CN109420174B

CN109420174B - Application of GPR18 and regulator thereof in preventing and treating immune system diseases

Info

Publication number: CN109420174B
Application number: CN201710771705.7A
Authority: CN
Inventors: 石彦
Original assignee: Tsinghua University
Current assignee: Bodihekang (Beijing) Biotechnology Co.,Ltd.
Priority date: 2017-08-31
Filing date: 2017-08-31
Publication date: 2021-07-13
Anticipated expiration: 2037-08-31
Also published as: WO2019041599A1; CN109420174A

Abstract

The invention discloses a GPR18 and application of a regulator thereof in preventing and treating immune system diseases. The invention provides the use of a modulator of GPR18 or GPR18 in any of the following: preparing products for preventing and/or treating diseases related to immune disorder and preparing products for regulating functions of peripheral immune system. The GPR18 modulators are substances capable of promoting or inhibiting GPR18 expression, or substances capable of increasing or decreasing GPR18 activity. Experiments have shown that the inhibitor O-1918 of the cannabinoid receptor GPR18 is able to inhibit metastasis of gut-derived dendritic cells (expressing CD103, CD11b and retinaldehyde dehydrogenase) to lymph nodes. The invention has important significance for preventing and/or treating diseases related to immune disorder, such as rheumatism, lupus erythematosus, enteritis and multiple sclerosis, and enhancing the immune function of the organism.

Description

Application of GPR18 and regulator thereof in preventing and treating immune system diseases

Technical Field

The invention belongs to the technical field of biology, and relates to GPR18 and application of a regulator thereof in preventing and treating immune system diseases.

Background

Microorganisms enter the animal body from skin, oral cavity, respiratory tract, etc., and they evolve together with the host to influence each other and inhibit and regulate the functions of the other. The research on the regulation and control mechanism between the symbiotic bacteria and the host has great significance to human health. Of the most concern to the immunologist is the effect of the gut flora on host metabolism and infection resistance. One of the issues is how the symbiotic bacteria regulate host immune function. Most literature studies focus on local immune regulation in the gut. This may be in the iceberg corner alone, and the effect of the commensal bacteria on the immune system may not be more than sufficient in the gut. For example, sterile animals have significant peripheral immunodeficiency, manifested primarily by hypoplasia of the systemic secondary lymphoid organs (i.e., lymph nodes). Since lymph nodes are the origin of immune responses, how symbionts regulate these widely distributed immune organs remotely has also become an important issue. However, to date, the association of these commensals with the development of the peripheral immune system has not been elucidated.

Lymph node development is a delicate and complex process. At the time of the embryo, the primordia of the lymph nodes appear in epithelial cell clusters. Upon stimulation with retinoic acid from nearby nerve endings, lymphoid tissue-inducing cells (LTi) begin to initiate the development of primitive lymphoid structures. After the mouse is born, LTi cells no longer remain, while peripheral lymph nodes continue to become larger and contain an increasing number of cells. One to two weeks after birth, the diafiltered lymphocytes form distinct T and B cell zones, almost identical to the lymph nodes of adult mice. In contrast, lymph node development in sterile animals was completely halted after birth. The developmental problems described above have led to a deficiency in the immune response. The immune response of the sterile mouse is weak after the sterile mouse is infected by intestinal pathogenic bacteria Shigella flexneri. Symptoms of salmonella infection are also more severe in germ-free mice. As a major site of immune response, it is conceivable that structural defects in lymph nodes may lead to a disturbance in the immune response. Then how do intestinal colonies initiate lymph node development after birth?

Disclosure of Invention

The invention aims to provide GPR18 and application of a regulator thereof in preventing and treating immune system diseases.

The application provided by the invention is specifically as follows:

firstly, the method comprises the following steps: use of a modulator of GPR18 or GPR18 in any one of (a) to (D) below:

(A) preparing a product for preventing and/or treating diseases related to immune disorders;

(B) preparing a product for modulating peripheral immune system function;

(C) preventing and/or treating diseases related to immune disorder;

(D) modulating peripheral immune system function.

Wherein, the regulator of GPR18 is a substance capable of promoting or inhibiting GPR18 expression or a substance capable of increasing or decreasing GPR18 activity.

Secondly, the method comprises the following steps: use of GPR18 or an agent capable of promoting GPR18 expression or capable of increasing GPR18 activity in any one of the following (E) to (H):

(E) preparing a product for driving the migration of gut-derived dendritic cells to lymph nodes;

(F) preparing a product for promoting lymph node development and/or lymph node function;

(G) driving gut-derived dendritic cells to metastasize to lymph nodes;

(H) promote lymph node development and/or promote lymph node function.

Thirdly, the method comprises the following steps: use of an agent capable of inhibiting GPR18 expression or capable of reducing GPR18 activity in any one of (E ') - (H') as follows:

(E') preparing a product for inhibiting the metastasis of gut-derived dendritic cells to lymph nodes;

(F') preparing a product for inhibiting lymph node development and/or inhibiting lymph node function;

(G') inhibiting metastasis of gut-derived dendritic cells to lymph nodes;

(H') inhibiting lymph node development and/or lymph node function.

Substances that inhibit GPR18 expression or that reduce GPR18 activity reduce immune activation and suppress autoimmune responses by inhibiting lymph node function.

The invention also claims a product having at least one of the functions shown in the following (a) to (d), wherein the active ingredient is GPR18 or a substance capable of promoting GPR18 expression or improving GPR18 activity; (a) preventing and/or treating diseases related to immune disorder; (b) modulating peripheral immune system function; (c) driving gut-derived dendritic cells to metastasize to lymph nodes; (d) promote lymph node development and/or promote lymph node function.

The invention also claims a product having at least one of the functions shown in (a ') - (d') as active ingredient, which is a substance capable of inhibiting the expression of GPR18 or reducing GPR18 activity; (a') the prevention and/or treatment of diseases associated with immune disorders; (b') modulating peripheral immune system function; (c') inhibiting migration of gut-derived dendritic cells to lymph nodes; (d') inhibiting lymph node development and/or inhibiting lymph node function.

In the present invention, all the immune disorder-related diseases mentioned above can be autoimmune diseases such as rheumatism, lupus erythematosus, enteritis, multiple sclerosis, ankylosing spondylitis and the like, and excessive immune inflammatory responses.

In the present invention, all of the above-mentioned gut-derived dendritic cells are specifically gut-derived dendritic cells expressing CD103, CD11b and retinal dehydrogenase.

In one embodiment of the present invention, all of the above driving/inhibiting the migration of gut-derived dendritic cells to lymph nodes is specifically driving/inhibiting the migration of gut-derived dendritic cells to mesenteric lymph nodes and/or non-mesenteric peripheral lymph nodes.

In the present invention, all substances described hereinbefore which are capable of inhibiting the expression of GPR18 or of reducing GPR18 activity are GPR18 inhibitors, such as O-1918.

Experiments have shown that the inhibitor O-1918 of the cannabinoid receptor GPR18 is able to inhibit metastasis of gut-derived dendritic cells (expressing CD103, CD11b and retinaldehyde dehydrogenase) to lymph nodes. Therefore, in practical application, the driver and antagonist of GPR18 (endogenous substances of intestinal fungus can be synthesized or used by a chemical method) can be taken and injected in the oral cavity to adjust the intensity and type of immune response of the organism and the number and function of each immune cell subtype, so as to achieve the aims of treating and/or preventing diseases related to immune disorder, such as rheumatism, lupus erythematosus, enteritis and multiple sclerosis, and enhancing immune function (such as immunotherapy and vaccination).

Drawings

FIG. 1 shows lymphocytes isolated from lymph nodes of adult normal mice, which are labeled and injected into normal (SPF) or sterile (GF) mice via the tail vein.

FIG. 2 shows lymph nodes of adult sterile mice lacking RALDH-positive DC cells.

FIG. 3 shows that the intestinal microorganisms are produced by the enzyme retinaldehyde dehydrogenase RALDH⁺Dendritic cells regulate lymph node development.

FIG. 4 shows CD103⁺CD11b⁺RALDH⁺Analysis of DC-like cell sources.

FIG. 5 shows intestinal CD103 driven by Candida tropicalis⁺CD11b⁺RALDH⁺The dendritic cells metastasize to lymph nodes. In the figure, RALDH at the ordinate⁺CD103⁺I.e. to CD103⁺CD11b⁺RALDH⁺A dendritic cell.

FIG. 6 shows that lipid extracts of Candida tropicalis are able to drive BMDC metastasis to lymph nodes.

FIG. 7 shows the results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the ninth fragment obtained by liquid chromatography of lipid extract of Candida tropicalis.

Figure 8 shows that some molecular structures in the ninth paragraph (upper, middle) are very close to endocannabinoids (lower, AEA).

FIG. 9 shows that AEA and fungal fat extracts are able to strongly drive CD103 compared to other endocannabinoids⁺CD11b⁺RALDH⁺Dendritic cell migration.

FIG. 10 shows that inhibitors of GPR55 and GPR18, O-1918, inhibit CD103⁺CD11b⁺RALDH⁺Migration of dendritic cells.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Inhibitor of GPR 18O-1918: product number 2288 from Tocris corporation.

Example 1 inhibitor of cannabinoid receptor GPR 18O-1918 inhibits metastasis of intestinal DC cells to lymph nodes

First, lymphocyte to lymph node reflux in sterile mice were deficient

Lymphocytes isolated from lymph nodes of adult normal C57BL/6 mice, labeled with CFSE, were injected into normal (SPF) or sterile (GF) C57BL/6 mice (at a dose of 2X 10 per mouse) via tail vein⁶Cell) 24 hours later, separate the various types of stranguriaBarns, fluorescence imaging after sectioning, or analysis by flow cytometry after cell suspension was taken.

The results are shown in FIG. 1. Left: reflux of injected lymphocytes to Inguinal Lymph Node (iLN), mesenteric lymph node (mLN) and spleen (spl), and analysis of lymphocyte subset reflux to iLN (bottom left). And (3) right: expression of MAdCAM-1 and PNAd on High Endothelial Venules (HEVs) within iLN in GF mice (converted) after caging with SPF, and SPF. This experiment showed that lymphocyte reflux to the lymph nodes was deficient in germ free mice due to the absence of expression of PNAd.

II, adult sterile mouse lymph node deletion RALDH positive DC cells

Lymph nodes from 5-week normal (SPF) and sterile (GF) C57BL/6 mice were stained and compared to see the presence or absence of RALDH-positive DC cells in both.

The results are shown in FIG. 2. The method comprises the following steps: adult sterile mice have a depletion in their lymph nodes of a class of dendritic cells of retinal dehydrogenase (RALDH) compared to normal mice. After the SPF mice are bred in cages, RALDH positive cells appear in peripheral lymph nodes of the sterile mice. The following: and (4) comprehensive data analysis.

Thirdly, the intestinal microorganisms are obtained by RALDH⁺Dendritic cells regulate lymph node development

Isolation of RALDH from lymph nodes of SPF grade C57BL/6 mice⁺Dendritic cells and RALDH^-Dendritic cells were injected via tail vein into sterile (GF) C57BL/6 mice (injection dose 2X 10)⁵Cells), the lymph nodes were sectioned seven days later and analyzed by flow cytometry.

The results are shown in FIG. 3. The method comprises the following steps: RALDH isolated from lymph nodes of SPF mice⁺Dendritic cells or RALDH^-Dendritic cells, injected into sterile mice via the tail vein, cause changes in lymph node volume and imaging of mature T, B regions. The following: lymph node size, and statistics of the areas of zone B and zone T. This experiment demonstrated that intestinal microorganisms were produced by RALDH⁺This class of dendritic cells regulates lymph node development.

IV, CD103⁺CD11b⁺RALDH⁺Analysis of DC-like cell origin

The effect of gut symbionts on immune organ development may not be direct. Within the lamina propria of the intestinal tract is a group of unconventional dendritic cells which are capable of expressing CD103, CD11b and retinal dehydrogenase (RALDH). They can come into contact with lymphocytes, and this process has a great influence on the fate of these lymphocytes. Apparently, there appears to be no connection between lymph node development and the above processes. Earlier stage work in our laboratory found that when the lymphocyte induced cells of the newborn mice disappear, if intestinal colonies appear in time, a CD103 will appear in lymph nodes⁺CD11b⁺DC-like cells. These cells isolated from normal mice, when injected into sterile mice via tail vein, promote the development of the latter lymph nodes and the entry of a large number of lymphocytes into the lymph nodes. These dendritic cells highly express retinal dehydrogenase and are capable of expressing a substance called peripheral lymphokine on the vascular epithelium at the entrance to the lymph node, which can also be considered as a marker for address localization. T and B cells develop lymph nodes by recognizing peripheral lymphokines entering the lymph nodes. This process is particularly evident at the time of birth of the mice. However, in adult mice, the lymph nodes still have a small number of such dendritic cells, which maintain long-term lymph node homeostasis. In vitamin a deficient mice, dendritic cells such as lymph nodes disappear, causing structural destruction. We have shown by means of a marker that such dendritic cells originate from the gut, and that the unconventional dendritic cells in the lamina propria of the gut (which express CD103, CD11b and retinal dehydrogenase) are of the same type as we have said above.

The specific method comprises the following steps: neonatal C57BL/6 mice were gavaged with FITC Dextran (FITC-Dextran 2000KD (Sigma)0.3 mg per g body weight) and after 6 hours the proportion of CD11b and CD103 single positive, Double Negative (DN) and Double Positive (DP) dendritic cells in peripheral lymph nodes was determined by flow cytometry.

As shown in FIG. 4, the FITC signal was strongest in CD11b and CD103 double positive dendritic cells, which were found to originate from the intestinal tract.

Fifth, candida tropicalis drives intestinal CD103⁺CD11b⁺RALDH⁺Metastasis of dendritic cells to lymph nodes

Adult SPF grade C57BL/6 mice were mixed with mixed antibiotics (antibacterial only and antifungal, 1g/L ampicillin, 1g/L neomycin, 1g/L metronidazole and 0.5g/L vancomycin, concentrations expressed as final concentrations in drinking water, Sigma) or the antifungal agent fluconazole (Sigma) (final concentrations in drinking water of 0.5g/L, Sigma) in drinking water and three weeks later were followed by flow cytometry of lymph nodes to analyze mesenteric lymph nodes (mLN) and CD103 in Peyer's Patches (PP) of mice⁺CD11b⁺RALDH⁺Percentage of dendritic cells.

Adult normal C57BL/6 mice were individually gavaged with three major intestinal fungi cultured, Candida tropicalis, Saccharomyces cerevisiae and Trichosporon trichotomum (10 per strain per mouse)⁸CFU)24 hours later, the lymph nodes were taken for flow cytometry analysis of CD103 in the mouse Inguinal (iLN) and mesenteric (mLN) lymph nodes⁺CD11b⁺RALDH⁺Percentage of dendritic cells. In the same manner, the experiment was performed on newborn C57BL/6 mice and sterile C57BL/6 mice.

The results are shown in FIG. 5. Upper left: adult SPF mice were mixed with a mixture of antibiotic or antifungal fluconazole (fluconazole) in drinking water, and three weeks later, mesenteric lymph nodes (mLN) and Peyer's Patch (PP) with CD103⁺CD11b⁺RALDH⁺Percentage of dendritic cells. Upper right: adult normal mice were CD103 in Inguinal (iLN) and mesenteric (mLN) lymph nodes after 24 hours of gavage with cultured Candida tropicalis (Candida tropicalis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Trichosporon (Trichosporon)⁺CD11b⁺RALDH⁺Percentage of dendritic cells. Left lower: similar to the upper right, results for newborn mice. Right lower: similar to the results for the upper right and lower left, adult germ-free mice. It can be seen that Candida tropicalis (C tropicalis) in intestinal microorganisms drives CD103⁺CD11b⁺RALDH⁺Metastasis of dendritic cells to lymph nodes.

Lipid extract of Candida tropicalis capable of driving intestinal CD103⁺CD11b⁺RALDH⁺Metastasis of dendritic cells to lymph nodes

Ribonucleic acid, protein and lipid of fungi and bacteria (the fungi include Candida tropicalis, Saccharomyces cerevisiae and Trichosporon trichomonad, and the bacteria are Escherichia coli) are obtained by separation and extraction. After reinjection of these isolates into mice, only the Candida tropicalis lipids were able to drive the transfer of dendritic cells to lymph nodes. The method comprises the following specific steps:

the chloroform-methanol extraction method is used for extracting lipids of fungi and bacteria, wherein the fungi include Candida tropicalis (Candida tropicalis), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Trichosporon (Trichosporon), and the bacteria include Escherichia coli (E coli). Ultrasonically crushing fungi or bacteria in chloroform-methanol (volume ratio of 2:1), centrifuging, washing with water to remove methanol, and blow-drying chloroform with nitrogen at low temperature to obtain lipid extract.

The lipid extracts of the fungi and bacteria obtained above were subjected to liquid chromatography analysis, as follows: a chromatographic column: the diameter is 1.5cm, and the length is 30 cm; the filler is silica gel (200-300 mesh, particle size 45-75 μm, pore diameter 40-70A, specific surface area 400-600 m)²Per gram, pore volume 0.60-0.85 ml/gram), filled at atmospheric pressure. Lipids extracted from 5g (wet weight) of fungi or bacteria were loaded with 100% chloroform (30ml) and then treated with chloroform: methanol was eluted at 10:0, 8:2, 6:4, 5:5, 3:7, 0:10 volumes, 30ml each for the first 5 gradients, each divided equally into two front and back sections, and 45ml for the last gradient (i.e. gradient No. 6), divided equally into three sections. Thereby forming 1 to 13 paragraphs. At room temperature, the samples were allowed to fall freely and all samples were blown dry with nitrogen.

The lipid extract was separated into fractions by liquid phase separation, each fraction was dissolved in the same volume of DMSO as the total non-fractionated lipids, and subjected to Transwell assay (lipid was added to the lower chamber, and the ratio of lipid to culture solution was 1:1000 to determine the activity of each fraction to attract migration of mouse bone marrow-derived dendritic cells (BMDC) (the chemotaxis of BMDC and CD10 are clear to those skilled in the art)3⁺CD11b⁺RALDH⁺Dendritic cells were similar, and BMDC was used in place of CD103 in this experiment⁺CD11b⁺RALDH⁺The reason for dendritic cells is that: the latter is difficult to obtain sufficient cell number for mass analysis, and moreover, the latter is not very active in culture and is not suitable for long-term experiments).

The results are shown in FIG. 6, A: liquid chromatography spectra of various fungal lipids; b: several segments of tropical yeast induce the migration of dendritic cells in vitro, the ninth segment being the strongest. The relevant activities in the lipids of visible fungi can be concentrated and purified by the liquid phase.

Seventhly, structure determination of endocannabinoid AEA analogue from candida tropicalis

And (3) separating the C tropicalis lipid extract in a liquid phase for a ninth section, and performing liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and performing automatic analysis (Full Scan mode) on data of a Waters QTOF mass spectrometer by Thermal X-caliber software to obtain the molecular composition of the lipid.

The results are shown in FIGS. 7 and 8.

In fig. 7, upper: c tropicalis lipid extract liquid phase separation ninth section liquid chromatography analysis; liquid chromatography results of N-arachidonic acid aminoethanol (AEA) standard. The following: AEA mass spectrometry results; c tropicalis lipid extract liquid phase separation of AEA homologues extracted from the LC-MS/MS results in the ninth stage.

In fig. 8, upper, middle: some molecular structures in the ninth paragraph (shown as formula I and formula II, respectively). The following: endocannabinoids (AEA is shown in formula III)) molecular structure.

The results show that: c tropicalis lipid extract the ninth stage contained the endogenous cannabinoid AEA homologue by liquid phase isolation. According to the LC-MS/MS results, the AEA homologue was closest to arachidonic acid N-propylamine (N-propyl arachidonoyl amine) or arachidonic acid isopropylamine (N-isopropyl arachidonoyl amine).

Eight, endocannabinoid AEA analogs from Candida tropicalis capable of driving intestinal CD103⁺CD11b⁺RALDH⁺Metastasis of dendritic cells to lymph nodes

Different endocannabinoids and C tropicalis lipid extracts were injected intra-abdominally into 4-week-old SPF grade C57BL/6 mice. DMSO stocks of C tropicalis lipid and cannabinoid were dissolved in PBS buffer for injection. DMSO at the same dose served as a blank. The treatment time was one hour. Flow cytometry detection of migratory DCs (i.e., CD 103) in mesenteric lymph nodes (mLNs)⁺CD11b⁺RALDH⁺Dendritic cells). The amount of C tropicalis lipid was 250. mu.g per mouse dissolved in DMSO fraction.

The results are shown in FIG. 9, where it can be seen that: lipid extracts of AEA and C tropicalis are able to drive CD103 more strongly than other endocannabinoids⁺CD11b⁺RALDH⁺Dendritic cell migration.

Nine, GPR18 inhibitor O-1918 can inhibit intestinal CD103⁺CD11b⁺RALDH⁺Metastasis of dendritic cells to lymph nodes

The lipid extract of capiciles was mixed with cannabinoid receptor inhibitors and dissolved in PBS buffer and injected intraperitoneally into large SPF grade C57BL/6 mice all four weeks old. One hour later, the mesenteric lymph node (mLN) of the mouse was stained and the migratory dendritic cell migratory DC (i.e., CD 103) was detected by flow cytometry⁺CD11b⁺RALDH⁺Dendritic cells). CD11c⁺MHCII^intresidual DC was used as control. The dose of C tropicalis lipid was identical to that described above (i.e. 250ug per mouse). Concentration of cannabinoid receptor inhibitor finally dissolved in PBS solution: otenanbant (CB1 inhibitor), AM630(CB2 inhibitor), O-1918(GPR55 and GPR18 inhibitor) were all 2 μ M; the final concentration of PSB-SB-487(GPR55 inhibitor) was 200 nM.

The results are shown in FIG. 10, where it can be seen that: inhibition of GPR18 (e.g., using GPR18 inhibitor O-1918) is effective in reducing the effects of C tropicalis lipids on CD103⁺CD11b⁺RALDH⁺Promotion of dendritic cell migration.

Claims

Use of the GPR18 inhibitor O-1918 for the manufacture of a product for inhibiting the metastasis of gut-derived dendritic cells to lymph nodes; the GPR18 inhibitor O-1918 is capable of inhibiting GPR18 expression or is capable of decreasing GPR18 activity.
2. Use according to claim 1, characterized in that: the gut-derived dendritic cells express CD103, CD11b, and retinal dehydrogenase.