilin2 3.75 NM_007515.2 Slc7a3 3.73 NM_017372.3 Lyz2 3.64 NM_010705.2 Lgals3 3.51 NM_013612.1 Slc11a1 3.45 NM_173372.1 Grm6 3.39 NM_009645.2 Aicda 3.35 NM_010176.2 Fah 3.29 NM_013599.2 Mmp9 3.28 NM_017370.1 Hp 3.18 NM_024253.4 Nkg7 3.10 NM_007547.2 Sirpa 3.10 NM_144559.1 Fcgr4 3.10 NM_021273.3 Ckb 2.98 NM_172449.1 Bzrap1 2.89 NM_009689.2 Birc5 2.85 NM_172723.4 Adap1 2.84 NM_011121.3 Plk1 2.83 NM_009778.1 C3 2.83 NM_014194.1 Klra7 2.81 NM_013652.2 Ccl4 2.80 XM_001475189.1 LOC100040592 2.76 NM_009910.2 Cxcr3 2.75 NM_017370.1 Hp 2.72 NM_009645.1 Aicda 2.72 NM_010615.1 Kif11 2.72 NM_009689.1 Birc5 2.71 NM_008823.3 Cfp 2.71 NM_009689.2 Birc5 2.70 NM_023223.1 Cdc20 2.70 NM_023223.1 Cdc20 2.70 NM_008685.2 Nfe2 2.69 NM_013538.3 Cdca3 2.67 NM_024245.3 Kif23 2.64 NM_134469.3 Fdps 2.63 NM_013538.4 Cdca3 2.63 NM_134471.3 Kif2c 2.62 NM_007681.2 Cenpa 2.61 NM_023716.2 Tubb2b 2.61 NM_153101.1 Mrgpra2 2.59 NM_021386.3 Cldn10 2.59 NM_025831.3 1300014I06Rik 2.57 NM_009270.3 Sqle 2.57 NM_012055.3 Asns 2.56 NM_020010.2 Cyp51 2.56 NM_008446.1 Kif4 2.53 NM_145150.1 Prc1 2.53 NM_053214.1 Myo1f 2.50 NM_025436.1 Sc4mol 2.50 NM_010620.1 Kif15 2.49 NM_009382.3 Thy1 2.48 NM_172301.3 Ccnb1 2.47 NM_017407.1 Spag5 2.46 NM_010129.1 Emp3 2.46 NM_015768.1 Prok2 2.46 NM_021407.1 Trem3 2.44 NM_145588.1 Kif22 2.44 NM_175384.3 Cdca2 2.43 NM_197959.1 3000004C01Rik 2.42 NM_012055.1 Asns 2.40 NM_028390.2 Anln 2.37 NM_009140.2 Cxcl2 2.37 NM_139206.1 Centd3 2.36 NM_011701.3 Vim 2.36 NM_023223.1 Cdc20 2.33 XM_001471776.1 LOC100038894 2.33 NM_178683.4 Depdc1b 2.32 NM_011497.3 Aurka 2.32 XM_359281.1 Lst1 2.31 NM_008519.1 Ltb4r1 2.31 NM_013517.3 Fcer2a 2.30 XM_980391.1 D230007K08Rik 2.29 NM_013658.2 Sema4a 2.28 NM_023878.2 Cldn10 2.28 NM_008252.2 Hmgb2 2.28 NM_013612.1 Slc11a1 2.27 NM_007532.2 Bcat1 2.27 NM_021293.2 Cd33 2.27 NM_001033484.1 Iqgap3 2.26 NM_010555.4 Il1r2 2.26 NM_009270.3 Sqle 2.25 NM_172659.2 Slc2a6 2.23 NM_009477.1 Upp1 2.22 NM_010436.2 H2afx 2.22 NM_133662.2 Ier3 2.21 NM_007671.2 Cdkn2c 2.20 NM_008247.2 Ppap2a 2.20 NM_008505.3 Lmo2 2.19 NM_007471.1 App 2.19 NM_011340.3 Serpinf1 2.19 NM_018767.2 Cd160 2.19 NM_009773.1 Bub1b 2.19 NM_010858.4 Myl4 2.18 NM_197959.1 3000004C01Rik 2.17 NM_133198.1 Pygl 2.15 NM_008446.1 Kif4 2.15 NM_013517.1 Fcer2a 2.15 NM_009860.2 Cdc25c 2.15 NM_009835.2 Ccr6 2.14 NM_021530.2 Slc4a8 2.13 NM_028232.1 Sgol1 2.10 NM_194069.1 D12Ertd647e 2.09 NM_025654.2 Rdm1 2.08 NM_146171.1 Ncapd2 2.08 NM_008288.2 Hsd11b1 2.08 NM_016692.1 Incenp 2.08 NM_144512.2 Slc6a13 2.08 NM_016745.2 Atp2a3 2.08 NM_178911.4 Pld4 2.08 NM_016662.4 Mxd3 2.08 NM_009174.3 Siah2 2.07 NM_025377.1 1110001A07Rik 2.07 NM_019972.2 Sort1 2.06 NM_011662.2 Tyrobp 2.06 NM_008021.4 Foxm1 2.05 NM_026515.2 2810417H13Rik 2.05 NM_146094.1 Fads1 2.05 NM_194068.1 D12Ertd647e 2.04 NM_010058.1 Dmwd 2.04 NM_009477.1 Upp1 2.04 NM_009955.2 Dpysl2 2.03 NM_001042652.1 Nusap1 2.03 NM_013870.2 Smtn 2.02 NM_009128.1 Scd2 2.02 NM_009860.1 Cdc25c 2.02 NM_198411.2 2610204M08Rik 2.01 NM_001081403.1 Klhl14 2.01 NM_016745.2 Atp2a3 2.00 NM_011090.1 Pira3 2.00 NM_009985.3 Ctsw 2.00

24h B _H cells and 48h B _H cells Reference sequence ID sign 48h B _H /
24h B _H ratio NM_010501.1 Ifit3 -26.50 NM_016850.2 Irf7 -13.14 NM_021384.3 Rsad2 -9.76 NM_008332.2 Ifit2 -8.45 XM_001480051.1 LOC100048346 -7.46 NM_011909.1 Usp18 -7.07 NM_021274.1 Cxcl10 -6.74 NM_011909.1 Usp18 -6.51 NM_145227.1 Oas2 -5.75 NM_020557.3 Tyki -5.15 Mx2 -5.12 NM_011854.1 Oasl2 -4.95 NM_025378.2 Ifitm3 -4.60 NM_001033335.2 Serpina3f -4.56 NM_001039160.2 Gvin1 -4.24 NM_010479.2 Hspa1a -4.09 NM_011520.3 Sdc3 -4.00 NM_011150.2 Lgals3bp -3.95 NM_009696.2 Apoe -3.85 NM_021430.2 Rilpl1 -3.82 NM_018738.3 Igtp -3.82 NM_010741.2 Ly6c1 -3.77 NM_008332.2 Ifit2 -3.68 NM_198034.2 Sidt1 -3.50 NM_019440.2 Iigp2 -3.44 NM_011854.2 Oasl2 -3.28 NM_008733.3 Nrap -3.22 NM_030150.2 Dhx58 -3.21 NM_008326.1 Irgm1 -3.18 NM_011407.1 Slfn1 -3.15 NM_001024721.1 BC094916 -3.06 NM_145226.2 Oas3 -3.04 NM_145209.3 Oasl1 -3.02 NM_146142.1 Tdrd7 -3.02 XM_001471686.1 LOC100038882 -2.95 NM_010846.1 Mx1 -2.89 NM_198095.2 Bst2 -2.86 NM_008330.1 Ifi47 -2.86 NM_172275.1 Trafd1 -2.85 NM_011852.2 Oas1g -2.85 NM_199146.1 AI451617 -2.83 NM_020583.4 Isg20 -2.80 NM_145973.2 Ell3 -2.79 NM_199015.1 D14Ertd668e -2.78 NM_145209.3 Oasl1 -2.76 NM_011163.3 Eif2ak2 -2.74 NM_145209.2 Oasl1 -2.65 NM_008372.3 Il7r -2.62 NM_172603.1 Phf11 -2.57 NM_172603.2 Phf11 -2.55 NM_133362.2 Erdr1 -2.50 NM_001039530.1 Parp14 -2.48 XM_001479238.1 LOC100047963 -2.45 NM_023386.3 Rtp4 -2.43 NM_133200.3 P2ry14 -2.42 NM_172275.1 Trafd1 -2.42 NM_178283.3 Asb13 -2.42 NM_027320.4 Ifi35 -2.42 NM_011852.2 Oas1g -2.41 NM_133211.3 Tlr7 -2.40 NM_009099.2 Trim30 -2.39 NM_011068.1 Pex11a -2.39 NM_001033122.3 Cd69 -2.36 NM_009911.2 Cxcr4 -2.36 NM_021415.3 Cacna1h -2.34 NM_026004.2 Nt5c3 -2.34 NM_007829.3 Daxx -2.34 NM_018851.2 Samhd1 -2.30 NM_008326.1 Irgm1 -2.28 NM_172275.1 Trafd1 -2.25 NM_007707.2 Socs3 -2.23 NM_010708.1 Lgals9 -2.19 NM_007602.3 Capn5 -2.18 NM_007870.2 Dnase1l3 -2.15 NM_001038587.1 Adar -2.12 Tor3a -2.10 NM_174992.2 BC004728 -2.10 NM_010362.2 Gsto1 -2.09 NM_133200.3 P2ry14 -2.08 NM_029803.1 Ifi27 -2.08 NM_172608.1 Tmem184b -2.07 NM_027919.3 Tha1 -2.03 NM_029005.1 Mlkl -2.03 NM_145391.1 Tapbpl -2.02 NM_174992.3 BC004728 -2.01 NM_019949.1 Ube216 -2.01

As compared to 48h B _L cells and gene expression in cells 48h B _H Reference sequence ID sign 48h B _H /
48h B _L ratio NM_021609.3 Ccbp2 2.57 NM_025436.1 Sc4mol 2.14 NM_020010.2 Cyp51 2.00 NM_009270.3 Sqle 1.99 XM_001475189.1 LOC100040592 1.88 NM_146184.4 B3gnt8 1.85 NM_025831.3 1300014I06Rik 1.85 NM_134469.3 Fdps 1.83 NM_001038492.1 Ctsa 1.80 NM_010058.1 Dmwd 1.80 NM_146083.1 Sfrs7 1.72 NM_013517.1 Fcer2a 1.71 NM_008219.3 Hbb-bh1 1.71 NM_010479.2 Hspa1a 1.70 NM_019684.1 Srpk3 1.69 NM_008245.3 Hhex 1.68 NM_153526.2 Insig1 1.68 NM_009824.1 Cbfa2t3h 1.67 NM_027427.1 Taf15 1.67 NM_199080.2 Ddx17 1.66 NM_007725.2 Cnn2 1.66 NM_145125.2 Brwd1 1.66 NM_007758.2 Cr2 1.66 NM_138630.1 Arhgap4 1.64 NM_023716.2 Tubb2b 1.64 NM_011090.1 Pira3 1.63 NM_013730.4 Slamf1 1.63 NM_010227.2 Flna 1.63 NM_025876.2 Cdk5rap1 1.63 NM_198683.1 Cugbp1 1.62 NM_009270.3 Sqle 1.62 NM_001039562.1 Ankrd37 1.60 NM_001039150.1 Cd44 1.60 NM_025305.1 Mrps7 1.60 NM_011161.4 Mapk11 1.60 NM_023258.3 Pycard 1.59 NM_007598.2 Cap1 1.59 NM_010735.1 Lta 1.59 NM_009032.2 Rbm4 1.59 NM_008533.2 Cd180 1.59 NM_008823.3 Cfp 1.59 NM_007717.1 Cmah 1.58 XM_001476916.1 LOC100046855 1.58 NM_011480.1 Srebf1 1.57 NM_007575.2 Ciita 1.57 NM_198683.1 Cugbp1 1.57 NM_009109.2 Ryr1 1.56 NM_177733.2 E2f2 1.56 NM_022994.2 Dap3 1.55 Wdr9 1.55 NM_009835.3 Ccr6 1.55 NM_028281.1 Pcbd2 1.55 NM_010721.1 Lmnb1 1.54 NM_011091.1 Pira4 1.54 NM_144923.2 Blvrb 1.54 NM_007393.1 Actb 1.54 NM_183222.2 Fcrl5 1.54 NM_016745.2 Atp2a3 1.53 NM_010497.2 Idh1 1.53 NM_007573.2 C1qbp 1.53 NM_011203.2 Ptpn12 1.53 NM_013710.3 Fgd2 1.53 NM_133774.4 Stard4 1.53 NM_153424.2 Nphp4 1.52 XM_131538.1 Dhcr24 1.52 NM_011701.3 Vim 1.52 NM_001036740.2 B3gnt8 1.52 Abi3 1.52 NM_008235.2 Hes1 1.51 NM_009970.1 Csf2ra 1.51 NM_145619.2 Parp3 1.51 NM_010193.3 Fem1b 1.51 NM_199035.1 Alg8 1.50 NM_027959.3 Pdia6 1.50 NM_013538.3 Cdca3 1.50 NM_028817.2 Acsl3 1.50 NM_178781.2 9330186A19Rik 1.50

As compared to the _L 48h B cell gene expression in the low 48h _H B cells Reference sequence ID sign 48h B _H /
48h B _L ratio NM_010501.1 Ifit3 -14.00 NM_021384.3 Rsad2 -8.07 NM_010501.2 Ifit3 -6.81 XM_001480084.1 LOC667370 -6.79 NM_016850.2 Irf7 -6.59 NM_008332.2 Ifit2 -5.48 NM_011407.1 Slfn1 -5.18 NM_021274.1 Cxcl10 -5.17 NM_008332.2 Ifit2 -5.03 NM_009843.3 Ctla4 -4.42 NM_015811.1 Rgs1 -4.22 NM_011854.1 Oasl2 -4.20 NM_008372.3 Il7r -4.13 NM_011909.1 Usp18 -4.11 NM_010846.1 Mx1 -4.00 XM_001480051.1 LOC100048346 -3.99 Mx2 -3.82 NM_020557.3 Tyki -3.79 NM_008218.2 Hba-a1 -3.27 NM_011150.2 Lgals3bp -3.21 NM_025378.2 Ifitm3 -3.15 NM_011909.1 Usp18 -3.15 NM_007687.2 Cfl1 -3.08 NM_145227.1 Oas2 -3.03 NM_001033335.2 Serpina3f -2.92 NM_015811.1 Rgs1 -2.88 NM_010330.3 Emb -2.88 NM_133200.3 P2ry14 -2.85 NM_007705.2 Cirbp -2.75 NM_030150.2 Dhx58 -2.75 NM_010185.2 Fcer1g -2.72 NM_008337.1 Ifng -2.63 XM_001471776.1 LOC100038894 -2.58 NM_015811.1 Rgs1 -2.53 NM_001004174.1 AA467197 -2.50 NM_010649.2 Klra4 -2.49 NM_001033468.1 Gpr114 -2.44 NM_024253.4 Nkg7 -2.42 NM_172275.1 Trafd1 -2.41 NM_013487.1 Cd3d -2.39 NM_018738.3 Igtp -2.35 NM_021430.2 Rilpl1 -2.30 NM_010654.2 Klrd1 -2.29 NM_009696.2 Apoe -2.29 NM_010169.3 F2r -2.28 NM_010689.2 Lat -2.25 NM_001025571.1 Sh2d2a -2.23 NM_009852.3 Cd6 -2.19 NM_009852.3 Cd6 -2.18 NM_021301.1 Slc15a2 -2.18 NM_010260.1 Gbp2 -2.17 NM_145209.2 Oasl1 -2.15 NM_012046.2 Spo11 -2.14 NM_009858.2 Cd8b1 -2.14 NM_007799.2 Ctse -2.13 NM_010638.4 Klf9 -2.13 NM_011068.1 Pex11a -2.12 NM_145209.3 Oasl1 -2.09 NM_199015.2 D14Ertd668e -2.09 NM_029005.1 Mlkl -2.09 NM_010583.2 Itk -2.09 NM_011454.1 Serpinb6b -2.08 NM_001033122.3 Cd69 -2.08 NM_011815.1 Fyb -2.07 NM_008326.1 Irgm1 -2.07 NM_010071.2 Dok2 -2.06 XM_001479238.1 LOC100047963 -2.06 NM_019440.2 Iigp2 -2.05 NM_133888.2 Smpdl3b -2.04 NM_008365.1 Il18r1 -2.04 NM_009985.3 Ctsw -2.03 NM_001008548.2 Pde2a -2.03 NM_007498.2 Atf3 -2.02 NM_009382.3 Thy1 -2.01 NM_178404.2 Zc3h6 -2.01 NM_001039160.2 Gvin1 -2.01 NM_178149.4 Pic3ip1 -2.01 NM_177155.3 Klri2 -2.00

As compared with CD23 ^lo 48h _H B cell gene expression and in the 48h CD23 ^hi B cell _H Reference sequence ID sign 48h CD23 ^hi B _H /
48h CD23 ^lo B _H ratio NM_013517.1 Fcer2a 4.99 NM_013517.3 Fcer2a 3.43 NM_013517.1 Fcer2a 2.76 NM_173372.1 Grm6 2.75 NM_212449.1 AU019823 2.34 NM_010807.3 Marcksl1 2.34 NM_007535.2 Bcl2a1c 2.28 NM_007536.2 Bcl2a1d 2.17 NM_054070.1 Afg3l1 2.09 NM_009871.2 Cdk5r1 2.01 NM_170702.2 Cd40 1.94 NM_028244.1 Rrp1b 1.93 NM_054070.2 Afg3l1 1.91 NM_013730.4 Slamf1 1.86 NM_028190.2 Luc7l 1.80 NM_170702.2 Cd40 1.74 NM_033608.2 Igsf9 1.73 NM_001048207.1 Gypc 1.73 NM_007573.2 C1qbp 1.72 NM_008321.1 Id3 1.72 NM_010215.2 Il4i1 1.71 NM_010259.2 Gbp1 1.69 NM_009856.1 Cd83 1.67 NM_008652.2 Mybl2 1.67 NM_170702.2 Cd40 1.65 NM_134126.2 Ift140 1.64 NM_028965.3 Snx11 1.58 NM_009302.2 Swap70 1.53

48h &_lt; / _RTI > CD23 ^hi B _H cells compared to 48h CD23 ^lo B _H cells Reference sequence ID sign 48h CD23 ^hi B _H /
48h CD23 ^lo B _H ratio NM_025427.2 1190002H23Rik -7.99 NM_010800.3 Bhlhb8 -7.72 NM_010800.2 Mist1 -7.60 NM_024169.3 Fkbp11 -7.26 NM_021415.3 Cacna1h -6.14 NM_025290.3 Rsph1 -5.82 NM_178446.2 Rbm47 -5.28 NM_011414.2 Slpi -5.19 NM_010800.3 Bhlhb8 -4.84 NM_010741.2 Ly6c1 -4.81 NM_015786.1 Hist1h1c -4.79 NM_009375.2 Tg -4.74 NM_014194.1 Klra7 -4.71 NM_178661.2 Creb3l2 -4.64 NM_008920.4 Prg2 -4.48 NM_029720.1 Creld2 -4.33 NM_024253.4 Nkg7 -4.21 NM_007585.3 Anxa2 -4.07 NM_029720.2 Creld2 -3.94 NM_145367.3 Txndc5 -3.84 NM_026432.2 Tmem66 -3.83 NM_178114.3 Amigo2 -3.67 NM_010649.2 Klra4 -3.65 XM_001471776.1 LOC100038894 -3.61 NM_027650.1 Speer3 -3.58 NM_024440.2 Derl3 -3.46 NM_028000.1 Ppapdc1b -3.43 NM_021273.3 Ckb -3.42 NM_009382.3 Thy1 -3.37 NM_172574.1 Pqlc3 -3.33 NM_010654.2 Klrd1 -3.28 NM_013842.2 Xbp1 -3.24 NM_153590.3 Klre1 -3.22 NM_008495.1 Lgals1 -3.12 NM_027222.2 2010001M09Rik -3.11 NM_009985.3 Ctsw -3.11 NM_018767.2 Cd160 -3.05 NM_013653.2 Ccl5 -2.98 NM_001039089.1 Sel1l -2.86 NM_176933.4 Dusp4 -2.85 NM_007706.3 Socs2 -2.81 NM_013652.2 Ccl4 -2.78 NM_007657.2 Cd9 -2.71 NM_001005846.2 Mcoln2 -2.64 NM_021897.1 Trp53inp1 -2.54 NM_025448.2 Ssr2 -2.53 NM_011261.2 Reln -2.52 NM_023716.2 Tubb2b -2.51 NM_007431.1 Akp2 -2.48 NM_010169.3 F2r -2.41 NM_010376.3 H13 -2.41 NM_009858.2 Cd8b1 -2.39 NM_133626.2 Rrbp1 -2.38 NM_172574.1 Pqlc3 -2.37 NM_009151.2 Selplg -2.36 NM_010376.3 H13 -2.36 NM_145537.1 Edem2 -2.32 NM_013863.4 Bag3 -2.24 NM_001081212.1 Irs2 -2.23 NM_027881.1 Osbpl3 -2.21 NM_001033126.2 Cd27 -2.19 NM_024444.1 Cyp4f18 -2.16 NM_009696.2 Apoe -2.16 NM_144525.2 1110039B18Rik -2.16 NM_010173.3 Faah -2.16 XM_001472253.1 LOC100044439 -2.10 NM_007421.1 Adssl1 -2.08 NM_016969.1 Myadm -2.04

Example  18. LPS In the sepsis-induced sepsis model, sodium Taurodeoxycholate After treatment Increased CD23 ^hi B _H  Quantum cells I had to  Cell proliferation when growing

In Example 17, the increase of CD11b ⁺ Gr-1 ⁺ cells and immunoregulatory B cells was confirmed when CD23 ^hi immunoregulatory B _H cells were transplanted (Fig. 16). The number of recipient mice used in the above example was 3 PBS group, CD23 ^lo immunoregulatory B _H cell group and CD23 ^hi Immune regulation B _H And 3 x 10 ⁵ transduced cells were used in the cell group. When CD23 ^hi B _H cells were quantitatively transferred from B cells and CD11b ⁺ Gr-1 ⁺ cells at 24 hours and 48 hours after LPS administration, significant quantities of CD23 ^lo B _H cells Respectively.

Example  19. LPS In the sepsis-induced sepsis model, sodium Taurodeoxycholate After treatment Increased  H- MDSC  Quantum cells I had to  B cell subtype analysis

14A, when the L-MDSC and H-MDSC prepared as described in Example 12 were quantitatively transferred, the CD23 high portion was significantly increased in the B cells at 24 hours after the H-MDSC was quantitated. The FACS plot image Respectively. FIG. 14B shows that when the H-MDSC is quantitated, the CD23 high region is significantly increased in B cells than in the case of L-MDSC.

Example  20. LPS And sodium Taurodeoxycholate  using In vitro  H- MDSC  Analysis of the effects of the manufacture and sepsis treatment of these cells

Cells were harvested from the femur and tibia bone marrow at sacrifice of normal C57BL / 6 mice between 8 and 12 weeks, and cells (1 x 10 ⁶ / ml) were suspended in Cellgro media (CellGenix, Germany) . 1 × 10 ⁶ / well of cells were mixed with 1 mg / ml of LPS and 50 mg / ml of HY2191 and cultured for 24 hours.

The degree of CD11b ⁺ Gr-1 ⁺ MDSC differentiation was compared between MDSC (LexMDSC) treated with LPS alone in bone marrow cells and MDSC (HexMDSC) treated with LPS and HY2191 at the same time in vitro in bone marrow cells . Flow cytometry (FASC) confirmed that the absolute numbers of CD11b ⁺ Gr-1 ⁺ cells in the HY2191 treated group were increased by 36% and 34%, respectively, when cultured for 24 hours (FIG. 17A). LexMDSC and HexMDSC that had been cultured for 24 hours were intravenously transduced into C57BL / 6 mice injected with LPS 10 mg / kg 24 hours before. Of these, only mice injected with HexMDSC survived, and all mice treated with PBS alone, LexMDSC, and Media were killed (FIG. 17B).

Claims (7)

The expression of Gr-1 ⁺ CD11b ⁺ surface plasmids and the anti-inflammatory activity of TLR (toll-like recetor) signaling inducers and GPCR19 agonist-treated transformed or amplified or TLR signaling inducers and GPCR19 agonists Regulatory B cells transformed or amplified by treatment with MDSC (Myeloid-Derive Suppressor Cell) having the CD23 ^hi surface pattern and having anti-inflammatory activity, wherein said immunoregulatory B cells express CD1d Immunoregulatory B cells with low expression of Cd69 and Cd27 genes.
The method of claim 1, wherein the immunoregulatory B cells are selected from the group consisting of Fcer2a, Grm6, AU019823, Marcksl1, Bcl2a1c, Bcl2a1d, Afg3l1, Cdk5r1, Cd40, Rrp1b, Slamf1, Luc7l, Igsf9, Gypc, C1qbp, Id3, Il4i1, Gbp1, Hfbb-bhl, Hspa1a, Srpk3, Hhex, Insigl, Cbfa2t3h, Taf15, Ddx17, Cnn2, Thb1, Thb2, Myf2, Ift140, Snx11, Swap70, Ccbp2, Sc4mol, Cyp51, Sqle, LOC100040592, B3gnt8, 1300014I06Rik, Fdps, Ctsa, Dmwd, Sfrs7, Rd1, Cr2, Arhgap4, Pira3, Flna, Cdk5rap1, Cugbp1, Ankrd37, Cd44, Mrps7, Mapk11, Pycard, Cap1, Lta, Rbm4, Cd180, Cfp, Cmah, LOC100046855, Srebf1, Ciita, Ryr1, E2f2, Dap3, Wdr9, The present invention provides a method of screening for a compound capable of inhibiting the expression of Ccr6, Pcbd2, Lmnb1, Pira4, Blvrb or Actb gene, Cdl6, Cdd6, Cdd6, Cdd6, Cdd6, Cdd6, Cdd6, Tmd6, Tmem66, Amigo2, Klra4, LOC100038894, Speer3, Derl3, Ppapdc1b, Ckb, Thy1, Pqlc3, Klrd1, Xbp1, Klre1, Lgals1, 2010001M09Rik, Mcoln2, Trp53inp1, Ssr2, Reln, Tubb2b, A Hf-a1, Lgal3bp, Ifit3, Cf1, Cp1, Cp1, Cp1, Cp1, Cp1, Cp1, Cp1, Cpd11, Cpd11, Cd3d, Igtp, Rilpl1, Apoe, Lat, Sh2d2a, Cd6, Slc15a2, Gbp2, Oasl1, Spo11, Ctse, Klf9, Pex11a, Gp11a, Og2, Serpina3f, Emb, P2ry14, Cirbp, Dhx58, Fcer1g, Ifng, AA467197, Gpr114, Immunoregulatory B cells characterized by low expression of D14Ertd668e, Mlkl, Itk or Serpinb6b genes.
2. The immunomodulatory B cell of claim 1, wherein said immunoregulatory B cells express CD21 and CD23.
2. The immunomodulatory B cell according to claim 1, wherein the TLR signaling inducer is a bacterial derived factor, a fungal derived factor, a viral derived factor, a self-derived factor, an allergen or a cancer cell-derived factor.
5. The immunoregulatory B cell according to claim 4, wherein the TLR signaling inducing factor is a lipopolysaccharide (LPS) or a peptidoglycan as a bacteria-derived factor.
The immunoregulatory B cell according to claim 1, wherein the GPCR19 agonist is a compound of the following formula 1, a salt thereof or a hydrate thereof:
Formula 1

Wherein R ₁ is hydrogen, hydroxy, C _1-5 linear or branched alkyl or halogen; R ₂ is hydrogen or? -Hydroxy; R ₃ is selected from the group consisting of hydroxy, NH (CH ₂ ) _m SO ₃ H where m is an integer 0, 1, 2, 3, 4 or 5 or NH (CH ₂ ) _n CO ₂ H , 3, 4 or 5); R ₄ and R ₅ is hydrogen; R ₆ is a ring formed of 3, 4, 5 or 6 atoms in size with a hydrogen or R ₅ and R ₆ attached to carbon; R < ₇ > is hydrogen or hydroxy; R ₈ , R ₉ and R ₁₀ are C _1-3 linear or branched alkyl; And R < ₁₁ > is hydrogen.
The use of an immunomodulatory B cell according to any one of claims 1 to 6 as an active ingredient for the treatment of sepsis, autoimmune disease, inflammatory disease, rheumatoid arthritis, atopic dermatitis, Alzheimer's disease, ulcerative colitis, Prevention of any inflammatory disease selected from the group consisting of Crohn's disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, Castleman's disease, autoimmune hepatitis, autoimmune uveitis, myasthenia gravis, alopecia areata and systemic lupus erythematosus Or a pharmaceutical composition for therapeutic use.

Images