Application of serotonin or its receptor agonists in the treatment of ILC2 cell-mediated immune diseases

Abstract

The present invention provides the use of serotonin or its receptor agonist in the treatment of ILC2 cell-mediated immune diseases. Specifically, the present invention provides the use of a substance for preparing a composition or preparation, wherein the composition or preparation is used to prevent and / or treat ILC2 cell-mediated immune diseases, wherein the substance is selected from the following group: serotonin, a serotonin receptor agonist, or a combination thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular, to the use of serotonin or a receptor agonist thereof in the treatment of ILC2 cell-mediated immune diseases. Background Art

[0002] Asthma is a common chronic airway inflammation involving multiple cells and cell components. This inflammation is often accompanied by increased airway reactivity, leading to recurrent symptoms such as wheezing, shortness of breath, chest tightness and / or coughing, which often occur at night and / or early in the morning. These symptoms are often accompanied by extensive and variable airflow obstruction, which can be reversed on its own or through treatment. About 300 million people worldwide suffer from asthma-related diseases, and the incidence of asthma has increased significantly in the past 50 years. Asthma is mainly divided into allergic asthma and non-allergic asthma. Due to the complexity of the causes of asthma and the individual differences among asthma patients, asthma treatment is very difficult. Currently, the commonly used drugs for the treatment of asthma are glucocorticoids and steroid hormones, which are only effective for the treatment of allergic asthma, but not for other types and mixed asthma patients. Therefore, it is urgent to control the occurrence and development of asthma and find more effective drugs to treat asthma.

[0003] However, little is known about the mechanisms that control ILC2 cell responses to promote homeostasis, especially the molecular pathways that prevent ILC2 activation in the context of allergic asthma.

[0004] Therefore, there is an urgent need in the art to develop a drug that can more effectively treat ILC2 cell-mediated immune diseases. Summary of the invention

[0005] The object of the present invention is to provide a drug that can more effectively treat ILC2 cell-mediated immune diseases.

[0006] A first aspect of the present invention provides a use of a substance for preparing a composition or preparation for preventing and / or treating an ILC2 cell-mediated immune disease, wherein the substance is selected from the group consisting of serotonin, a serotonin receptor agonist, or a combination thereof.

[0007] In another preferred embodiment, the ILC2 cell-mediated immune disease includes an allergic disease.

[0008] In another preferred embodiment, the allergic disease is selected from the following group: pulmonary allergic disease, skin allergic disease, intestinal allergic disease, or a combination thereof.

[0009] In another preferred embodiment, the allergic disease is selected from the group consisting of allergic asthma, allergic dermatitis, eczema, food allergy, or a combination thereof.

[0010] In another preferred embodiment, the ILC2 cell-mediated immune disease is selected from the following group: pulmonary allergic diseases, skin allergic diseases, intestinal allergic diseases, allergic asthma, eczema, food allergy, pulmonary fibrosis, chronic obstructive pulmonary disease, allergic dermatitis, enteritis, or a combination thereof.

[0011] In another preferred embodiment, the serotonin receptor is selected from the group consisting of 5-HTR2A, 5-HTR1B, 5-HTR1A, 5-HTR1F, or a combination thereof.

[0012] In another preferred embodiment, the serotonin receptor includes 5-HTR2A.

[0013] In another preferred embodiment, the serotonin receptor agonist is a substance that promotes the expression or activity of serotonin receptors.

[0014] In another preferred embodiment, the promoting the expression or activity of serotonin receptors refers to increasing the expression or activity of serotonin receptor genes or proteins by ≥10%, preferably ≥20%, and more preferably ≥70%.

[0015] In another preferred embodiment, the serotonin receptor agonist is selected from the following group: a small molecule compound, a vector expressing a serotonin receptor, or a combination thereof.

[0016] In another preferred embodiment, the vector expressing the serotonin receptor comprises a viral vector.

[0017] In another preferred embodiment, the viral vector is selected from the following group: an adeno-associated viral vector, a lentiviral vector, or a combination thereof.

[0018] In another preferred embodiment, the serotonin receptor agonist is selected from the following group: DOI, psilocybin, mescaline, 25I-NBOMe (25-NB derivative), or a combination thereof.

[0019] In another preferred embodiment, the serotonin receptor comprises a full-length protein or a protein fragment.

[0020] In another preferred embodiment, the serotonin receptor is derived from mammals, more preferably rodents (such as mice, rats), primates and humans.

[0021] In another preferred embodiment, the serotonin receptor also includes a derivative of the serotonin receptor.

[0022] In another preferred embodiment, the derivatives of the serotonin receptor include modified serotonin receptors, protein molecules whose amino acid sequences are homologous to natural serotonin receptors and have natural serotonin receptor activity, dimers or polymers of serotonin receptors, and fusion proteins containing serotonin receptor amino acid sequences.

[0023] In another preferred embodiment, the modified serotonin receptor is a PEGylated serotonin receptor.

[0024] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural serotonin receptor and has natural serotonin receptor activity" refers to a protein molecule whose amino acid sequence has ≥85% homology, preferably ≥90% homology, more preferably ≥95% homology, and most preferably ≥98% homology compared to the serotonin receptor; and has natural serotonin receptor activity.

[0025] In another preferred embodiment, the serotonin receptor is selected from the following group:

[0026] (A) a polypeptide whose amino acid sequence is shown in any one of SEQ ID NOs.: 1-6;

[0027] (B) a PP2A protein derivative or an active fragment thereof formed by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs.: 1-6;

[0028] (C) A serotonin receptor derivative or an active fragment thereof having a sequence homology ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the amino acid sequence shown in any one of SEQ ID NOs.: 1-6.

[0029] In another preferred embodiment, the composition or preparation is also used for one or more purposes selected from the following group:

[0030] (i) Inhibit the activation of peripheral blood ILC2 cells;

[0031] (ii) inhibiting ILC2 cell proliferation and cytokine production;

[0032] (iii) inhibiting the function and proliferation of ILC2 cells;

[0033] (iv) attenuated papain-induced lung inflammation;

[0034] (v) inhibiting ILC2 cell responses;

[0035] (vi) Inhibit ILC2 cell-mediated immune responses.

[0036] In another preferred embodiment, the composition includes a pharmaceutical composition.

[0037] In another preferred embodiment, the composition comprises (a) serotonin, a serotonin receptor agonist, or a combination thereof; and (b) a pharmaceutically acceptable carrier.

[0038] In another preferred embodiment, the composition contains 0.001-99 wt %, preferably 0.1-90 wt %, more preferably 1-80 wt % of component (a), based on the total weight of the composition.

[0039] In another preferred embodiment, the composition further comprises other drugs for preventing and / or treating ILC2 cell-mediated immune diseases.

[0040] In another preferred embodiment, the other drugs for preventing and / or treating ILC2 cell-mediated immune diseases include glucocorticoids, β2 receptor agonists, leukotriene receptor antagonists, theophylline drugs, and anticholinergic drugs.

[0041] In another preferred embodiment, the composition or preparation can be used alone or in combination for preventing and / or treating ILC2 cell-mediated immune diseases.

[0042] In another preferred embodiment, the combined use includes: combined use with other drugs for ILC2 cell-mediated immune diseases.

[0043] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semisolid.

[0044] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.

[0045] In another preferred embodiment, the composition is an oral preparation.

[0046] In another preferred embodiment, the composition (such as a pharmaceutical composition) is administered to a mammal by the following means: oral administration, intravenous injection, or local administration.

[0047] In another preferred embodiment, the mammal includes a human or a non-human mammal.

[0048] In another preferred embodiment, the non-human mammal includes a rodent (such as a mouse or a rat) or a primate (such as a monkey).

[0049] The second aspect of the present invention provides a composition comprising:

[0050] (a1) serotonin, serotonin receptor agonists, or a combination thereof;

[0051] (b) a pharmaceutically acceptable carrier.

[0052] In another preferred embodiment, the serotonin receptor agonist is selected from the following group: DOI, psilocybin, mescaline, 25I-NBOMe (25-NB derivative), or a combination thereof.

[0053] In another preferred embodiment, the composition further comprises (a2) other drugs for preventing and / or treating ILC2 cell-mediated immune diseases.

[0054] In another preferred embodiment, the composition further comprises other drugs for preventing and / or treating ILC2 cell-mediated immune diseases.

[0055] In another preferred embodiment, the other drugs for preventing and / or treating ILC2 cell-mediated immune diseases include glucocorticoids, β2 receptor agonists, leukotriene receptor antagonists, theophylline drugs, and anticholinergic drugs.

[0056] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0057] In another preferred embodiment, in the composition, the component (a1) accounts for 1-99wt%, preferably 10-90wt%, and more preferably 30-70wt% of the total weight of the composition.

[0058] In another preferred embodiment, in the composition, the component (a2) accounts for 1-99wt%, preferably 10-90wt%, and more preferably 30-70wt% of the total weight of the pharmaceutical composition.

[0059] In another preferred embodiment, the weight ratio of component (a1) to component (a2) is 1:100 to 100:1, preferably 1:10 to 10:1.

[0060] In another preferred embodiment, the composition may contain a single compound or a mixture of multiple compounds.

[0061] In another preferred embodiment, the composition is used to prepare a drug or preparation for treating or preventing ILC2 cell-mediated immune diseases.

[0062] In another preferred embodiment, the pharmaceutical dosage form is an oral or parenteral dosage form.

[0063] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0064] In another preferred embodiment, the non-oral dosage form is an injection or injection.

[0065] In another preferred embodiment, the total content of the component (a1) and the component (a2) is 1 to 99 wt %, more preferably 5 to 90 wt %, of the total weight of the composition.

[0066] The third aspect of the present invention provides a medicine kit, comprising:

[0067] (i) a first container, and an active ingredient (a) serotonin, a serotonin receptor agonist, or a combination thereof, or a drug containing the active ingredient (a) contained in the first container;

[0068] (ii) an optional second container, and the active ingredient (b) contained in the second container, other drugs for preventing and / or treating ILC2 cell-mediated immune diseases, or drugs containing the active ingredient (b); and

[0069] (iii) instructions for preventing and / or treating ILC2 cell-mediated immune diseases by administering the active ingredient (a) and the active ingredient (b) in combination.

[0070] In another preferred embodiment, the first container and the second container may be the same or different.

[0071] In another preferred embodiment, the drug in the first container is a single preparation containing serotonin, a serotonin receptor agonist, or a combination thereof.

[0072] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for preventing and / or treating ILC2 cell-mediated immune diseases.

[0073] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

[0074] In another preferred embodiment, the kit further comprises instructions for preventing and / or treating ILC2 cell-mediated immune diseases by administering the active ingredient (a) and the active ingredient (b) in combination.

[0075] In another preferred embodiment, the preparation containing the active ingredient (a) serotonin, a serotonin receptor agonist, or a combination thereof or the preparation containing other drugs for preventing and / or treating ILC2 cell-mediated immune diseases comprises capsules, tablets, suppositories, or intravenous injections.

[0076] In another preferred embodiment, in the preparation containing the active ingredient (a) serotonin, a serotonin receptor agonist, or a combination thereof, the concentration of the serotonin, the serotonin receptor agonist, or a combination thereof is 0.0001-100 mg / kg body weight, preferably 0.1-50 mg / kg body weight, and more preferably 1-20 mg / kg body weight.

[0077] A fourth aspect of the present invention provides a method for inhibiting ILC2 cell proliferation or activation, comprising the steps of:

[0078] ILC2 cells are cultured in the presence of serotonin, a serotonin receptor agonist, or a combination thereof, thereby inhibiting the proliferation or activation of the ILC2 cells.

[0079] In another preferred embodiment, the cells are cells cultured in vitro.

[0080] In another preferred embodiment, the ILC2 cells are pulmonary ILC2 cells.

[0081] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0082] In another preferred embodiment, the method is therapeutic.

[0083] In another preferred embodiment, the effective concentration of the serotonin, serotonin receptor agonist, or a combination thereof is 0.0001-100 mg / kg body weight, preferably 1-50 mg / kg body weight, and more preferably 5-20 mg / kg body weight.

[0084] A fifth aspect of the present invention provides a method for screening potential therapeutic agents for ILC2 cell-mediated immune diseases, comprising:

[0085] (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing a serotonin receptor agonist for a period of time T1, and detecting the expression level E1 of the serotonin receptor agonist in the culture system of the test group;

[0086] And in a control group without the test compound and with other conditions being the same, detecting the expression amount E2 of the serotonin receptor agonist in the culture system of the control group; and

[0087] (b) Comparing E1 and E2, if E1 is significantly higher than E2, it indicates that the test compound is a potential therapeutic agent for ILC2 cell-mediated immune diseases.

[0088] In another preferred embodiment, the “significantly higher than” refers to E1 / E2 ≥ 2, preferably ≥ 3, more preferably ≥ 4.

[0089] In another preferred embodiment, the cells include ILC2 cells.

[0090] In another preferred embodiment, the cells are cells cultured in vitro.

[0091] In another preferred embodiment, the method is an in vitro method.

[0092] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0093] In another preferred embodiment, the method comprises step (c): administering the potential therapeutic agent determined in step (a) to a mammal, thereby determining its effect on an ILC2 cell-mediated immune disease in the mammal.

[0094] In another preferred embodiment, the mammal includes a human or a non-human mammal.

[0095] In another preferred embodiment, the non-human mammals include rodents and primates, preferably, mice, rats, rabbits and monkeys.

[0096] A sixth aspect of the present invention provides a method for preventing and / or treating an ILC2 cell-mediated immune disease, comprising the steps of:

[0097] Serotonin, a serotonin receptor agonist, or a combination thereof, the composition of the second aspect of the present invention, or the kit of parts of the third aspect of the present invention is administered to a subject in need thereof.

[0098] In another preferred embodiment, the administration comprises oral administration.

[0099] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0100] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0101] In another preferred embodiment, the dosage of serotonin, serotonin receptor agonist, or combination thereof is 0.0001-100 mg / kg body weight, preferably 1-50 mg / kg body weight, and most preferably 5-20 mg / kg body weight.

[0102] In another preferred embodiment, the administration frequency of the serotonin, serotonin receptor agonist, or a combination thereof is 1-150 times / month, preferably once a day.

[0103] In another preferred embodiment, the administration time of the serotonin, the serotonin receptor agonist, or the combination thereof is 5-100 days, preferably 10-50 days, and most preferably 14-42 days.

[0104] In another preferred embodiment, the administration includes simultaneous administration or sequential administration.

[0105] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 It shows that depression is closely related to type II immune response, and the neurotransmitter 5-HT can inhibit the activation of human peripheral blood ILC2 cells.

[0107] AC: Mice were treated intraperitoneally with SSRI from day 1 to day 10 and with papain from day 6 to day 10.

[0108] Mice were immunized by intrabronchial administration.

[0109] A: The number of eosinophils.

[0110] B: Number of ILC2 cells and Ki67 + The number of ILC2 cells.

[0111] C:IL-5 + ,IL-13 + ,IL-5 + IL-13 + The number of ILC2 cells.

[0112] D: Human ILC2 cells were cultured in vitro and treated with 5-HT, and cytokines IL-5 and IL-13 were detected by Elisa

[0113] Figure 2 It was shown that 5-HT inhibits ILC2 cell proliferation and cytokine production. C57BL / 6 mice were immunized with papain to induce a long-term lung inflammation model, and lung inflammatory ILC2 cells were isolated, cultured with cytokines IL-2, IL-7, and IL-33 in vitro for 3 days, and treated with 5-HT.

[0114] A: RNA-seq scatter plot.

[0115] B: GSEA analysis showed significantly changed signaling pathways.

[0116] C: The proliferation of cells after 5-HT treatment was detected using Ki67 flow cytometry antibody, and the flow cytometry analysis graph is shown in the figure.

[0117] D: Statistical chart of Figure C

[0118] E: Collect culture supernatant and detect cytokines IL-5 and IL-13 by Elisa

[0119] F: IL-5 and IL-13 flow cytometry antibodies were used to detect the secretion of cytokines after 5-HT treatment, and the results were statistically analyzed.

[0120] G: Annexin V flow cytometry antibody was used to detect cell apoptosis after 5-HT treatment. The flow cytometry analysis is shown in the figure.

[0121] H: FVD dye was used to detect the death of ILC2 cells cultured for 3 days

[0122] Figure 3 It was shown that 5-HT inhibited the function and proliferation of ILC2 cells by regulating the p-ERK-GATA3 signaling pathway. C57BL / 6 mice were immunized with papain to induce a long-term lung inflammation model. Lung inflammatory ILC2 cells were isolated and cultured with cytokines IL-2, IL-7, and IL-33 in vitro.

[0123] A: The expression of cytokines IL-5, IL-13, and transcription factor GATA3 was detected by QPCR.

[0124] B: GATA3 protein expression in cultured cells was detected by flow cytometry after 72 h of culture.

[0125] C: Statistical chart of B.

[0126] D: ILC2 cells were treated with different signal pathway inhibitors, and cytokines IL-5 and IL-13 were detected by Elisa.

[0127] E: The cells were treated with PBS or 5-HT for 30 min, and the expression of p-ERK was detected by flow cytometry.

[0128] F: ILC2 cells cultured in vitro were first treated with U0126, MG132, etc., and then treated with 5-HT.

[0129] The expression of GATA3 was detected by

[0130] G: ILC2 cells cultured in vitro were first treated with FR180204, then treated with 5-HT, and flow cytometry was performed 12 hours later.

[0131] Figure 4 It was shown that 5-HT inhibited ILC2 cell function and thus attenuated papain-induced lung inflammation.

[0132] in,

[0133] A: Schematic diagram of papain-induced lung inflammation model and treatment methods.

[0134] B: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0135] C: HE & PAS staining of lung tissue sections.

[0136] D: Lung tissue homogenate was obtained and the amount of cytokines IL-5 and IL-13 in the supernatant was detected by Elisa.

[0137] E: Alveolar lavage fluid was collected and the amount of cytokines IL-5 and IL-13 in the supernatant was detected by Elisa.

[0138] F: Flow cytometry detection of lung tissue lin - CD90.2 + ST2 + ILC2 cells as shown.

[0139] G: Statistics of the flow cytometry data in Figure F.

[0140] H: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The proportion of ILC2 cells secreting cytokines IL-5 and IL-13 was detected by flow cytometry and counted.

[0141] I: Pulmonary inflammatory ILC2 cells were isolated and treated with PBS or 5-HT. They were treated with PMA for 15 min and the expression of p-ERK was detected by flow cytometry and statistically analyzed.

[0142] J: The expression level of GATA3 protein in ILC2 cells was detected by flow cytometry, and the expression level of GATA3 mRNA was detected by QPCR.

[0143] Figure 5 It was shown that 5-HT inhibited papain-induced lung inflammation independently of T and B cells.

[0144] A: The number and proportion of eosinophils in bronchoalveolar lavage fluid.

[0145] B: HE & PAS staining of lung tissue sections.

[0146] C: The number and proportion of ILC2 cells in lung tissue.

[0147] D: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The proportion of ILC2 cells secreting cytokines IL-5 and IL-13 was detected by flow cytometry and counted.

[0148] E: The number of ILC2 cells secreting cytokines IL-5 and IL-13.

[0149] Figure 6It was shown that 5-HT inhibited ILC2 cell function and thus attenuated papain-induced lung inflammation.

[0150] in,

[0151] A: Schematic diagram of IL-33-induced lung inflammation model and treatment method.

[0152] B: Flow cytometry was used to detect and count the number and proportion of ILC2 cells in lung tissue.

[0153] C: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry and their numbers were counted.

[0154] D: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0155] E: HE & PAS staining of lung tissue sections.

[0156] F: Schematic diagram of A. alternata-induced lung inflammation model and treatment methods.

[0157] G: Flow cytometry was used to detect the number and proportion of ILC2 cells in lung tissue and the numbers were counted.

[0158] H: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0159] Figure 7 It was shown that 5-HT had no effect on ILC1 and ILC3 cells.

[0160] A: Flow cytometry gating diagram of ILC1 and ILC3 cells isolated from the colon lamina propria.

[0161] B: Statistical graph of the proportions of ILC1, ILC2, and ILC3 cells in A.

[0162] CE: The mice in the PBS or 5-HT treatment groups were divided into 2 groups and fed with 3% DSS and normal water, respectively (5 mice in each group). The inflammation in the colon was detected on the 7th day.

[0163] C: Mouse body weight statistics.

[0164] D: Flow cytometry detection of CD45 + Lin - CD127 + Rorγt + ILC3 cells produce the cytokine IL-17A + and IFN-γ +situation.

[0165] E: The production of cytokine IL-17A in D + and IFN-γ + Statistical graph of the proportion of ILC3 cells.

[0166] Figure 8 It shows that 5-HT acts on the HTR2A receptor on the surface of ILC2 cells and inhibits the immune response mediated by ILC2 cells.

[0167] A: QPCR detection of the expression of different mouse HTR receptors in ILC2 cells.

[0168] B: QPCR detected the expression of mouse HTR2A in ILC2 cells and other cell subsets.

[0169] C: QPCR detected the expression of HTR2A in different cell subsets in human peripheral blood PBMC.

[0170] D: Isolation of Htr2a f / f and PLZF cre Htr2a f / f ILC2 cells in mouse lung tissue, QPCR detected the expression of HTR2A in ILC2 cells.

[0171] E: Isolation of Htr2a f / f and PLZF cre Htr2a f / f Mouse lung tissue ILC2 cells, western blot detected the expression of HTR2A in ILC2 cells.

[0172] FH: papain induces Htr2a f / f and PLZF cre Htr2a f / f Mouse lung inflammation model and detection of corresponding indicators

[0173] F: Flow cytometry detection of ILC2 cells and Ki67 in lung tissue + The number of ILC2 cells was counted.

[0174] G: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0175] H: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0176] I: ILC2 cells were isolated from a papain-induced long-term lung inflammation model, cultured in vitro, and treated with 5-HT to detect the proliferation of ILC2 cells.

[0177] JL: Papain induced lung inflammation model in C57 / B6 mice and detected the corresponding indicators

[0178] J: Eosinophils in bronchoalveolar lavage fluid were detected and their number and proportion were counted.

[0179] K: Flow cytometry was used to detect ILC2 cells in lung tissue, and the number and proportion were counted.

[0180] L: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0181] Fig. 9 It showed that TPH1 was not involved in the regulation of ILC2 cell function.

[0182] AC: Papain was administered intrabronchially for 5 consecutive days to induce lung inflammation and detect

[0183] A: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0184] B: Flow cytometry was used to detect ILC2 cells in lung tissue, and the number and proportion were counted.

[0185] C: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0186] D: Lung ILC2 cells in the papain-induced long-term model were isolated and cultured in vitro to detect the proliferation of ILC2 cells in WT and KO mice.

[0187] E: Isolation of ILC2 cells from a papain-induced long-term lung inflammation model, in vitro culture, and detection of IL-5 in WT and KO mice + , IL-13 + and IL-5 + IL-13 + The proportion of ILC2 cells.

[0188] F: The amount of 5-HT in lung tissue homogenate and bronchoalveolar lavage fluid of WT and KO mice was detected.

[0189] GI: Isolation of ILC2 cells from a papain-induced long-term lung inflammation model and transfer to Rag2- / - Iq - / - mice, and induced lung inflammation with papain.

[0190] G: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0191] H: Flow cytometry was used to detect ILC2 cells in lung tissue, and the number and proportion were counted.

[0192] I: Isolate lung ILC2 cells, stimulate them with PMA and Innomycin, detect ILC2 cells secreting cytokines IL-5 and IL-13 by flow cytometry, and count the number and proportion.

[0193] Fig.10 It was shown that 5-HT can alleviate the type II immune response mediated by ILC2 cells.

[0194] A: Diagram of mouse immunization and 5-HT treatment pattern.

[0195] B: Flow cytometry was used to detect and count the number and proportion of ILC2 cells in lung tissue.

[0196] C: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0197] D: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0198] E: HE & PAS staining of lung tissue sections.

[0199] Fig.11 It was shown that 5-HT can alleviate the type II immune response mediated by ILC2 cells.

[0200] A: Diagram of mouse immunization and DOI treatment pattern.

[0201] B: Flow cytometry was used to detect and count the number and proportion of ILC2 cells in lung tissue.

[0202] C: Lung ILC2 cells were isolated and stimulated with PMA and Innomycin. The ILC2 cells secreting cytokines IL-5 and IL-13 were detected by flow cytometry, and the number and proportion were counted.

[0203] D: Detect eosinophils in alveolar lavage fluid and count their number and proportion.

[0204] E: HE & PAS staining of lung tissue sections. DETAILED DESCRIPTION

[0205] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that serotonin, serotonin receptor agonists, or a combination thereof can significantly prevent and / or treat ILC2 cell-mediated immune diseases. On this basis, the inventors completed the present invention.

[0206] As used herein, the structural formula of DOI is:

[0207]

[0208] As used herein, the structural formula of psilocybin is:

[0209]

[0210] As used herein, the structural formula of mescaline is:

[0211]

[0212] As used herein, the structural formula of 25I-NBOMe (25-NB derivative) is:

[0213]

[0214] As used herein, the structural formula of iBET151 is:

[0215]

[0216] Serotonin

[0217] Serotonin (5-HT) (Young, 2007) is a monoamine neurotransmitter that is involved in regulating various physiological functions, including mood, sleep, appetite and pain. In addition to its important role in the central nervous system, the role of 5-HT in the peripheral immune system has also been gradually discovered. 5-HT can regulate the functions of many cells such as mast cells, eosinophils, monocytes and macrophages, dendritic cells (DCs), T cells, lung epithelial cells and lung fibroblasts. At present, 15 5-HT receptors have been identified. Enterochromaffin cells (EC) are the main source of peripheral 5-HT. After 5-HT is released from EC cells into the plasma, it is rapidly absorbed by resting platelets through 5-HT-specific transporters. After inflammatory signal stimulation, 5-HT can be released from circulating platelets and tissue-specific mast cells to exert its function. Once platelets release 5-HT, it can participate in peripheral inflammation through its chemical attractants and immune cell activity. 5-HT is produced by intestinal chromaffin cells. When the intestine is infected with bacteria or stimulated by compounds, 5-HT can activate immune cells in the intestine, such as macrophages, and promote intestinal inflammation. In addition to its role in enteritis, 5-HT also plays a role in other immune-related diseases. In an autoimmune arthritis mouse model, Tph KO mice lead to an imbalance of Treg / Th17 and show a stronger inflammatory response.

[0218] Serotonin receptors

[0219] Serotonin receptors, also known as serotonin receptors or 5-HT receptors, are a group of G protein-coupled receptors and ligand-gated ion channels found in the central nervous system and peripheral nervous system. They regulate the transmission of both excitatory and inhibitory neurotransmitters. Serotonin receptors can be divided into seven subfamilies: 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, 5-HT7. At least fourteen receptor subtypes have been discovered, including G protein-coupled receptors and a ligand-gated ion channel. Serotonin receptors (or 5-HT receptors) are located in the cell membrane of animal neurons and other cell types and mediate the effects of serotonin as an endogenous ligand and a wide range of drugs and psychedelic drugs. With the exception of the 5-HT3 receptor, a ligand-gated ion channel (LGIC), all other serotonin receptors are G protein-coupled receptors (GPCRs) that activate intracellular second messenger cascades. (Also called seven transmembrane receptors or seven helix receptors). Serotonin receptors regulate many neurotransmitters, including glutamate, gamma-aminobutyric acid (GABA), dopamine, epinephrine / norepinephrine, and acetylcholine, as well as many other hormones, including oxytocin, prolactin, vasopressin, cortisol, adrenocorticotropic hormone, and substance P, among others. Serotonin receptors influence a variety of biological and neurological processes, such as aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and temperature regulation. Serotonin receptors are targets for a variety of drugs, including many antidepressants, antipsychotics, anorectics, antiemetics, gastrokinetics, and antimigraine drugs.

[0220] Specifically, the present invention relates to a serotonin receptor and a variant thereof. In a preferred embodiment of the present invention, the amino acid sequence of the serotonin receptor is shown in any one of SEQ ID NO.: 1 to 6. The serotonin receptor or the vector expressing the serotonin receptor of the present invention can prevent and / or treat ILC2 cell-mediated immune diseases.

[0221] The serotonin receptor or the vector expressing the serotonin receptor of the present invention can also be used for one or more purposes selected from the following group:

[0222] (i) Inhibit the activation of peripheral blood ILC2 cells;

[0223] (ii) inhibiting ILC2 cell proliferation and cytokine production;

[0224] (iii) inhibiting the function and proliferation of ILC2 cells;

[0225] (iv) attenuated papain-induced lung inflammation;

[0226] (v) inhibiting ILC2 cell responses;

[0227] (vi) Inhibit ILC2 cell-mediated immune responses.

[0228] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology with the sequences shown in SEQ ID NO.: 1-6 of the present invention.

[0229] Among them, SEQ ID NO.: 1-3 is a human serotonin receptor:

[0230] >NP_001365853.1 5-hydroxytryptamine receptor 2A isoform 1[Homosapiens]

[0231] MDILCEENTSLSSTTNSLMQLNDDTRLYSNDFNSGEANTSDAFNWTVDSENRTNLSCEGCLSPSCLSLLHLQEKNWSALLTAVVIILTIAGNILVIMAVSLEKKLQNATNYFLMSLAI ADMLLGFLVMPVSMLTILYGYRWPLPSKLCAVWIYLDVLFSTASIMHLCAISLDRYVAIQNPIHHSRFNSRTKAFLKIIAVWTISVGISMPIPVFGLQDDSKVFKEGSCLLADDNFVLI GSFVSFFIPLTIMVITYFLTIKSLQKEATLCVSDLGTRAKLASFSFLPQSSLSSEKLFQRSIHREPGSYTGRRTMQSISNEQKACKVLGIVFFLFVVMWCPFFITNIMAVICKESCNED VIGALLNVFVWIGYLSSAVNPLVYTLFNKTYRSAFSRYIQCQYKENKKPLQLILVNTIPALAYKSSQLQMGQKKNSKQDAKTTDNDCSMVALGKQHSEEASKDNSDGVNEKVSCV(SEQ ID NO.1)

[0232] >NP_000515.2 5-hydroxytryptamine receptor 1A[Homo sapiens]

[0233] MDVLSPGQGNNTTSPPAPFETGGNTTGISDVTVSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVLPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGWRTPEDRSDPDACTISKDHGYTIYSTFGAFYIPLLLMLVLYGRIFRAARFRIRKTVKKVEKTGADTRHGASPAPQPKKSVNGESGSRNWRLGVESKAGGALCANGAVRQGDDGAALEVIEVHRVGNSKEHLPLPSEAGPTPCAPASFERKNERNAEAKRKMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPTLLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCRQ(SEQ ID NO.2)

[0234] >NP_000854.1 5-hydroxytryptamine receptor 1B[Homo sapiens]

[0235] MEEPGAQCAPPPPAGSETWVPQANLSSAPSQNCSAKDYIYQDSISLPWKVLLVMLLALITLATTLSNAFVIATVYRTRKLHTPANYLIASLAVTDLLVSILVMPISTMYTVTGRWTLGQVVCDFWLSSDITCCTASILHLCVIALDRYWAITDAVEYSAKRTPKRAAVMIALVWVFSISISLPPFFWRQAKAEEEVSECVVNTDHILYTVYSTVGAFYFPTLLLIALYGRIYVEARSRILKQTPNRTGKRLTRAQLITDSPGSTSSVTSINSRVPDVPSESGSPVYVNQVKVRVSDALLEKKKLMAARERKATKTLGIILGAFIVCWLPFFIISLVMPICKDACWFHLAIFDFFTWLGYLNSLINPIIYTMSNEDFKQAFHKLIRFKCTS(SEQ ID NO.3)

[0236] SEQ ID NO.: 4 - 6 are murine serotonin receptors:

[0237] >NP_766400.1 5-hydroxytryptamine receptor 2A[Mus musculus]

[0238] MEILCEDNISLSSIPNSLMQLGDDSRLYPNDFNSRDANTSEASNWTIDAENRTNLSCEGYLPPTCLSILHLQEKNWSALLTTVVIILTIAGNILVIMAVSLEKKLQNATNYFLMSLAIADMLLGFLVMPVSMLTILYGYRWPLPSKLCAVWIYLDVLFSTASIMHLCAISLDRYVAIQNPIHHSRFNSRTKAFLKIIAVWTISVGISMPIPVFGLQDDSKVFKEGSCLLADDNFVLIGSFVAFFIPLTIMVITYFLTIKSLQKEATLCVSDLSTRAKLSSFSFLPQSSLSSEKLFQRSIHREPGSYAGRRTMQSISNEQKACKVLGIVFFLFVVMWCPFFITNIMAVICKESCNENVIGALLNVFVWIGYLSSAVNPLVYTLFNKTYRSAFSRYIQCQYKENRKPLQLILVNTIPTLAYKSSQLQVGQKKNSQEDAEPTANDCSMVTLGNQHSEEMCTDNIETVNEKVSCV(SEQ ID NO.:4)

[0239] >NP_032334.2 5-hydroxytryptamine receptor 1A[Mus musculus]

[0240] MDMFSLGQGNNTTTSLEPFGTGGNDTGLSNVTFSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTTDLMVSVLVPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGWRTPEDRSNPNECTISKDHGYTIYSTFGAYIPLLML VLYGRIFRAARFRIRKTVKKVEKKGAGTSFGTSSAPPKKSLNGQPGSGDCRRSAENRAVGTPCANGAVRQGEDDATLEVIEVHRVGNSKGHLPLPSESGATSYVPACLERKNERTAEAKRKMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPELLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCR(SEQ ID NO.: 5)

[0241] >NP_034612.1 5-hydroxytryptamine receptor 1B[Mus musculus]

[0242] MEEQGIQCAPPPPAASQTGVPLTNLSHNCSADGYIYQDSIALPWKVLLVALLALITLATTLSNAFVIATVYRTRKHLHTPANYLIASLAVTDLLVSILVMPISTMYTVTGRWTLGQVVCDFWLSSDITCCTASMHLCVIALDRYWAITDAVEYSAKRTPKRAAIMIVLVWVFSISISLPPFFWRQAKAEEEMLDC FVNTDHVLYTVYSTVGAFAYLPTLLLIALYGRIYVEARSRILKQTPNKTGKRLTRAQLITDSPGSTSSVTSINSRAPDVPSESGSPVYVNQVKVRVSDALLEKKLMAARERKATKTLGIILGAFIVCWLPFFIISLVMPICKDACWFHMAIFDFFNWLGYLNSLINPIIYTMSNEDFKQAFHKLIRFCAG(SEQ ID NO.: 6)

[0243] The “same or similar functions” mainly refer to: “(a) preventing and / or treating ILC2 cell-mediated immune diseases; and / or (i) inhibiting peripheral blood ILC2 cell activation; and / or (ii) inhibiting ILC2 cell proliferation and cytokine production; and / or (iii) inhibiting ILC2 cell function and proliferation; and / or (iv) attenuating papain-induced lung inflammation; and / or (v) inhibiting ILC2 cell response; and / or (vi) inhibiting ILC2 cell-mediated immune response”.

[0244] The protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein. The protein of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production scheme, the protein of the present invention can be glycosylated, or can be non-glycosylated. The protein of the present invention may also include or not include an initial methionine residue.

[0245] The present invention also includes fragments and analogs of serotonin receptors having serotonin receptor activity. As used herein, the terms "fragment" and "analog" refer to proteins that substantially retain the same biological function or activity of the natural serotonin receptor of the present invention.

[0246] Mutant protein fragments, derivatives or analogs of the present invention can be (i) substituted mutant proteins with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) mutant proteins with substituent groups in one or more amino acid residues, or (iii) mutant proteins formed by fusion of mature mutant proteins with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) mutant proteins formed by fusion of additional amino acid sequences to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art. In the present invention, the conservatively substituted amino acids are preferably produced by amino acid substitution according to Table I.

[0247] Table I

[0248] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0249] The present invention also includes polypeptides or proteins with the same or similar functions that have 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology with the natural serotonin receptor of the present invention. The protein variant may be a derivative sequence obtained by replacing, deleting or adding at least one amino acid by several (usually 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10) substitutions, deletions, or additions of at least one amino acid, and adding one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the protein, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and adding one or several amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The present invention includes analogs of natural serotonin receptors, and the difference from the natural serotonin receptors may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known biological techniques. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

[0250] Modifications (usually without changing the primary structure) include: chemical derivatization forms of in vivo or in vitro proteins such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modification during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). In addition, the mutant proteins of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of in vivo or in vitro mutant proteins such as acetylation or carboxylation. Modifications also include glycosylation, such as those mutant proteins produced by glycosylation modification during the synthesis and processing of mutant proteins or in further processing steps. This modification can be accomplished by exposing the mutant protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins that have been modified to increase their resistance to proteolysis or optimize their solubility.

[0251] The present invention also provides a polynucleotide sequence encoding a serotonin receptor protein. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes: DNA, genomic DNA or artificially synthesized DNA, and the DNA may be single-stranded or double-stranded. The polynucleotide encoding a mature polypeptide includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and a non-coding sequence. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and / or non-coding sequences. The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. The variant of this polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0252] In a preferred example of the present invention, the DNA sequence encoding the human serotonin receptor protein encodes the serotonin receptor shown in any one of SEQ ID NOs.: 1-3, the polynucleotide sequence encoding the human serotonin receptor protein is shown in any one of SEQ ID NOs.: 7, 9-10, and the nucleotide sequence of the mouse serotonin receptor gene is shown in any one of SEQ ID NOs.: 8, 11-12.

[0253] SEQ ID NO.: 1

[0254] >NP_001365853.1 5-hydroxytryptamine receptor 2A isoform 1[Homosapiens]

[0255] MDILCEENTSLSSTTNSLMQLNDDTRLYSNDFNSGEANTSDAFNWTVDSENRTNLSCEGCLSPSCLSLLHLQEKNWSALLTAVVIILTIAGNILVIMAVSLEKKLQNATNYFLMSLAIADMLLGFLVMPVSMLTILYGYRWPLPSKLCAVWIYLDVLFSTASIMHLCAISLDRYVAIQNPIHHSRFNSRTKAFLKIIAVWTISVGISMPIPVFGLDNFQDKVFKESCLLADV LIGSFVSFFIPLTIMVITYFLTIKSLQKEATLCVSDGLTRAKLASFSFLPQSSLSSEKLFQRSIHREPGSYTGRRTMQSISNEQKACKVLGIVFFLFVVMWCPFFITNIMAVICKESCNEDVIGALLNVFFWIGYLSSAVNPLVYTLFNKTYRSAFSRYIQCQYKENKKPLQLILVNTIPALAYKSSQLQMGQKKNSKQDAKTTDNDCSMVALGKQHSEEASKDNSDGVNEKVSCV

[0256] SEQ ID NO.:2

[0257] >NP_000515.2 5-hydroxytryptamine receptor 1A[Homo sapiens]

[0258] MDVLSPGQGNNTTSPPAPFETGGNTTGISDVTVSYQVITSLLLGTLIFCAVLGNACVVAAIALERSLQNVANYLIGSLAVTDLMVSVLVPMAALYQVLNKWTLGQVTCDLFIALDVLCCTSSILHLCAIALDRYWAITDPIDYVNKRTPRRAAALISLTWLIGFLISIPPMLGWRTPEDRSDPDACTISKDHGYTIYSTFGAFYIPLLLM LVLYGRIFRAARFRIRKTVKKVEKTGADTRHGASPAPQPPKKSVNGESGSRNWRLGVESKAGGALCANGAVRQGDDGAALEVIEVHRVGNSKEHLPLPSEAGPTPCAPASFERKNERNAEAKRKMALARERKTVKTLGIIMGTFILCWLPFFIVALVLPFCESSCHMPTLLGAIINWLGYSNSLLNPVIYAYFNKDFQNAFKKIIKCKFCRQ

[0259] SEQ ID NO.:3

[0260] >NP_000854.1 5-hydroxytryptamine receptor 1B[Homo sapiens]

[0261] MEEPGAQCAPPPPAGSETWVPQANLSSAPSQNCSAKDYIYQDSISLPWKVLLVMLLALITLATTLSNAFVIATVYRTRKHLHTPANYLIASLAVTDLLVSILVMPISTMYTVTGRWTLGQVVCDFWLSSDITCCASILHLCVIALDRYWAITDAVEYSAKRTPKRAAVMIALVWVFSISISLPPFFWRQAKAEEE VSECVVNTDHILYTVYSTVGAFYFPTLLLLIALYGRIYVEARSRILKQTPNRTGKRLTRAQLITDSPGSTSSVTSINSRVPDFVPSESGSPVYVNQVKVRVSDALLEKKLMAARERKATKTLGIILGAFIVCWLPFFIISLVMPICKDACWFHLAIFDFFTWLGYLNSLINPIIYTMSNEDFKQAFHKLIRFKCTS

[0262] >NM_000524.4 Homo sapiens 5-hydroxytryptamine receptor 1A(HTR1A),mRNA

[0263]

[0264] >NM_000863.3 Homo sapiens 5-hydroxytryptamine receptor 1B(HTR1B),mRNA

[0265]

[0266] >NM_000621.5 Homo sapiens 5-hydroxytryptamine receptor 2A(HTR2A),transcriptvariant 1,mRNA

[0267]

[0268] >NM_008308.4 Mus musculus 5-hydroxytryptamine(serotonin)receptor 1A(Htr1a),mRNA

[0269]

[0270] >NM_000863.3 Homo sapiens 5-hydroxytryptamine receptor 1B(HTR1B),mRNA

[0271]

[0272] >NM_172812.3Mus musculus 5-hydroxytryptamine(serotonin)receptor 2A(Htr2a),mRNA

[0273]

[0274] According to the nucleotide sequence described herein, the technical personnel can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include but are not limited to: PCR, DNA artificial synthesis, etc., and the specific methods can be found in J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR.

[0275] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

[0276] The proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0277] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0278] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0279] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0280] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0281] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0282] Serotonin receptor agonists

[0283] In the present invention, the serotonin receptor agonist includes substances that can increase the activity and / or content of serotonin receptors in vivo or in vitro.

[0284] In the present invention, the serotonin receptor agonist also includes substances that promote the expression or activity of serotonin receptors.

[0285] Among them, the expression level of serotonin receptors can be increased by the following methods: the tissue itself secretes a large amount of serotonin receptor protein or artificially overexpresses serotonin receptor protein, or artificially delivers serotonin receptor protein (for example, using a viral vector, such as an adeno-associated viral vector) or a serotonin receptor agonist.

[0286] In the present invention, the serotonin receptor agonist is not particularly limited, and any agonist that can promote the expression of serotonin receptors or enhance the activity of serotonin receptors is within the protection scope of the present invention.

[0287] In a preferred embodiment, the serotonin receptor agonist comprises a small molecule compound.

[0288] Compound pharmaceutical composition and medicine kit

[0289] The present invention provides a compound pharmaceutical composition containing active ingredients (a) serotonin, serotonin receptor agonists, or a combination thereof; and (b) a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under aseptic conditions. The pharmaceutical combination of the present invention can also be prepared in powder form for aerosol inhalation. The dosage form of the pharmaceutical composition of the present invention is an injection. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared into a sustained-release preparation. The pharmaceutical composition of the present invention is preferably an injection preparation. In addition, the pharmaceutical composition of the present invention can also be used with other therapeutic agents. In addition, the pharmaceutical composition of the present invention can also include additional components, which are selected from the following group: other components for preventing and / or treating ILC2 cell-mediated immune diseases.

[0290] The effective amount of the active ingredient of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.01 μg-1 mg / kg body weight per day, preferably 0.1-10 μg / kg body weight, and more preferably 0.5-5 μg / kg body weight. A satisfactory effect can be obtained. The optimal dose will be appropriately adjusted according to the course of the disease and the condition. For example, several divided doses may be administered per day, or the dose may be reduced proportionally, depending on the urgency of the treatment condition.

[0291] The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

[0292] The present invention also provides a drug kit for (a) preventing and / or treating ILC2 cell-mediated immune diseases; and / or (i) inhibiting peripheral blood ILC2 cell activation; and / or (ii) inhibiting ILC2 cell proliferation and cytokine production; and / or (iii) inhibiting ILC2 cell function and proliferation; and / or (iv) attenuating papain-induced lung inflammation; and / or (v) inhibiting ILC2 cell response; and / or (vi) inhibiting ILC2 cell-mediated immune response, the drug kit comprising:

[0293] (i) a first container, and an active ingredient (a) serotonin, a serotonin receptor agonist, or a combination thereof, or a medicament containing the active ingredient (a) in the first container; and

[0294] (ii) an optional second container, and the active ingredient (b) contained in the second container, other drugs for preventing and / or treating ILC2 cell-mediated immune diseases, or drugs containing the active ingredient (b); and

[0295] (ii) instructions for administering the active ingredient (a) to (a) prevent and / or treat ILC2 cell-mediated immune diseases; and / or (i) inhibit peripheral blood ILC2 cell activation; and / or (ii) inhibit ILC2 cell proliferation and cytokine production; and / or (iii) inhibit ILC2 cell function and proliferation; and / or (iv) reduce papain-induced lung inflammation; and / or (v) inhibit ILC2 cell response; and / or (vi) inhibit ILC2 cell-mediated immune response.

[0296] The pharmaceutical compositions and kits of the present invention are suitable for (a) preventing and / or treating ILC2 cell-mediated immune diseases; and / or (i) inhibiting peripheral blood ILC2 cell activation; and / or (ii) inhibiting ILC2 cell proliferation and cytokine production; and / or (iii) inhibiting the function and proliferation of ILC2 cells; and / or (iv) attenuating papain-induced lung inflammation; and / or (v) inhibiting ILC2 cell responses; and / or (vi) inhibiting ILC2 cell-mediated immune responses.

[0297] The preparation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained-release manner. The preferred mode is to take it once a day, because it is convenient for patients to adhere to it, thereby significantly improving the compliance of patients to take the medicine.

[0298] When taking the drug, the total daily dose in most cases should be lower than (or equal to or slightly higher than) the daily dose of each single drug in a few cases. Of course, the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.

[0299] Treatment

[0300] The present invention also provides a method for using the two active ingredients of the present invention or corresponding drugs to (a) prevent and / or treat ILC2 cell-mediated immune diseases; and / or (i) inhibit peripheral blood ILC2 cell activation; and / or (ii) inhibit ILC2 cell proliferation and cytokine production; and / or (iii) inhibit ILC2 cell function and proliferation; and / or (iv) reduce papain-induced lung inflammation; and / or (v) inhibit ILC2 cell response; and / or (vi) inhibit ILC2 cell-mediated immune response, which comprises administering to a mammal an effective amount of the active ingredient (a) serotonin, a serotonin receptor agonist, or a combination thereof, or administering a pharmaceutical composition containing the active ingredient (a); and (b) other drugs for preventing and / or treating ILC2 cell-mediated immune diseases, or a pharmaceutical composition containing the active ingredient (b).

[0301] When the two active ingredients of the present invention are used for the above purposes, they can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media). For example, these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% (by weight) of the active ingredient mixed with a carrier.

[0302] The two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or local administration. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration.

[0303] From the standpoint of ease of administration, the preferred pharmaceutical composition is a liquid composition, especially an injection.

[0304] In addition, the two active ingredients or drugs of the present invention can also be used in combination with other components or drugs (e.g., iBET151, IL-2 inhibitors, IL-5 inhibitors, IL-7 inhibitors, IL-13 inhibitors, IL-25 inhibitors, IL-33 inhibitors, IL-4Rα inhibitors and TSLP inhibitors, etc.) that (a) prevent and / or treat ILC2 cell-mediated immune diseases; and / or (i) inhibit peripheral blood ILC2 cell activation; and / or (ii) inhibit ILC2 cell proliferation and cytokine production; and / or (iii) inhibit ILC2 cell function and proliferation; and / or (iv) attenuate papain-induced lung inflammation; and / or (v) inhibit ILC2 cell responses; and / or (vi) inhibit ILC2 cell-mediated immune responses.

[0305] The main advantages of the present invention include:

[0306] (1) The present invention first discovered that serotonin, serotonin receptor agonists, or a combination thereof can significantly (a) prevent and / or treat ILC2 cell-mediated immune diseases; and / or (i) inhibit peripheral blood ILC2 cell activation; and / or (ii) inhibit ILC2 cell proliferation and cytokine production; and / or (iii) inhibit ILC2 cell function and proliferation; and / or (iv) attenuate papain-induced lung inflammation; and / or (v) inhibit ILC2 cell responses; and / or (vi) inhibit ILC2 cell-mediated immune responses.

[0307] (2) The present invention discovered for the first time that the neurotransmitter receptor 5-HTR2A is specifically and highly expressed in ILC2 cells under steady state, and that the ligand of 5-HTR2A, the neurotransmitter serotonin (5-HT), and 5-HTR2A agonists such as DOI can inhibit ILC2 activation both in vitro and in vivo.

[0308] (3) The present invention first discovered that by using a papain or IL-33-induced lung inflammation model and administering 5-HT in the early stages of induction, 5-HT was found to strongly inhibit the allergic inflammatory response in the lung by inhibiting the secretion of cytokines IL-5 and IL-13 by ILC2, thereby regulating ILC2 activation.

[0309] (4) The present invention first discovered, through the use of microRNA sequencing analysis, that there was heterogeneity in the lung ILC2s of mice treated with IL-33 and IL-33 plus 5-HT. After 5-HT treatment, ILC2 proliferation was significantly inhibited and cytokine secretion was significantly reduced.

[0310] (5) The present invention discovered for the first time that 5-HT mainly affects the phosphorylation of ERK protein in ILC2 cells, thereby regulating the stability of the main transcription factor GATA3, thereby inhibiting the production of cytokines IL-5 and IL-13. 5-HT can significantly inhibit the function of ILC2 cells, and thus can effectively alleviate lung inflammation in mice. These data indicate that the 5-HT signaling pathway provides a new regulatory mechanism for type II inflammatory response, that is, the innate immune system rapidly inhibits type II cytokine response and regulates lung inflammation through neurotransmitters, highlighting the importance of neurotransmitters in allergic inflammation. Moreover, this neurotransmitter is effective in treating lung inflammation in mice, which can provide new ideas for the treatment of asthma with small molecule drugs.

[0311] (6) The present invention discovered for the first time a new type II immune response regulation mechanism, further revealing the function of the neurotransmitter 5-HT in the type II immune response mediated by ILC2 cells. This provides a new understanding of the body's neuro-immune regulation and provides drug targets and theoretical basis for the treatment of asthma.

[0312] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0313] Unless otherwise specified, all materials and reagents used in the examples are commercially available products.

[0314] Experimental Materials and Methods

[0315] 1. Materials and Reagents

[0316] DMEM and RPMI 1640 culture media were purchased from Thermo Fisher, fetal bovine serum, double antibody, and Glutamine were purchased from Gibco BRL; cell stimulants PMA and Ionomycin were purchased from Sigma-Aldrich; Golgi transport blocker was purchased from BD Pharmingen; 1xPBS was purchased from Gibco, and 20xPBS was purchased from Shanghai Biotechnology Co., Ltd.; papain was purchased from Sigma-Aldrich; the anesthetic isoflurane was purchased from Reward; the reverse transcription kit was purchased from TakaRa; SYBP GREEN was purchased from Toyobo; the nuclear cytokine detection kit was purchased from ebioscience; the phosphorylated ERK inhibitor U0126 was purchased from MCE, and the proteasome pathway degradation inhibitor MG132 was purchased from Sigma.

[0317] 2. Allergens and antibodies

[0318] Papain was from Sigma and was mainly used to induce type II immune response mediated by ILC2 cells; the fungal component Alternaria was purchased from Beijing Broad Biotechnology Co., Ltd. The antibodies were mainly flow cytometry antibodies. CD5 (53-7.3), CD45R (RA3-6B2), Gr-1 (RB6-8C5), NK1.1 (PK136), TCRγ / δ (UC7-13D5) were purchased from BioLegend, CD3 (2C11), CD4 (RM4-5), CD11b (M1 / 70), CD11c (N418), CD127 (A7R34), CD45.2 (104), FcεR1 (MAR-1), IL-5 (TRFK5), IL-13 (eBio13A), KLRG1 (2F1), NKp46 (29A1.4), and RORγt (AFKJS-9) were purchased from eBioscience, Thy1.2 (53-2.1) and GATA3 (L50-823) were purchased from BD Biosciences, and IL-33R (DJ8) was purchased from MD Bioproducts.

[0319] 3.5-HT and cytokines

[0320] The mouse 5-HT used in the experiment was purchased from selleck; the cytokines used for cell culture were IL-2 / IL-7 / IL-33, mouse IL2 and IL7 were purchased from Peprotech, IL-33 was purchased from Biolegend, human IL2 and IL7 were purchased from Peprotech, and IL-33 was purchased from R&D.

[0321] 4. Mouse Model Induction

[0322] (1) Papain-induced short-term lung inflammation model: 6-8w mice were selected and anesthetized with isoflurane through an anesthesia machine. After the mice were anesthetized, 40ul 1xPBS containing 5ug of the allergen papain (papain) was instilled through the bronchus. The same treatment was given at a fixed time point every day for 5 consecutive days. On the 6th day, the mice were treated and the alveolar lavage fluid of the mice was isolated to detect the infiltration of inflammatory cells such as eosinophils and the production of cytokines IL-5 and IL-13. At the same time, the lung tissue was isolated to detect the activation and proliferation of ILC2 cells and to perform tissue pathological section staining to observe the infiltration of inflammatory cells and the secretion of mucus by goblet cells. In the experiment on the effect of 5-HT on lung inflammation, different doses of 5-HT were given through the bronchus at the same time as the allergen on the first day. The experiment on the treatment of lung inflammation with 5-HT was to give 5-HT 100mg / kg through the bronchus on the 6th day after papain induction for 5 days, and lung inflammation was detected on the 7th and 9th days respectively. The experiment of 5-HTR2A agonist for the treatment of lung inflammation was carried out by administering DOI 2 mg / kg through the bronchi on the 6th day after papain induction for 5 days, and detecting lung inflammation on the 7th day.

[0323] (2) Cytokine IL-33 and fungal-induced lung inflammation model: For IL-33-induced airway inflammation, recombinant mouse cytokine IL-33 (50 ng / mouse; BioLegend) or PBS was administered intrabronchially to mice on days 1, 2, and 3. 24 hours after the last immunization, the mice were killed and samples were collected for detection of lung inflammation. For A. alternata-induced lung inflammation, mice were given 10 μg A. alternata (grerlabs) intrabronchially for 4 consecutive days. Mice were killed 24 hours after the last immunization, and BAL fluid and lung tissue were collected for relevant detection and analysis.

[0324] (3) DSS-induced acute colitis model in mice: Weigh a certain amount of DSS and dissolve it in the sterile water that mice drink to make a 3% solution. First feed normal sterile water for 5 consecutive days, record the weight changes of mice every day, and record the first day of feeding water as day 0. On the 5th day, change to 3% DSS solution, continue to record the weight of mice, observe the status of mice, and when the weight of mice drops below 85% on the 7th day, treat the mice, record the length of the colon, isolate the lymphocytes of the colon lamina propria, and detect inflammation in the colon.

[0325] 5. Isolation of Monocytes from Lung Tissue

[0326] The mice were killed, the left atrial appendage was cut open, and the blood was perfused from the right atrium with a 10ml syringe to remove the lungs. The lungs were removed and washed in 1xPBS, and then the lung tissue was minced with scissors, and collagenase I was added and digested in a shaking incubator at 37°C for 30 minutes. The digested tissue was placed in a 6-well plate, fresh culture medium was added, and the tissue was ground with a 5ml syringe and received in a 15ml centrifuge tube. Centrifuged at 500g for 5min, the supernatant was removed, and the cells were reselected with 40% percol, and gently added to the upper layer of 80% percol. The lung mononuclear cells were separated by density gradient centrifugation.

[0327] 6. Flow Cytometry

[0328] (1) Surface staining: Pipette a single cell suspension of no more than 3×106 cells into a 5 ml flow cytometry centrifuge tube (BDfalcon). After centrifugation, remove the supernatant, wash the cells with 1xPBS, and then add Fixable ViabilityDye dye diluted 1:1000. Stain on ice for 15 min to remove the interference of dead cells on flow cytometry detection. After staining, add 2 ml cell surface staining buffer (Stain Buffer, 1x PBS solution containing 2% FBS and 2 mM EDTA) to wash, centrifuge at 350g at 4℃ for 5 min, carefully remove the supernatant, gently flick the cells to mix well, and proceed to the next step of cell surface marker staining. The staining antibody is diluted according to the antibody usage ratio. At the same time, add Fc blocker, stain on ice for about 30 min, wash with 2 ml Stain Buffer, centrifuge at 350g at 4℃ for 5 min, carefully remove the supernatant, gently flick the cells to mix well, add an appropriate amount of Stain Buffer to re-select the cells, and filter them before testing on the machine.

[0329] (2) Intracellular cytokine staining method: stimulate cells with PMA+Ionomycin for 6 hours, add Golgi transport inhibitor BFA in the last 2 hours of stimulation, collect treated cells for intracellular cytokine expression detection, and after cell death and cell surface staining, wash cells with Stain Buffer, and then fix and permeabilize cells with ebioscience intracellular staining kit, on ice for 30 minutes. Dilute 10x perm buffer with ddH2O, fix cells, wash cells with 1xperm, and then dilute the intracellular cytokines to be stained to the corresponding ratio according to the instructions, add to the fixed permeabilized cells for staining, and on ice for 30 minutes. After the cells are stained, wash with 1xperm, reselect cells with Stain Buffer and filter before testing on the machine.

[0330] (3) Nuclear transcription factor staining method: The cell surface stained samples were stained with the eBioscience Foxp3 Staining Buffer Set membrane permeabilization staining kit for nuclear transcription factors. After the last step of cell surface staining, wash the cells with 1xPBS, add 100ul Foxp3 A solution (prepared with black bottle buffer diluent at a ratio of 1:3), and fix the membrane at 4℃ for 30min; then add 2ml 1x Foxp3 B solution (10xperm buffer diluted to 1x with ddH2O) to wash the cells, centrifuge at 4℃, 500g, 5min, the entire staining process must be completed in the 1x Foxp3 B solution system; add the transcription factor antibody containing Fc Blocker (prepared according to the antibody usage method), stain in the dark, and keep on ice for 30min. After the staining is completed, add 2ml 1x Foxp3 B solution to wash the sample once, add an appropriate amount of Stain Buffer and then test on the machine.

[0331] 7. Flow sorting

[0332] ILC2 cells were isolated using the EasySep Mouse ILC2 Enrichment Kit from STEM CELL. The cells enriched with the EasySep Mouse ILC2 Enrichment Kit were washed with 1xPBS, and then the Fixable Viability Dye dye was added to stain the cells to remove the interference of dead cells. Antibody surface staining was then performed. The main antibodies were lineage cocktail antibody, CD127, ST2, and CD45.2. Antibody surface staining was performed according to the method described in detail above. After staining, the cells were resuspended in an appropriate amount of RPMI 1640 culture medium containing 1% FBS and 1mM EDTA, and filtered through a 0.45um filter membrane before being put on the machine. Pay attention to aseptic operation during the entire staining and sorting process. The cell sorting instruments mainly use BDbioscience's Aria II and Aria sorp.

[0333] 8. In vitro culture of ILC2 cells

[0334] The flow sorted cells were used for the next culture. First, the cells were centrifuged, then the supernatant was removed, and 1 ml of 1640 containing 10% FBS was added to resuspend the cells, and then transferred to a 1.5 ml EP tube. Centrifugation was continued at 500g for 5 min, and the supernatant was removed. The cells were resuspended in 1640 containing 10% FBS and cytokines mIL-2, mIL-7 (20 ng / ml), and mIL-33 (10 ng / ml), and then spread into a 96-well round bottom plate, with 200ul complete medium and 3000 ILC2 cells per well. After treatment with different reagents such as 5-HT, MG132, etc., relevant tests were performed.

[0335] 9. ELISA detection of cytokines

[0336] In order to detect the intensity of lung inflammation in the mouse model and the activation of ILC2 cells cultured in vitro, we used the IL-5 and IL-13 Elisa detection kits of ebioscience. First, we coated a 96-well plate (Nunc) with our own coating buffer (antibody coating solution: NaHCO3 0.738g, Na2CO3 0.397g, add water to 250mL, pH = 9.5) overnight (4°C), and blocked it with 1xPBS with 1% BSA for 1h the next day. At the same time, we diluted the collected samples to the appropriate concentration and completed the dilution of the standards. Then, we washed the plate three times with 1xPBS containing Tween-20, added the samples and standards to the corresponding wells of the 96-well plate, incubated at room temperature for 2h, washed the plate three times with 1xPBS containing Tween, and then added the horseradish peroxidase (HRP)-labeled secondary antibody (detection Antibody), continue incubation at room temperature for 1-2h, wash the plate 3 times with 1xPBS containing Tween-20, add HRP, room temperature for 40min, wash the plate 5 times with 1xPBS containing Tween-20, add the color developing solution in the kit, and then perform color development reaction. After timely termination with 2M concentrated sulfuric acid, use a microplate reader to read the OD value at 450nm.

[0337] 10. RNA Extraction and Reverse Transcription

[0338] Phenol / chloroform method was used for extraction. First, cells were collected into 1.5ml EP tubes, centrifuged, supernatant removed, appropriate amount of TRizol was added and vortexed to fully lyse cells. Lysed cells can be directly used for the next step of extraction or frozen at -80℃ and extracted together after all samples were collected. 200ul chloroform was added to the sample and vortexed to mix thoroughly. After mixing, the sample was allowed to stand at room temperature for 10min and then placed on ice for 10min. When the sample was clearly stratified, it was centrifuged at 13000rpm for 30min in a precooled 4℃ centrifuge. After centrifugation, the sample was taken out and the solution was divided into 3 layers, namely the supernatant layer (containing RNA), the middle layer (containing protein) and the organic layer. The supernatant aqueous phase was gently aspirated with an RNase free pipette tip (be careful not to aspirate the middle layer) and placed in a new imported 1.5ml In the EP tube, add an equal volume of ice-cold isopropanol solution, add 5ul glycogen, gently invert and mix, and place at -20℃ for precipitation for more than 1 hour; wash the precipitated RNA once with 75% ethanol (anhydrous ethanol solution prepared with DEPC water), remove the supernatant, air-dry, add appropriate amount of DEPC water, place in a 4℃ refrigerator to dissolve, then measure the concentration and store in a -80℃ refrigerator. Using TAKARA's PrimeScript RT Master Mix Perfect Real Time Kit (Cat#RR036A), mix the mixture containing RNA, centrifuge briefly to make the reaction solution sink to the bottom of the PCR tube, and invert it with a PCR instrument. Mix the mixture containing RNA, centrifuge briefly to make the reaction solution sink to the bottom of the PCR tube, and invert it with a PCR instrument.

[0339] 11. Real-time PCR

[0340] The SRBR PCR Master Mix (ABI) system was used to perform real-time fluorescence quantitative detection of the mRNA level of the target molecule on the cDNA obtained by reverse transcription. Three replicate wells were set for each sample when detecting the target gene. The HPRT gene was used as an internal reference for experiments and analysis. The real-time fluorescence quantitative PCR used the SYBR GREEN method, the reagents were purchased from TaKaRa, and the test instrument was the QuanStudio6 Flex 384-well real-time fluorescence quantitative PCR instrument (Life). The data were analyzed by QuantStudio TM The results were analyzed using the 2-ΔΔCt method using Real-Time PCR Software.

[0341] 12. RNA-seq

[0342] Pure ILC2 cells were isolated and sorted from immunized mice, cultured in vitro with serum-containing 1640 medium, and cytokines IL-2, IL-7, and IL-33 were added. After treatment with 300ug / ml 5-HT for 72h, the cells were harvested, centrifuged at 500g, 4°C, the supernatant was removed, and PBS was added for washing once, then centrifuged again, the supernatant was removed, and TRIzolTM Reagent (Invitrogen) was added for subsequent RNA extraction. The library was established using VAHTS Total RNA-seq (H / M / R) Library PrepKit from Novo Biotech Co., Ltd. After the cDNA library was established, it was sent to Novo for sequencing, and the sequencing instrument was IIlumina HiSeq-PE150.

[0343] 13. High Performance Liquid Chromatography

[0344] 50ul of mouse peripheral blood was collected by orbital blood sampling, and allowed to stand at room temperature for more than 3h. The blood was centrifuged at 12000rpm for 20min, and the supernatant was taken (avoiding red blood cells). A certain amount of perchloric acid was then added to precipitate protein (the final concentration of perchloric acid was 0.4M). After adding perchloric acid and mixing, the blood was placed on ice for 20min, and centrifuged at 4℃ at 20000rpm for 20min. The supernatant was taken, diluted with double distilled water, and then tested on a machine (experimental testing instrument thermo A).

[0345] Example 1 Depression is closely related to type II immune response, and the neurotransmitter 5-HT can inhibit the activation of human peripheral blood ILC2 cells

[0346] In the type II cytokine inflammatory response, the dynamic and complex interaction between ILC and the nervous system is still unclear, which has become a hot spot and difficulty in current research. The present invention uses papain to induce type II immune response mediated by ILC2 cells, and treats them with 5-HT reuptake inhibitors (SSRI: Escitalopram), a common drug for clinical depression patients. It is found that the type II immune response of mice in the drug-treated group is significantly weakened, mainly manifested by a significant decrease in eosinophils ( Figure 1 , A), ILC2 cell proliferation was significantly reduced ( Figure 1 , B), the ability of ILC2 cells to produce cytokines was inhibited ( Figure 1 , C). The experimental results show that depression is closely related to type II immune response, and the depression-related neurotransmitter 5-HT plays an important role in it. To further explore the function of 5-HT, we isolated ILC2 cells from healthy human PBMC and cultured them in vitro, and treated them with 5-HT. We found that 5-HT can inhibit the production of cytokines IL-5 and IL-13 by ILC2 cells ( Figure 1, D). The above experiments indicate that the depression-related neurotransmitter 5-HT may regulate type II immune response by inhibiting ILC2 cells.

[0347] Example 2 5-HT inhibits ILC2 cell proliferation and cytokine production

[0348] Through the above experiments, we found that 5-HT inhibited the production of cytokines IL-5 and IL-13 by human peripheral blood ILC2 cells. In order to further explore the mechanism of 5-HT on ILC2 cell activation, we isolated activated ILC2 cells from mouse models, cultured them with cytokines IL-2, IL-7, and IL-33 in vitro, and treated them with 5-HT. After 3 days, the cells were harvested, RNA was extracted, and a cDNA library was established to make RNA-seq ( Figure 2 , A). RNA-seq analysis revealed that the changes in genes in the cell proliferation-related signaling pathways in the 5-HT treatment group were more obvious. In addition, the expression of cytokines at the mRNA level in the 5-HT treatment group was also reduced to a certain extent ( Figure 2 , B). We isolated ILC2 cells from a papain-induced long-term lung inflammation model, added cytokines IL-2, IL-7, and IL-33 in vitro and cultured them for 3 days. We also treated them with 5-HT and collected the cells 3 days later. We then performed flow cytometry analysis to detect the proliferation of ILC2 cells and found that 5-HT could inhibit the proliferation of ILC2 cells in a dose-dependent manner ( Figure 2 , C, D). In addition, we collected the supernatant after culture, detected the production of cytokines by Elisa, collected cells, and used flow cytometry to detect ILC2 cells that produced cytokines IL-5 and IL-13, and made corresponding statistical graphs of their proportions ( Figure 2 , E, F). The results showed that the production of cytokines by ILC2 cells decreased in a 5-HT concentration gradient-dependent manner. In the 5-HT treatment group, the proportion of IL-5+, IL-13+ and double-positive cells was significantly reduced. The above experiments showed that 5-HT affects the activation of ILC2 cells. In addition to proliferation, we detected the death and apoptosis of ILC2 cells ( Figure 2 , G, It was found that 5-HT did not affect the cell stability and apoptosis of ILC2. When the 5-HT concentration increased, there was no significant change in ILC2 cell apoptosis.

[0349] Example 3 5-HT inhibits the function and proliferation of ILC2 cells by regulating the p-ERK-GATA3 signaling pathway

[0350] To further explore the molecular mechanism of 5-HT regulating ILC2 cells, activated ILC2 cells were isolated from mouse models, cultured with cytokines IL-2, IL-7, and IL-33 in vitro, and treated with 5-HT. After 3 days, the cells were harvested and RNA was extracted. The mRNA expression levels of cytokines IL-5, IL-13, and GATA3 in the 5-HT treatment group were detected by QPCR. It was found that 5-HT did not affect the mRNA expression levels of IL-5, IL-13, and GATA3 ( Figure 3 , A). GATA3 can specifically bind to the promoter region of cytokines such as IL-5 and IL-13, promote cytokine transcription, and participate in Th2 cell activation. ILC2 cells are very similar to Th2 cells and also mainly produce cytokines IL-5 and IL-13. Therefore, GATA3 also plays a key role in the development, cell plasticity and activation of ILC2 cells. We speculate that 5-HT may affect the function of ILC2 cells by regulating the expression of GATA3. Therefore, we used flow cytometry to detect the expression of GATA3 protein level and found that cells treated with 5-HT for 72 hours showed reduced GATA3 expression and showed a certain 5-HT dose dependence ( Figure 3 , B, C). In summary, the in vitro experimental results prove that 5-HT inhibits the activation of ILC2 cells by affecting the stability of GATA3 protein levels. p-ERK p-P38, calcium flow, HDAC activity, and pyruvate metabolism can all regulate ILC2 cell activation. First, we treated cells with inhibitors of these signaling pathways to explore the molecular mechanism by which 5-HT regulates GATA3 protein stability. We found that when ILC2 cells were treated with p-ERK inhibitors, the inhibition of 5-HT on ILC2 cell function disappeared ( Figure 3 , D), indicating that 5-HT affects ILC2 cell function through the p-ERK signaling pathway. We isolated papain-induced long-term lung inflammation model ILC2 cells, cultured them in vitro, and treated them with different concentrations of 5-HT to detect the expression of p-ERK in ILC2 cells. As the dose of 5-HT increased, 5-HT significantly inhibited the expression of p-ERK in ILC2 cells ( Figure 3, E). Does 5-HT affect GATA3 protein stability by affecting ERK protein phosphorylation? U0126 is an inhibitor of phosphorylated ERK. In cells treated with U0126, the expression of p-ERK and GATA3 protein levels were significantly reduced. At this time, treatment with 5-HT could not inhibit the expression of GATA3 protein levels. The experimental results prove that 5-HT inhibits GATA3 protein stability by regulating ERK phosphorylation. The cells were first treated with MG132, an inhibitor of the proteasome pathway, and then treated with 5-HT. It was found that after treatment with MG132, 5-HT no longer inhibited the expression of cell GATA3, indicating that 5-HT affects GATA3 protein stability through proteasome degradation. The cells were treated with MG132 and U0126 at the same time, and then treated with 5-HT. It was found that 5-HT could not inhibit the expression of GATA3, proving that 5-HT affects GATA3 protein stability through proteasome degradation mediated by the p-ERK signaling pathway ( Figure 3 , F), at the same time, after we treated ILC2 cells with another ERK inhibitor, 5-HT did not affect GATA3 protein expression ( Figure 3 , G).

[0351] Example 4 5-HT inhibits ILC2 cell function and thus reduces papain-induced lung inflammation To further explore the function of 5-HT in asthma, we constructed a papain-induced mouse lung inflammation model ( Figure 4 , A), papain is a protease extracted from plants. Continuous immunization of mice by nasal or bronchial drops will cause damage to mouse epithelial cells and produce a large number of cytokines such as IL-25, IL-33, TSLP, etc. These cytokines will rapidly activate downstream innate lymphocytes ILC2 cells. The activated ILC2 cells will release cytokines IL-5 and IL-13 to rapidly mediate lung inflammation. Mice were immunized according to the above immunization method, and the lung inflammation response of mice was detected on the 6th day. Eosinophils are the detection index of the intensity of inflammation in this model. We detected the infiltration of the cells in the lungs and the pathological conditions of the lung tissues and found that 5-HT can significantly inhibit the number and proportion of eosinophils ( Figure 4 , B). Immunohistochemical staining also demonstrated that 5-HT significantly inhibited the infiltration of inflammatory cells in the lungs and the secretion of mucus by goblet cells ( Figure 4 , C). In addition, we found that the amount of cytokines IL-5 and IL-13 in lung tissue homogenate and bronchoalveolar lavage fluid was significantly reduced ( Figure 4, D, E). We isolated mouse lung monocytes, stained them, and then used flow cytometry to detect and count the number and proportion of ILC2 cells in the papain-induced mouse lung inflammation model. We found that the number and proportion of ILC2 cells in mice treated with the neurotransmitter 5-HT were significantly reduced, and with the increase in the dose of 5-HT, they became dose-dependent ( Figure 4 , F, G). In addition, 5-HT can dose-dependently inhibit the production of cytokines IL-5 and IL-13 by ILC2 cells, as shown by a significant decrease in the number and proportion of ILC2 cells producing cytokines IL-5 and IL-13 ( Figure 4 , H). In the 5-HT treatment group, the expression of p-ERk in ILC2 cells was significantly decreased, the expression of GATA3 protein level was decreased, and the mRNA level did not change significantly ( Figure 4 , I, J). The experimental results show that 5-HT inhibits the function of ILC2 cells in the papain-induced mouse lung inflammation model, thereby reducing lung inflammation.

[0352] Example 5 5-HT inhibits papain-induced lung inflammation independently of T and B cells

[0353] Existing data show that the TPH1 gene can regulate the activation of Th2 cells through antigen presenting cells DC cells, and Th2 cells play an important role in the occurrence of asthma. In order to further exclude that the regulation of 5-HT on ILC2 cells in the papain-induced lung inflammation model is independent of T and B cells, we expanded Rag-deficient mice, immunized the mice with papain, modeled the disease, and treated them with 5-HT at the same time. The alveolar lavage fluid of the mice was isolated and the number and proportion of inflammatory cells eosinophils in the alveolar lavage fluid were detected. After 5-HT treatment, the number and proportion of eosinophils were significantly inhibited ( Figure 5 , A). We further demonstrated through paraffin sections and immunohistochemical staining that 5-HT affects the infiltration of inflammatory cells in the lungs and the secretion of mucus by goblet cells ( Figure 5 , B). The above experiments prove that the inhibitory function of 5-HT on papain-induced lung inflammation is through regulating the activation of ILC2 cells, and this regulation is independent of T and B cells. On the 6th day, the mouse lung monocytes were isolated, and the number and proportion of ILC2 cells were detected by flow cytometry. It was found that 5-HT can reduce the number and proportion of ILC2 cells ( Figure 5 , C), at the same time, we re-stimulated the isolated lung monocytes with PMA and Inomycin, and detected and analyzed the number and proportion of ILC2 cells that secreted cytokines IL-5 and IL-13 ( Figure 5, D, E), the results showed that 5-HT significantly inhibited the production of IL-5 and IL-13 by ILC2 cells. The above experiments proved that the effect of 5-HT on the activation of ILC2 cells is independent of T and B cells.

[0354] Example 6 5-HT can inhibit ILC2 cell response in IL-33 and fungal induced type II immune response

[0355] IL-33 is currently the most effective cytokine for activating ILC2 cells, so we used the cytokine IL-33-induced lung inflammation model ( Figure 6 , A), further exploring the function of 5-HT in regulating lung inflammation, in the IL-33-induced lung inflammation model, the number and proportion of ILC2 cells in the lungs of mice treated with 5-HT were significantly reduced ( Figure 6 , B), the number of activated ILC2 cells, i.e., ILC2 cells secreting cytokines IL-5 and IL-13, was significantly reduced ( Figure 6 , C), the above proves that in the IL-33-induced lung inflammation model, ILC2 cell activation is inhibited by 5-HT, and the production of cytokines is significantly reduced. Through the detection of eosinophils in alveolar lavage fluid, as well as HE and PAS staining of lung tissue pathological sections, it was found that the inflammatory cell infiltration in the lungs of mice treated with 5-HT was significantly reduced, the mucus secretion of goblet cells was significantly reduced, and the lung inflammation was significantly alleviated ( Figure 6 , D, E). In addition, we established a fungal infection model that is closer to clinical asthma ( Figure 6 , F), in this model, it can be clearly seen that 5-HT inhibits the number and proportion of ILC2 cells ( Figure 6 , G), the production of cytokines IL-5 and IL-13 by ILC2 cells was also significantly reduced ( Figure 6 , H). The above experiments prove that in the cytokine and fungus-induced lung inflammation model, 5-HT can also reduce ILC2 cell activation and cytokine secretion, thereby alleviating lung inflammation.

[0356] Example 7 5-HT does not affect ILC1 and ILC3 cells

[0357] Innate lymphoid cells (ILCs) are mainly divided into three subgroups: type I innate lymphoid cells (ILC1), type II innate lymphoid cells (ILC2), and type III innate lymphoid cells (ILC3). Type I innate lymphoid cells mainly play an important role in antiviral infection and anti-tumor immunity, type II innate lymphoid cells mainly play a role in the clearance of allergic diseases such as asthma, dermatitis, and parasitic infections, and type III innate lymphoid cells are closely related to intestinal inflammation. We have experimentally proved that 5-HT affects the function of ILC2 cells, thereby regulating lung inflammation. Whether 5-HT also has a regulatory effect on other subgroups of the ILC family, ILC1 and ILC3, we will next explore the regulation of 5-HT on ILC1 and ILC3 cells. ILC1 and ILC3 cells exist in large numbers in the intestinal tissue. We first treated mice with 5-HT or PBS by intraperitoneal perfusion, isolated colon tissue, and detected the effect of 5-HT treatment on ILC1, ILC2, and ILC3 cells in the colon tissue of mice. The results showed that after 5-HT treatment, the proportion of ILC1, ILC2, and ILC3 in the colon tissue of mice was not affected ( Figure 7 , A, B), indicating that 5-HT does not affect ILC1 and ILC3 in the resting state. To further explore the effect of 5-HT on the function of ILC1 and ILC3 cells under inflammatory conditions, we constructed a DSS-induced mouse enteritis model. Starting from day 0, 5-HT or PBS was given by intraperitoneal injection for 5 consecutive days. The weight changes of mice were detected every day. The weight of mice was counted on day 7 and the mice were processed for relevant tests. It was found that compared with the control group, the weight of mice in the DSS group was significantly reduced. After 5-HT or PBS treatment in the DSS group, there was no difference in the weight of mice, which proved that 5-HT did not affect DSS-induced enteritis ( Figure 7 , C). In addition, we detected the production of cytokines IL-17 and IFN-r by colon ILC3 cells by flow cytometry and found that there was no difference in the proportion of ILC3 cells producing cytokines IL-17 and IFN-r between the PBS group and the 5-HT treatment group, that is, 5-HT did not affect the function of ILC3 cells ( Figure 7 , D, E).

[0358] Example 8 5-HT acts on the HTR2A receptor on the surface of ILC2 cells to inhibit the immune response mediated by ILC2 cells

[0359] To further explore the molecular mechanism by which 5-HT inhibits the proliferation and function of ILC2 cells, we isolated ILC2 cells from mouse lung tissue and detected the expression of 5-HTR mRNA in ILC2 cells. We found that HTR2A was expressed in lung tissue. The highest expression was in ILC2 ( Figure 8, A), at the same time, we isolated different cell subsets from mouse lung tissue and spleen, detected the expression of HTR2A mRNA levels in different cell subsets, and found that HTR2A was specifically highly expressed in ILC2 cells ( Figure 8 , B), Due to certain differences between humans and mice, we isolated PBMC cells from healthy human peripheral blood, sorted different immune cell subsets by flow cytometry, and detected the expression of HTR2A mRNA levels. We found that HTR2A is also specifically highly expressed in ILC2 cells in human peripheral blood ( Figure 8 , C). The experimental results suggest that 5-HT may function through HTR2A. We produced Htr2a f / f Mice, with PLZF Cre The mice were bred to obtain mice with HTR2A knockout in ILC cells, and ILC2 cells were isolated to detect the knockout of HTR2A ( Figure 8 , D, E). We treated the mice with papain and found that the number of ILC2 cells in HTR2A knockout mice increased significantly ( Figure 8 , F), the number and proportion of ILC2 cells producing cytokines IL-5 and IL-13 increased significantly ( Figure 8 , G), the number and proportion of eosinophils in bronchoalveolar lavage fluid also increased significantly ( Figure 8 , H). In addition, through in vitro culture experiments, we found that after HTR2A knockout, the inhibitory effect of 5-HT on ILC2 cell proliferation was significantly weakened. ( Figure 8 , I). 2,5-dimethoxy-4-iodoamphetamine (DOI) is a specific inhibitor of HTR2A. We first induced a short-term lung inflammation model with papain and simultaneously treated the mice with 5-HTR2A agonist DOI to detect lung inflammation in mice. In the DOI-treated group, the number and proportion of eosinophils in the bronchoalveolar lavage fluid were significantly reduced ( Figure 8 , J), the number and proportion of ILC2 cells in lung monocytes were also significantly reduced ( Figure 8 , K). The number and proportion of ILC2 cells secreting cytokines IL-5 and IL-13 in lung tissue were significantly reduced ( Figure 8 , L), and the effect of DOI is significantly better than that of 5-HT. The above experiments show that 5-HT acts on the HTR2A receptor on the surface of ILC2 cells and inhibits the immune response mediated by ILC2 cells. DOI provides a new idea for the treatment of asthma.

[0360] Example 9 TPH1 is not involved in the regulation of ILC2 cell function

[0361] TPH1 (Tryptophan hydroxylase 1) is the rate-limiting enzyme for peripheral 5-HT synthesis. We treated TPH1 WT and KO mice with papain and found that there was no difference in the number and proportion of eosinophils in the bronchoalveolar lavage fluid and ILC2 cells in the lung tissue between WT and KO mice, and the function of ILC2 cells was not different ( Fig. 9 , AC). In addition, we isolated ILC2 cells from WT and KO mice and cultured them in vitro and found that TPH1 knockout did not affect the proliferation and function of ILC2 cells ( Fig. 9 , D, E). In addition, we treated mice with papain and found that after TPH1 knockout and papain treatment, the amount of 5-HT in the lung tissue and bronchoalveolar lavage fluid of mice increased significantly, indicating that 5-HT from other sources may regulate the function of ILC2 cells ( Fig. 9 , F). To further explore the function of TPH1 in ILC2 cells, we performed a transfer experiment in which ILC2 cells isolated from WT and KO mice were transferred to Rag2 - / - Iq - / - Mice were treated with papain to induce lung inflammation. It was found that there was no difference in the number of eosinophils in the bronchoalveolar lavage fluid and the number of ILC2 cells in the lung tissue between WT and KO mice, and there was no difference in the function of ILC2 cells ( Fig. 9 , GI). The above experimental results indicate that TPH1 is not involved in the regulation of ILC2 cell function.

[0362] Example 10 5-HT can alleviate type II immune response mediated by ILC2 cells

[0363] 5-HT can inhibit the activation of ILC2 cells and reduce lung inflammation in different lung inflammation models. Moreover, activated ILC2 cells isolated from the body and cultured with 5-HT in vitro can also inhibit the production of cytokines by ILC2, indicating that 5-HT still has an inhibitory function on activated ILC2. From an application perspective, 5-HT is very likely to treat lung inflammation. In order to explore the therapeutic efficacy of 5-HT, we immunized mice with papain for 5 consecutive days to construct a short-term lung inflammation model, treated them with 5-HT on the 6th day, and tested the lung inflammation of mice on the 7th and 9th days respectively ( Fig.10 , A). It was found that the number and proportion of ILC2 cells in the lung tissue of the 5-HT treatment group were significantly reduced on the 7th and 9th days ( Fig.10 , B), In addition, we detected the number and proportion of ILC2 cells producing IL-5 and IL-13 by flow cytometry, and the results showed that the number and proportion of ILC2 cells producing IL-5 and IL-13 in the lungs of 5-HT-treated mice were also significantly reduced ( Fig.10, C). In addition, the number and proportion of eosinophils in bronchoalveolar lavage fluid were found to be significantly reduced ( Fig.10 , D), HE and PAS staining further showed that lung inflammation was significantly reduced ( Fig.10 , E). In summary, 5-HT inhibits the function of activated ILC2 cells, reduces the production of cytokines IL-5 and IL-13, thereby reducing the infiltration of inflammatory cells in the lungs and mucus secretion, achieving the effect of treating lung inflammation in mice, and is expected to provide new ideas for the treatment of asthma.

[0364] Example 11 DOI can alleviate type II immune response mediated by ILC2 cells

[0365] Given that 5-HT has 14 receptors in the body and a relatively wide range of action, it is critical to find small molecule compounds with more specific targets that can treat lung inflammation. In vivo and in vitro experiments have shown that the 5-HTR2A agonist DOI can significantly inhibit ILC2 cell activation and reduce lung inflammation in mice. Its effect is better than 5-HT. In vitro culture experiments have also shown that DOI inhibits activated ILC2 cells from producing cytokines IL-5 and IL-13. It is speculated that the 5-HTR2A agonist DOI is likely to treat lung inflammation. We selected a papain-induced lung inflammation model, immunized mice for 5 consecutive days, treated them with DOI on the 6th day, and tested the lung inflammation of mice on the 7th day ( Fig.11 , A). In the DOI-treated group, the number of eosinophils in bronchoalveolar lavage fluid was significantly reduced, and the number and proportion of ILC2 cells in lung tissue were also significantly reduced ( Fig.11 , B), In addition, we detected the number and proportion of ILC2 cells secreting cytokines IL-5 and IL-13 by flow cytometry. The results showed that the number and proportion of ILC2 cells producing IL-5 and IL-13 in the lungs of mice treated with DOI were significantly reduced ( Fig.11 , C). HE and PAS staining further showed that the infiltration of inflammatory cells in the lungs was significantly reduced, and the mucus secreted by goblet cells was also significantly reduced ( Fig.11 , D).

[0366] In summary, DOI treats lung inflammation in mice by inhibiting the function of ILC2 cells. This small molecule compound is a selective 5-HTR2A agonist with a relatively specific target, and is expected to provide a new drug target for the treatment of clinical asthma.

[0367] Summary and discussion

[0368] 1. Through the papain-induced mouse lung inflammation model, we found that SSRI, a common drug for depression patients, can inhibit the function of ILC2 cells and relieve mouse lung inflammation. In addition, through in vitro culture experiments, we found that the ability of human ILC2 cells treated with depression-related neurotransmitter 5-HT to produce IL-5 and IL-13 was significantly weakened. The above experimental results show that 5-HT is an important neurotransmitter that links depression and lung tissue inflammation, and it affects lung inflammation by regulating ILC2 cells.

[0369] 2. Through flow cytometry sorting and ILC2 cell in vitro culture system, it was found that 5-HT inhibited the proliferation of ILC2 cells in mouse lung tissue and the production of cytokines IL-5 and IL-13. Through further mechanism exploration, it was found that 5-HTR2A was specifically highly expressed in ILC2 cells, and 5-HT mainly functions through its cell surface receptors, affecting the downstream p-ERK signaling pathway and then affecting the degradation of the GATA3 proteasome pathway, regulating the proliferation of ILC2 cells and cytokine production.

[0370] 3. Through in vivo experiments, it was further found that 5-HT inhibited the type II immune response induced by papain, IL-33, fungi, etc. by regulating the proliferation and function of ILC2 cells. In addition, 5-HT and HTR2A receptor-specific agonist DOI can significantly alleviate lung inflammation in mice. This further proves the importance of depression-related neurotransmitter 5-HT to type II immune response at the animal level. At present, small molecule drugs are safer and more effective to use, so 5-HT and DOI as small molecules can be used in combination with SSRI, a commonly used drug for depression, to better treat asthma.

[0371] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences <120> Application of serotonin or its receptor agonists in the treatment of ILC2 cell-mediated immune diseases <130> P2020-2329 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 471 <212> PRT <213> Homo sapiens <400> 1 Met Asp Ile Leu Cys Glu Glu Asn Thr Ser Leu Ser Ser Thr Thr Asn 1 5 10 15 Ser Leu Met Gln Leu Asn Asp Asp Thr Arg Leu Tyr Ser Asn Asp Phe 20 25 30 Asn Ser Gly Glu Ala Asn Thr Ser Asp Ala Phe Asn Trp Thr Val Asp 35 40 45 Ser Glu Asn Arg Thr Asn Leu Ser Cys Glu Gly Cys Leu Ser Pro Ser 50 55 60 Cys Leu Ser Leu Leu His Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80 Thr Ala Val Val Ile Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile 85 90 95 Met Ala Val Ser Leu Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe 100 105 110 Leu Met Ser Leu Ala Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120 125 Pro Val Ser Met Leu Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130 135 140 Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu Asp Val Leu Phe Ser Thr 145 150 155 160 Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala 165 170 175 Ile Gln Asn Pro Ile His His Ser Arg Phe Asn Ser Arg Thr Lys Ala 180 185 190 Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Ser Val Gly Ile Ser Met 195 200 205 Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Ser Lys Val Phe Lys Glu 210 215 220 Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Val Leu Ile Gly Ser Phe 225 230 235 240 Val Ser Phe Phe Ile Pro Leu Thr Ile Met Val Ile Thr Tyr Phe Leu 245 250 255 Thr Ile Lys Ser Leu Gln Lys Glu Ala Thr Leu Cys Val Ser Asp Leu 260 265 270 Gly Thr Arg Ala Lys Leu Ala Ser Phe Ser Phe Leu Pro Gln Ser Ser 275 280 285 Leu Ser Ser Glu Lys Leu Phe Gln Arg Ser Ile His Arg Glu Pro Gly 290 295 300 Ser Tyr Thr Gly Arg Arg Thr Met Gln Ser Ile Ser Asn Glu Gln Lys 305 310 315 320 Ala Cys Lys Val Leu Gly Ile Val Phe Phe Leu Phe Val Val Met Trp 325 330 335 Cys Pro Phe Phe Ile Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser 340 345 350 Cys Asn Glu Asp Val Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile 355 360 365 Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr Thr Leu Phe Asn 370 375 380 Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys 385 390 395 400 Glu Asn Lys Lys Pro Leu Gln Leu Ile Leu Val Asn Thr Ile Pro Ala 405 410 415 Leu Ala Tyr Lys Ser Ser Gln Leu Gln Met Gly Gln Lys Lys Asn Ser 420 425 430 Lys Gln Asp Ala Lys Thr Thr Asp Asn Asp Cys Ser Met Val Ala Leu 435 440 445 Gly Lys Gln His Ser Glu Glu Ala Ser Lys Asp Asn Ser Asp Gly Val 450 455 460 Asn Glu Lys Val Ser Cys Val 465 470 <210> 2 <211> 422 <212> PRT <213> Homo sapiens <400> 2 Met Asp Val Leu Ser Pro Gly Gln Gly Asn Asn Thr Thr Ser Pro Pro 1 5 10 15 Ala Pro Phe Glu Thr Gly Gly Asn Thr Thr Gly Ile Ser Asp Val Thr 20 25 30 Val Ser Tyr Gln Val Ile Thr Ser Leu Leu Leu Gly Thr Leu Ile Phe 35 40 45 Cys Ala Val Leu Gly Asn Ala Cys Val Val Ala Ala Ile Ala Leu Glu 50 55 60 Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Ile Gly Ser Leu Ala Val 65 70 75 80 Thr Asp Leu Met Val Ser Val Leu Val Leu Pro Met Ala Ala Leu Tyr 85 90 95 Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Val Thr Cys Asp Leu Phe 100 105 110 Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Ser Ile Leu His Leu Cys 115 120 125 Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asp Pro Ile Asp Tyr 130 135 140 Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Ala Leu Ile Ser Leu Thr 145 150 155 160 Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro Pro Met Leu Gly Trp Arg 165 170 175 Thr Pro Glu Asp Arg Ser Asp Pro Asp Ala Cys Thr Ile Ser Lys Asp 180 185 190 His Gly Tyr Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile Pro Leu 195 200 205 Leu Leu Met Leu Val Leu Tyr Gly Arg Ile Phe Arg Ala Ala Arg Phe 210 215 220 Arg Ile Arg Lys Thr Val Lys Lys Val Glu Lys Thr Gly Ala Asp Thr 225 230 235 240 Arg His Gly Ala Ser Pro Ala Pro Gln Pro Lys Lys Ser Val Asn Gly 245 250 255 Glu Ser Gly Ser Arg Asn Trp Arg Leu Gly Val Glu Ser Lys Ala Gly 260 265 270 Gly Ala Leu Cys Ala Asn Gly Ala Val Arg Gln Gly Asp Asp Gly Ala 275 280 285 Ala Leu Glu Val Ile Glu Val His Arg Val Gly Asn Ser Lys Glu His 290 295 300 Leu Pro Leu Pro Ser Glu Ala Gly Pro Thr Pro Cys Ala Pro Ala Ser 305 310 315 320 Phe Glu Arg Lys Asn Glu Arg Asn Ala Glu Ala Lys Arg Lys Met Ala 325 330 335 Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu Gly Ile Ile Met Gly 340 345 350 Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Ile Val Ala Leu Val Leu 355 360 365 Pro Phe Cys Glu Ser Ser Cys His Met Pro Thr Leu Leu Gly Ala Ile 370 375 380 Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Leu Asn Pro Val Ile Tyr 385 390 395 400 Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala Phe Lys Lys Ile Ile Lys 405 410 415 Cys Lys Phe Cys Arg Gln 420 <210> 3 <211> 390 <212> PRT <213> Homo sapiens <400> 3 Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pro Pro Pro Ala Gly Ser 1 5 10 15 Glu Thr Trp Val Pro Gln Ala Asn Leu Ser Ser Ala Pro Ser Gln Asn 20 25 30 Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp Ser Ile Ser Leu Pro Trp 35 40 45 Lys Val Leu Leu Val Met Leu Leu Ala Leu Ile Thr Leu Ala Thr Thr 50 55 60 Leu Ser Asn Ala Phe Val Ile Ala Thr Val Tyr Arg Thr Arg Lys Leu 65 70 75 80 His Thr Pro Ala Asn Tyr Leu Ile Ala Ser Leu Ala Val Thr Asp Leu 85 90 95 Leu Val Ser Ile Leu Val Met Pro Ile Ser Thr Met Tyr Thr Val Thr 100 105 110 Gly Arg Trp Thr Leu Gly Gln Val Val Cys Asp Phe Trp Leu Ser Ser 115 120 125 Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu His Leu Cys Val Ile Ala 130 135 140 Leu Asp Arg Tyr Trp Ala Ile Thr Asp Ala Val Glu Tyr Ser Ala Lys 145 150 155 160 Arg Thr Pro Lys Arg Ala Ala Val Met Ile Ala Leu Val Trp Val Phe 165 170 175 Ser Ile Ser Ile Ser Leu Pro Pro Phe Phe Trp Arg Gln Ala Lys Ala 180 185 190 Glu Glu Glu Val Ser Glu Cys Val Val Asn Thr Asp His Ile Leu Tyr 195 200 205 Thr Val Tyr Ser Thr Val Gly Ala Phe Tyr Phe Pro Thr Leu Leu Leu 210 215 220 Ile Ala Leu Tyr Gly Arg Ile Tyr Val Glu Ala Arg Ser Arg Ile Leu 225 230 235 240 Lys Gln Thr Pro Asn Arg Thr Gly Lys Arg Leu Thr Arg Ala Gln Leu 245 250 255 Ile Thr Asp Ser Pro Gly Ser Thr Ser Ser Val Thr Ser Ile Asn Ser 260 265 270 Arg Val Pro Asp Val Pro Ser Glu Ser Gly Ser Pro Val Tyr Val Asn 275 280 285 Gln Val Lys Val Arg Val Ser Asp Ala Leu Leu Glu Lys Lys Lys Leu 290 295 300 Met Ala Ala Arg Glu Arg Lys Ala Thr Lys Thr Leu Gly Ile Ile Leu 305 310 315 320 Gly Ala Phe Ile Val Cys Trp Leu Pro Phe Phe Ile Ile Ser Leu Val 325 330 335 Met Pro Ile Cys Lys Asp Ala Cys Trp Phe His Leu Ala Ile Phe Asp 340 345 350 Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Ile Ile 355 360 365 Tyr Thr Met Ser Asn Glu Asp Phe Lys Gln Ala Phe His Lys Leu Ile 370 375 380 Arg Phe Lys Cys Thr Ser 385 390 <210> 4 <211> 471 <212> PRT <213> Mus musculus <400> 4 Met Glu Ile Leu Cys Glu Asp Asn Ile Ser Leu Ser Ser Ile Pro Asn 1 5 10 15 Ser Leu Met Gln Leu Gly Asp Asp Ser Arg Leu Tyr Pro Asn Asp Phe 20 25 30 Asn Ser Arg Asp Ala Asn Thr Ser Glu Ala Ser Asn Trp Thr Ile Asp 35 40 45 Ala Glu Asn Arg Thr Asn Leu Ser Cys Glu Gly Tyr Leu Pro Pro Thr 50 55 60 Cys Leu Ser Ile Leu His Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80 Thr Thr Val Val Ile Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile 85 90 95 Met Ala Val Ser Leu Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe 100 105 110 Leu Met Ser Leu Ala Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120 125 Pro Val Ser Met Leu Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130 135 140 Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu Asp Val Leu Phe Ser Thr 145 150 155 160 Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala 165 170 175 Ile Gln Asn Pro Ile His His Ser Arg Phe Asn Ser Arg Thr Lys Ala 180 185 190 Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Ser Val Gly Ile Ser Met 195 200 205 Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Ser Lys Val Phe Lys Glu 210 215 220 Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Val Leu Ile Gly Ser Phe 225 230 235 240 Val Ala Phe Phe Ile Pro Leu Thr Ile Met Val Ile Thr Tyr Phe Leu 245 250 255 Thr Ile Lys Ser Leu Gln Lys Glu Ala Thr Leu Cys Val Ser Asp Leu 260 265 270 Ser Thr Arg Ala Lys Leu Ser Ser Phe Ser Phe Leu Pro Gln Ser Ser 275 280 285 Leu Ser Ser Glu Lys Leu Phe Gln Arg Ser Ile His Arg Glu Pro Gly 290 295 300 Ser Tyr Ala Gly Arg Arg Thr Met Gln Ser Ile Ser Asn Glu Gln Lys 305 310 315 320 Ala Cys Lys Val Leu Gly Ile Val Phe Phe Leu Phe Val Val Met Trp 325 330 335 Cys Pro Phe Phe Ile Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser 340 345 350 Cys Asn Glu Asn Val Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile 355 360 365 Gly Tyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr Thr Leu Phe Asn 370 375 380 Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys 385 390 395 400 Glu Asn Arg Lys Pro Leu Gln Leu Ile Leu Val Asn Thr Ile Pro Thr 405 410 415 Leu Ala Tyr Lys Ser Ser Gln Leu Gln Val Gly Gln Lys Lys Asn Ser 420 425 430 Gln Glu Asp Ala Glu Pro Thr Ala Asn Asp Cys Ser Met Val Thr Leu 435 440 445 Gly Asn Gln His Ser Glu Glu Met Cys Thr Asp Asn Ile Glu Thr Val 450 455 460 Asn Glu Lys Val Ser Cys Val 465 470 <210> 5 <211> 421 <212> PRT <213> Mouse (Mus musculus) <400> 5 Met Asp Met Phe Ser Leu Gly Gln Gly Asn Asn Thr Thr Thr Ser Leu 1 5 10 15 Glu Pro Phe Gly Thr Gly Gly Asn Asp Thr Gly Leu Ser Asn Val Thr 20 25 30 Phe Ser Tyr Gln Val Ile Thr Ser Leu Leu Leu Gly Thr Leu Ile Phe 35 40 45 Cys Ala Val Leu Gly Asn Ala Cys Val Val Ala Ala Ile Ala Leu Glu 50 55 60 Arg Ser Leu Gln Asn Val Ala Asn Tyr Leu Ile Gly Ser Leu Ala Val 65 70 75 80 Thr Asp Leu Met Val Ser Val Leu Val Leu Pro Met Ala Ala Leu Tyr 85 90 95 Gln Val Leu Asn Lys Trp Thr Leu Gly Gln Val Thr Cys Asp Leu Phe 100 105 110 Ile Ala Leu Asp Val Leu Cys Cys Thr Ser Ser Ile Leu His Leu Cys 115 120 125 Ala Ile Ala Leu Asp Arg Tyr Trp Ala Ile Thr Asp Pro Ile Asp Tyr 130 135 140 Val Asn Lys Arg Thr Pro Arg Arg Ala Ala Ala Leu Ile Ser Leu Thr 145 150 155 160 Trp Leu Ile Gly Phe Leu Ile Ser Ile Pro Pro Met Leu Gly Trp Arg 165 170 175 Thr Pro Glu Asp Arg Ser Asn Pro Asn Glu Cys Thr Ile Ser Lys Asp 180 185 190 His Gly Tyr Thr Ile Tyr Ser Thr Phe Gly Ala Phe Tyr Ile Pro Leu 195 200 205 Leu Leu Met Leu Val Leu Tyr Gly Arg Ile Phe Arg Ala Ala Arg Phe 210 215 220 Arg Ile Arg Lys Thr Val Lys Lys Val Glu Lys Lys Gly Ala Gly Thr 225 230 235 240 Ser Phe Gly Thr Ser Ser Ala Pro Pro Pro Lys Lys Ser Leu Asn Gly 245 250 255 Gln Pro Gly Ser Gly Asp Cys Arg Arg Ser Ala Glu Asn Arg Ala Val 260 265 270 Gly Thr Pro Cys Ala Asn Gly Ala Val Arg Gln Gly Glu Asp Asp Ala 275 280 285 Thr Leu Glu Val Ile Glu Val His Arg Val Gly Asn Ser Lys Gly His 290 295 300 Leu Pro Leu Pro Ser Glu Ser Gly Ala Thr Ser Tyr Val Pro Ala Cys 305 310 315 320 Leu Glu Arg Lys Asn Glu Arg Thr Ala Glu Ala Lys Arg Lys Met Ala 325 330 335 Leu Ala Arg Glu Arg Lys Thr Val Lys Thr Leu Gly Ile Ile Met Gly 340 345 350 Thr Phe Ile Leu Cys Trp Leu Pro Phe Phe Ile Val Ala Leu Val Leu 355 360 365 Pro Phe Cys Glu Ser Ser Cys His Met Pro Glu Leu Leu Gly Ala Ile 370 375 380 Ile Asn Trp Leu Gly Tyr Ser Asn Ser Leu Leu Asn Pro Val Ile Tyr 385 390 395 400 Ala Tyr Phe Asn Lys Asp Phe Gln Asn Ala Phe Lys Lys Ile Ile Lys 405 410 415 Cys Lys Phe Cys Arg 420 <210> 6 <211> 386 <212> PRT <213> Mus musculus <400> 6 Met Glu Glu Gln Gly Ile Gln Cys Ala Pro Pro Pro Pro Ala Ala Ser 1 5 10 15 Gln Thr Gly Val Pro Leu Thr Asn Leu Ser His Asn Cys Ser Ala Asp 20 25 30 Gly Tyr Ile Tyr Gln Asp Ser Ile Ala Leu Pro Trp Lys Val Leu Leu 35 40 45 Val Ala Leu Leu Ala Leu Ile Thr Leu Ala Thr Thr Leu Ser Asn Ala 50 55 60 Phe Val Ile Ala Thr Val Tyr Arg Thr Arg Lys Leu His Thr Pro Ala 65 70 75 80 Asn Tyr Leu Ile Ala Ser Leu Ala Val Thr Asp Leu Leu Val Ser Ile 85 90 95 Leu Val Met Pro Ile Ser Thr Met Tyr Thr Val Thr Gly Arg Trp Thr 100 105 110 Leu Gly Gln Val Val Cys Asp Phe Trp Leu Ser Ser Asp Ile Thr Cys 115 120 125 Cys Thr Ala Ser Ile Met His Leu Cys Val Ile Ala Leu Asp Arg Tyr 130 135 140 Trp Ala Ile Thr Asp Ala Val Glu Tyr Ser Ala Lys Arg Thr Pro Lys 145 150 155 160 Arg Ala Ala Ile Met Ile Val Leu Val Trp Val Phe Ser Ile Ser Ile 165 170 175 Ser Leu Pro Pro Phe Phe Trp Arg Gln Ala Lys Ala Glu Glu Glu Met 180 185 190 Leu Asp Cys Phe Val Asn Thr Asp His Val Leu Tyr Thr Val Tyr Ser 195 200 205 Thr Val Gly Ala Phe Tyr Leu Pro Thr Leu Leu Leu Ile Ala Leu Tyr 210 215 220 Gly Arg Ile Tyr Val Glu Ala Arg Ser Arg Ile Leu Lys Gln Thr Pro 225 230 235 240 Asn Lys Thr Gly Lys Arg Leu Thr Arg Ala Gln Leu Ile Thr Asp Ser 245 250 255 Pro Gly Ser Thr Ser Ser Val Thr Ser Ile Asn Ser Arg Ala Pro Asp 260 265 270 Val Pro Ser Glu Ser Gly Ser Pro Val Tyr Val Asn Gln Val Lys Val 275 280 285 Arg Val Ser Asp Ala Leu Leu Glu Lys Lys Lys Leu Met Ala Ala Arg 290 295 300 Glu Arg Lys Ala Thr Lys Thr Leu Gly Ile Ile Leu Gly Ala Phe Ile 305 310 315 320 Val Cys Trp Leu Pro Phe Phe Ile Ile Ser Leu Val Met Pro Ile Cys 325 330 335 Lys Asp Ala Cys Trp Phe His Met Ala Ile Phe Asp Phe Phe Asn Trp 340 345 350 Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Ile Ile Tyr Thr Met Ser 355 360 365 Asn Glu Asp Phe Lys Gln Ala Phe His Lys Leu Ile Arg Phe Lys Cys 370 375 380 Ala Gly 385 <210> 7 <211> 5415 <212> DNA <213> Homo sapiens <400> 7 agagagctac tccattcccg tggaaccag gagtcccttg gtgcagacag ctcttcctac 60 tttcccatgc agttctttg tgcgacttg aggggctcgt gatgatttc taaatgtgtg 120 cctgctgagg cgagccgcac agggagggag gaacccagcc gagccgtgcc agaggaagcc 180 aacaggatcc tagcagtgcg ggagctggct cagctcttgc atgcagtttt tgagtcagc 240 aaaaacagaaa catchattact atcattatt gctggtgga catchagaag agggactct 300 acaccagttt aattactgtg agagatgcag cgagtcacag ataacaat gtatctcatg 360 tgtggaccct gagacaaat gatattac ttcccgagcg ctcaaaaaaa accctgcaac 420 ctctatgcta aaagttcatt ctgcttttt gtcctcggtt tggtgagaaa atataaaac 480 caacagtgg actctcctaa aattgtgaat gagaaaact tacagccacc acagttcagt 540 tctttaacta tcattgtaat aatggagac aaaaatccag ccccgggaga acaccatgta 600 caccagccctc agtgttacag agtgtggta catchaggtg aatggtgagc agaaactata 660 acctgttagt ccttctacac ctcatctgct acaagttctg gcttagacat ggatattctt 720 tgtgaagaaa atacttcttt gagctcaact acgaactccc taatgcaatt aaatgatgac 780 accaggctct acagtaatga ctttaactcc ggagaagcta acacttctga tgcatttaac 840 tggacagtcg actctgaaaa tcgaaccaac ctttcctgtg aagggtgcct ctcaccgtcg 900 tgtctctcct tacttcatct ccaggaaaaa aactggtctg ctttactgac agccgtagtg 960 attattctaa ctattgctgg aaacatactc gtcatcatgg cagtgtccct agagaaaaag 1020 ctgcagaatg ccaccaacta tttcctgatg tcacttgcca tagctgatat gctgctgggt 1080 ttccttgtca tgcccgtgtc catgttaacc atcctgtatg ggtaccggtg gcctctgccg 1140 agcaagcttt gtgcagtctg gatttacctg gacgtgctct tctccacggc ctccatcatg 1200 cacctctgcg ccatctcgct ggaccgctac gtcgccatcc agaatcccat ccaccacagc 1260 cgcttcaact ccagaactaa ggcatttctg aaaatcattg ctgtttggac catatcagta 1320 ggtatatcca tgccaatacc agtctttggg ctacaggacg attcgaaggt ctttaaggag 1380 gggagttgct tactcgccga tgataacttt gtcctgatcg gctcttttgt gtcatttttc 1440 attcccttaa ccatcatggt gatcacctac tttctaacta tcaagtcact ccagaaagaa 1500 gctactttgt gtgtaagtga tcttggcaca cgggccaaat tagcttcttt cagcttctc 1560 cctcagagtt ctttgtcttc agaaaagctc ttccagcggt cgatccatag ggagccaggg 1620 tcctacacag gcaggagc tatgcagtcc atcagcaatg agcaaaaggc atgcaaggtg 1680 ctgggcatcg tcttcttcct gtttgtggtg atgtggtgcc ctttcttcat caaaacatc 1740 atggccgtca tctgcaaaga gtcctgcaat gaggatgtca ttggggccct gctcaatgtg 1800 tttgtttgga tcggttatct ctcttcagca gtcaacccac tagtctacac actgttcaac 1860 aagacctata ggtcagcctt ttcacggtat attcagtgtc agtacaagga aaacaaaaaa 1920 ccattgcagt taattttagt gaacaata ccggctttgg cctacaagtc tagccaactt 1980 2040 atggttgctc taggaaagca gcattctgaa gaggcttcta aagacaatag cgacggagtg 2100 aatgaaaagg tgagctgtgt gtgataggct agttgccgtg gcaactgtgg aaggcacact 2160 gagcaagttt tcacctatct ggaaaaaaaa aaatatgaga ttggaaaaaa ttagacaagt 2220 ctagtggaac caacgatcat atctgtatgc ctcattttat tctgtcaatg aaaagcgggg 2280 ttcaatgcta caaaatgtgt gcttggaaaa tgttctgaca gcatttcagc tgtgagcttt 2340 ctgatactta tttataacat tgtaaatgat atgtctttaa aatgattcac ttttattgta 2400 taattatgaa gccctaagta aatctaaatt aacttctatt ttcaagtgga aaccttgctg 2460 ctatgctgtt cattgatgac atgggattga gttggttacc tattgctgta aataaaaata 2520 gctataaata gtgaaaattt tattgaatat aatggcctct taaaaattat ctttaaaact 2580 tactatggta tatattttga aaggagaaaa aaaaagccac taaggtcagt gttataaaat 2640 ctgtattgct aagataatta aatgaaatac ttgacaacat ttttcattcc tgctttttca 2700 tagataccat ttgaaatat tcacaaggtt gctggcattt gctgcatttc aagttaattc 2760 tcagaagtga aaaagacttc aaatgttatt caataactat tgctgctttc tcttctactt 2820 cttgtgcttt actctgaatt tccagtgtgg tcttgttaa tatttgttcc tctaggtaaa 2880 ctagcaaaag gatgatttaa cattaccaaa tgcctttcta gcaattgctt cctaaaaca 2940 gcactatcga ggtatttggt aacttgctgt gaaatgactg catcatgcat gcactcttttt 3000 gagcagtaaa tgtatattga tgtaactgtg tcaggattga ggatgaactc agttttccgg 3060 ctactgacag tggtagagtc ctaggacatc tctgtaaaaa gcaggtgact ttcctatgac 3120 actcatcagg taaactgatg ctttcagatc catcggttta tactatttat taaaaccatt 3180 ctgcttggtt ccacaatcat ctattgagtg tacatttatg tgtgaagcaa atttctagat 3240 atgagaaata taaaaataat taaaaaaaa tccttgcctt caaacgaaat ggctcggcca 3300 ggcacggagg ctcgtgcatg taatcctagc actttgggag gctgagatgg gaggatcact 3360 tgaggccaag agttgagac caacctgggt aaaaagtga gacctccctg tctctacaaa 3420 aaaaatcaaa aaattatctg atccttgtgg cacacaactg tggtcccagc tacaggggag 3480 gctgagacgc aaggatcact tgagcccaga agctcaaggc tgcagtgagc caagttcaca 3540 ccactgccat ttcctcctgg gcacagagt gagaccctt cacaaaaaa aagaaagaaa 3600 aaaagaaaat gactaactg aaatggtctt agtcaaaaa tatatacaga cttatttta 3660 gatcatttc aacttatttc cttcttacct gttcagaaa caagttttgtt tcaactgtga 3720 gagtagtttt aaaaataagt tttcctgatt ggttaccaat aaacaccaaaaaaagg 3780 agggggaaaaa tatcacttga ggcttacat tgcattaat ttagaatac gcacctcaat 3840 ctcaagccag tttataacta ccatagtttt gagcaaaattt ttctctat ttctccaaaa 3900 actcactcat ttcagtatta ctcattgaca ctgggctccc taatagaaaa gcagatatta 3960 taatattca ttgaatggt tactaagag aaaagagat taacacagt taatccacct 4020 tgatattata acactcagg ttaattgca tgttgttatt agaatcaatg ttatatat 4080 gattacaatg ctgaagcatt tctattgaat gagaatagt gtttgcccta aacctttggg 4140 aaaaaaatac agatttgaa actaaagat gtgggaagtg tcattgtgta atttggaat 4200 gcataaaatt ctcattatgt cctcatagat atgttgaag atggttcact gtttcaaga 4260 tagaatgtat aatatgctgc tagtaccaag gctgcatttg aactgaacta ctccttatga 4320 tataccatac taactgatga aaatataaat gttaaaatgt ctccactcta atgtattttg 4380 ccttcacttg taactctgca catttcatta tatattaggg aaaagattac agattctacc 4440 cccatatact caatcttgat cagaatgtac attaatgttg cctgatagat gtcttttgac 4500 tacctatata ttacaaatgt gtatgtgttt ttttcttcca taagtatcta ttcaagtgta 4560 tactgtatag gcaaatgtgt agctgtatat atgtacagac atgttctcat gcactttatt 4620 caatatttat atatttatat ggacgaaaag caagtcaatg aaaacactca gtatatacac 4680 atgtgtgcat gtttgtatca tgtttattta aatgtgaatt catgttctaa tatttctatg 4740 accttccatt gctgcaaagc agattaaagt gtaacaaaaa tatcagctct ttaaataaac 4800 tggaaattaa tctaattttg tggaagaact ttaagacaag tctatgagga tggcgaaaac 4860 aatggtccac atatatagtt tcttgatcct aaataaaatg gtacctttgg gccctttgtt 4920 tagtctggca atttctgac tactatggct ttttaaacag actcacacct aagtgaaaca 4980 cagttgtcaa atgatttatg gatctgtgat taaaggattc acttttaatg aatactatta 5040 aaacggagta acaaaccttg ctctacttta agttgaatga tagattcctc tatattcaaa 5100 aaaaagcaat taactgccat aattagggaa aactgtttaa agcatactgc atttacttgg 5160 ccacagcgcc atctcatggt aattttttaa agtgctaccg tagccaaatt gaactaagtc 5220 actgtactgc tttcagcaaa gggtgctcct cccatttgtg catcaatgag acatatttat 5280 aaagtgctaa attattctgt gccatatgta acaaatacag tgaagattat tttatgaact 5340 tattttaatc aaggcgatgc taaaagtttt caagaaagga taaataactg taaataaagt 5400 agactcaaaa atgaa 5415 <210> 8 <211> 2937 <212> DNA <213> Mouse (Mus musculus) <400> 8 attcttttgg agctccggga gcctcttgat acagacagcg tttcctactt tccctgcggc 60 tcttttgtgt gactttgaga ggggctccct agtgactgat tcctctctgt gcgctcgcct 120 cagcaagcag ccaggctctg ggagggaaga cccaaccaag ctcagcagta gcagaagcag 180 ccagcacaga cttcaaccgt gctggaaccg gcatgcggtt tttgaagtca gcagaaacag 240 aaatcaatt actatcatct tatgctgcta gaagctcaaa gagaggggat tccacactgg 300 cctagtcacc atgcgacagg caagtcacag gatagcggac cctggcgaca atgaatgtaa 360 gttcctcggc tgctgtcctt tactgctgtg ggattttctt tctgcttaaa gtcacgcttg 420 gctagaatgc cgtcattgcc gcgagaattt ctgatgtgga aagttctctg ctgcgcttcg 480 ggatgcgttt tccttcgtt aactgagcca gagatacaag ctgaaattcc tgaccatcgc 540 tgcggcgatt ctgcctgaga ctaaaaaggg ttaaccctta tgatggcatt aacattgcgt 600 ggattttaa ttgacttcct taattaatat agagggcaca cagccctccc tcctcgtttt 660 ggatctcatg ctgttttaac tttgtgatgg ctgaactctt gaaagcagca tatccaaccc 720 gagaattggc tgaaagattc tcaccggata caaaactttt cttccttaac caggaacacg 780 tttgtgtctc caaatgctcc acactgcttt ttttgccttt gcttccgtga gaacttacag 840 ctccgccgtg gactctccct agcactgtga agcgaggcat aatcaagagc catcacactt 900 ctgtaactct tactatggaa gaggagaaag cagccagagg agccacacag gtctccgctt 960 cagcatgccc tagctccagg acgtaaagat gaatggtgag ccccggctat gactcgctag 1020 tctctccaca cttcatctgc tacaacttcc ggcttagaca tggaaattct ctgtgaagac 1080 aatatctccc tgagctcaat tccaaactcc ttaatgcaat taggtgacga ctcgaggctc 1140 taccctaatg acttcaactc cagggatgct aacacttccg aagcctcgaa ctggacaatt 1200 gatgctgaaa acagaaccaa cctctcctgc gaagggtacc tcccaccgac atgcctctcc 1260 attcttcatc tccaggaaaa aaactggtct gctttattga caactgtcgt gattattctc 1320 accattgcgg gaaacatact ggtcatcatg gcagtgtccc tagagaaaaa gctgcagaat 1380 gccaccaact atttcctgat gtcacttgcc atagctgata tgctgctggg tttccttgtc 1440 atgcccgtgt ccatgttaac catcctgtat gggtaccggt ggcctttgcc cagcaagctc 1500 tgtgccgtct ggatttacct ggatgtgctc ttctccacgg cgtccatcat gcacctctgc 1560 gccatctccc tggaccgcta cgtggctatc cagaacccca ttcaccatag ccgcttcaac 1620 tccagaacca aagccttcct gaaaatcatt gcggtgtgga ccatatccgt aggtatatcc 1680 atgccaatcc cagtcttcgg gctacaggat gattcgaagg tctttaagga agggagctgc 1740 ctgctcgccg atgacaactt tgtcctcata ggctcttttg tggcattttt catcccccta 1800 accatcatgg tgatcaccta cttcctgact atcaagtcac ttcagaaaga agccaccttg 1860 tgtgtgagtg acctcagcac tcgggccaaa ttatcctcct tcagcttcct ccctcagagt 1920 tctctgtcat cagaaaagct cttccagcgg tccatccaca gagagccagg ctcctacgca 1980 ggccgaagga cgatgcagtc catcagcaac gagcaaaaag catgcaaggt gctgggcatc 2040 gtgttctttc tgtttgttgt aatgtggtgc ccattcttca tcaccaatat catggccgtc 2100 atctgcaaag aatcctgcaa tgaaaatgtc attggagccc tgctcaatgt gtttgtctgg 2160 attggttatc tctcctcagc cgtcaaccca ctggtatata cgttgttcaa taaaacttat 2220 aggtccgcct tctcacggta cattcagtgc cagtacaagg agaacagaaa gccgctgcag 2280 ttaattttag tgaacactat accaacattg gcctacaagt ctagtcagct ccaggtggga 2340 caaaaaaaga actcacagga agatgctgag ccgacagcta atgactgctc catggttaca 2400 ctagggaacc aacactcgga agagatgtgt acagacaata ttgaaaccgt gaatgaaaag 2460 gttagctgtg tgtgatggac cggatgctgg gacgattgcc cagggtgtgt gaacaagttt 2520 acacccacgt gtgtgggggc aggataagga ggctcagatc aaattagact actccagtgg 2580 accaactata atgtccggac agcatttgaa ctaggagtgt tgtgatgctt ttaacattgc 2640 caatgagatc tttaaaatca ttggccttta ttgtacaatt gtaatgaggc ataaaatcca 2700 tctaattaac ttctattgtc aaatagaaac cgggctgcca tgttgttgat gggtagcatg 2760 ggagtaagtt ggtgacatat tgttgtatat aaaaatagct ataaatactg aaaataatga 2820 atagcctctt taaaaacaaa aatgagtgtt tcaaattcac cacaacatat attttgaaaa 2880 gaaaaataga cagtattatg acatttgtat tgctaataca attcattgaa atacttg 2937 <210> 9 <211> 4572 <212> DNA <213> Homo sapiens <400> 9 gttgacaaaa agagactcga atgcaagac gctgagctag agggagag gggcggggac 60 ccagaggaaa gaggcactcc tcggggttgg ggaagtatta ggaggggagg gttagagtgg 120 gaggaagga gcctggcttt cgaagcgact cacagaggga taaataagg gaagtgagga 180 gagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagggagg 240 gagagagggagggagggagggagggagggagggagggagggagggagggag 300 cacagagtga ccgtggagga tggcttct cggttctaga tattctggg attggact 360 gtttgctagt ggggactc cagctccggc agccagttcg ggagcggcaa agtaaaatgg 420 acagcgacag acagacgttc cagccaccctc tccgccgccg ggagatcctg gagctgctt 480 caggccaact ccagttccc agctggagct tctgaacgcg ctggactgcg agagcccagg 540 gagcgcctga aagctgctcc tcggagatc ccttcgccga agcagtaaga acttcctgct 600 tgggtctctg cattcccttc ctccgaact tcccaggaga agggcggaag accccagggg 660 aaggggcgag gcgaatctc gcgctgcttt ttctccctc ccccttcccg cgccgggcgc 720 gcaggcatgg atgtgctcag ccctggtcag ggcaacaaca ccacatcacc accggctccc 780 tttgagaccg gcggcaacac tactggtatc tccgacgtga ccgtcagcta ccaagtgatc 840 acctctctgc tgctgggcac gctcatcttc tgcgcggtgc tgggcaatgc gtgcgtggtg 900 gctgccatcg ccttggagcg ctccctgcag aacgtggcca attatcttat tggctctttg 960 gcggtcaccg acctcatggt gtcggtgttg gtgctgccca tggccgcgct gtatcaggtg 1020 ctcaacaagt ggacactggg ccaggtaacc tgcgacctgt tcatcgccct cgacgtgctg 1080 tgctgcacct catccatctt gcacctgtgc gccatcgcgc tggacaggta ctgggccatc 1140 acggacccca tcgactacgt gaacaagagg acgccccggc gcgccgctgc gctcatctcg 1200 ctcacttggc ttattggctt cctcatctct atcccgccca tgctgggctg gcgcaccccg 1260 gaagaccgct cggaccccga cgcatgcacc attagcaagg atcatggcta cactatctat 1320 tccacctttg gagctttcta catcccgctg ctgctcatgc tggttctcta tgggcgcata 1380 ttccgagctg cgcgcttccg catccgcaag acggtcaaaa aggtggagaa gaccggagcg 1440 gabacccgcc atggagcatc tcccgccccg cagcccaaga agatgtga tggagtcg 1500 gggagcagga actggaggct gggcgtggag agcaggctg ggggtgctct gtgcgccaat 1560 ggcgcggtga ggcaggtga cgatggcgcc gccctggagg tgatcgaggt gcaccgagtg 1620 ggcaactcca agagcactt gcctctgccc agcgaggctg gtcctacccc ttgtgcccc 1680 gcctctttcg agaggaaaaa tgagcgcaac gccgaggcga agcgcagat gccctggcc 1740 cgagagagga agacagtgaa gacgctgggc atcatcatgg gcaccttcat cctctgctgg 1800 ctgcccttct tcatcgtggc tcttgttctg cccttctgcg agagcagctg ccacatgccc 1860 accctgttgg gcgccataat caatggctg ggctactcca actctctgct taaccccgtc 1920 atttacgcat acttcaaca ggactttca aacgcgttta agagatcat tagtgtag 1980 ttctgccgcc agtgatgacg gaggagtagc cggccagtcg aggctacagg atccgtccca 2040 ttcactatgc ttccccaac cctagggaat caacacttaa gatatcgc cactctccct 2100 ctttctctct gctccgctca cggcttgcag acctgtccc ctcccactt cctgctccac 2160 ggcagggccc tttgtgcaaa ggagacccag cggaggagcg ttgagagccc aggaaattca 2220 gagagtttgt gagaagcgac attggctcag acttcgcctg tatcatcagt tttgatccca 2280 gtaattgcct cttctctctt ctatctccta aatctttgcg gtggatgttt aatgcttagt 2340 tcaaggcaga aaatccaaaa aataaataaa ctgtacacac agccgccgcg cccactaagg 2400 ggctccaccc catcacccaa cttctagaac ttggacttga ggtttgggaa tttgctccat 2460 ctctcccgcc ctgtggccct tgaaatattc acctttttct ccccattttt atcttttctt 2520 tttttcattg cttccctctc tgccctcgcc catccttatg cacgaatctg ctggtccgga 2580 gaggcggaag aggctgcttt tccccgtgcc agcggtgccc ccgggattcc ccgtcttctc 2640 gcagacttga gattaactct ctcgtgcagt gcacaacccc tatttccttt gtttccttgg 2700 actcaaagca agtggctaga aggtatcttt tagagtggac ttgtaagtac aaggccgggc 2760 cagtggggga gagcaaggag gaggagggtg tgcagaccac tttgtttatg cttgccttgg 2820 tgtcagcact caccaaagaa actgacaatc atacagtgga gagagcggac gctgtttgta 2880 caggtagctg gcgagtgtgt acgacctgac atcgccactt acttactgcg ggaggcgcca 2940 gccggccgct gtttgcgctc tggggaagct gagaggcctg gaatccgcgc gcctccccag ccatctgtgc tctcgcagcg cgctaggcgc tcccggctgc tgacttacag gatctgggtt 3060 cctccgccta cgctctgggg cgccctgaag cggcgggtaga cacctgaggt ttttgcctta 3120. gccagtggcg caagatgccg cggaactctt gggattggag agaccaaga ggctttaaag gggcgacacg caggggcggc aaagaggaa ggttccccag ggcgcgcgtg cccaaggctc 3240 cagctggctc ccccagtaaa acctgaaaga gcaatttt ccttacgatg tagaattttt cctcctaatt cacgctcttg tccttttttgg atcggggaat aattctgttt agcgtgtccc 3360. 3420. aacactcgct gcatttgctt aacactcgct gcatttgctt ccaagacaca aagttagcag aactttgttt tccaaatagg aattctgggc tttcaatgga atgcccagat agtccatagg ttagcactat ctgtgacaac aggcagatag tttaatagc agggtcctaa aggactagtt aggaggtggt cttcctgggg aggctgagtc ttttatgggga ttaattgctc cctagcgtga ccattctgct gtaccaaggg tctgcggaaa ccaccttttt gccctttgaa ttgtgcagca ttccaggctt 3660 tctctccttc agttattctt gaagccttgc cactcctgcc gagaaactgg ttaaaatctt 3720 tgctgcccca gaaagggaag ttatttaggt actctggatg taaatttcca actacatgat 3780 ggagatctag aggttgtca ttgtttgatc tagcctccaa ttttcttatg aataatggtc 3840 ttgttttcca gaaaattcac aaagtaattg atccctactc ttttctctct tcagctgtac 3900 ctttcgaatg ttttcatttg ctgttttgag aaccgtatta catttacaga ttgcaaaaat 3960 tgccagtgaa aagttcattt gtttataatt aagaaagtga aaggacagct ctgtcagttg 4020 aagccatttt attgtttgtc agactgaaca aagctttcct ttggtgtctg cttctgtatt 4080 ctaaactgga agtagacatc atatcccagc taaacaggga aagttttaga actttgggaa 4140 tagttgtct atgcatgtat aatgctgaga agggtctgca gtgaagaaga taagatagac 4200 ttctccagca caactattac tcatttgata attcagtttc atttttgctc acataatcta 4260 agtaaagcca tatcagaaac caagtttttc tgtatgttac actaaatttt aagaacaaag 4320 tctatattta aaataatcca aaccccaaat ttgctcatcc aaattatgtc ttggccagca 4380 agttttacat tagagtttt gagacttcca tttatttgtg tctttacag aattgtgtga 4440 caatgacttt height tcttcggc height caatgactt height 4500 aacatccaac tggagcataa ttgtcaggc atagagaat ttagagaat aaatagctg 4560 catalog aa 4572 <210> 10 <211> 2568 <212> DNA <213> Homo sapiens <400> 10 aaacctgcga tcgccacggt ccttccgccc tctccttcgt ccgctccatg cccaagagct 60 gcgctccgga gctggggcga ggagagccat ggaggaccg ggtgctcagt gcgctccacc 120 gccgcccgcg ggctccgaga cctgggttcc tcaagccaac ttacctcg ctccctccca 180 aaactgcagc gccaaggact accattacca ggactccatc tcctaccct ggaaagtact 240 gctggttatg ctattggcgc tcacctt ggccaccacg ctctccaatg cctttgtgat 300 tgccacagtg taccggaccc ggaaactgca caccccggct aactacctga tcgcctct 360 ggcggtcacc gacctgcttg tgtccatcct ggtgatgccc atcagcacca tgtacactgt 420 caccggccgc tggacactgg gccaggtggt ctgtgacttc tggctgtcgt cggacatcac 480 ttgttgcact gcctccatcc tgcacctctg tgtcatcgcc ctggaccgct actgggccat 540 cacggacgcc gtggagtact cagctaaaag gactcccaag agggcggcgg tcatgatcgc 600 gctggtgtgg gtcttctcca tctctatctc gctgccgccc ttcttctggc gtcaggctaa 660 ggccgaagag gaggtgtcgg aatgcgtggt gaacaccgac cacatcctct acacggtcta 720 ctccacggtg ggtgctttct acttccccac cctgctcctc atcgccctct atggccgcat 780 ctacgtagaa gcccgctccc ggattttgaa acagacgccc aacaggaccg gcaagcgctt 840 gacccgagcc cagctgataa ccgactcccc cgggtccacg tcctcggtca cctctattaa 900 ctcgcgggtt cccgacgtgc ccagcgaatc cggatctcct gtgtatgtga accaagtcaa 960 agtgcgagtc tccgacgccc tgctggaaaa gaagaactc atggccgcta gggagcgcaa 1020 agccaccaag accctaggga tcattttgg agcctttat gtgtgttggc tacccttctt 1080 catcatctcc ctagtgatgc ctatctgcaa agatgcctgc tggttccacc tagccatctt 1140 tgacttcttc acatggctgg gctatctcaa ctccctcatc aaccccataa tctataccat 1200 gtccaatgag gactttaaac aagcattcca taaactgata cgttttaagt gcacaagttg 1260 acttgccgtt tgcagtgggg tcgcctaagc gacctttggg gaccaagttg tgtctggttc 1320 cacaggtagg tcgaatcttc tttcgcggtt tctgggtccc agcgaggctc tctctcctgg 1380 gcaagggcaa tggatcctga gaagccagaa tagtcctgag agagagctct gaaaggagaa 1440 gtgttgaaac taaatgtaga gcttccctgc ccaggaggag gctcacttcc tcccctcaag 1500 ccccgggctc agcactgacc ctgcggcagc caatcccaaa gggggttgca acttttaaaa 1560 attgataatg gaagggaatc cctgccctgc tttggtatcg tggataatgc ccactagaag 1620 cagtgtactt gtaattgttg tctgaagcct gtctgagaca gatctacata cagcctggca 1680 gtacttgaac tagacgctta atgccctgtg tttttggggg gagaactttg tgttacagct 1740 taatttaaga acagttactt tggcatcatt cagtcttcac tttttgtcta tttaaacttg 1800 gttggagaaa cttgtggatt tggtgcttca aaccctatgt gtggcttgga tggcgcagag 1860 aaaccttgaa gagttaacag caaaattctg atgctgagat ctctattttt attatacttg 1920 aaactatatg ggggtgggtg ggtgggaatg ggagatgagg agtgttaaac tgagaatcaa 1980 cacctatgat tgtttgtttt ctgcagattt acaattttgt aattcctgtt tagcgattgt 2040 caagccacaa ctctaacaaa caaaccatta tgtgtgctag tgccgaagtc tgcagactgc 2100 tttatttttt ctcttaattt catgtacctg tcactttaca catttaaatc cccataaatg 2160 aagggtatga tgggtgactc agcccacact gctgctatat ttcttactaa tgcaattggt 2220 aaaaccgatt agtattggaa atatactgtt tcttaacaag aaaagtgtct ttatttctta 2280 tccaatttag tgagatgtga aggagactga tgacatgggg atagttctta cacaattgag 2340 gaatggggtg ggggcaatag gaggatgtat attttgactt gtaaaaaaat cttaaaatgc 2400 atgaaacttt tatctgatag tcatttgcac tctccttccc atctgtgatt ccttgtgtgc 2460 taacatataa agaaaccaag agaactatct tccttctcca gaaaccttaa aaatacagtt 2520 aagggcccta aaaacgatat tgaaaagaaa ataaacttgt ttcttttt 2568 <210> 11 <211> 4441 <212> DNA <213> Mouse (Mus musculus) <400> 11 gagaaggaaa ggggaactgg aagggaaggc cagtgggagg aagccgggag aaggaagctg 60 ggaaaagaga agcagactgg agaaggcagg agagatggag agacagagag aaggaactag 120 gagatgaggg tcatcgagtg acccgggagg atggggtttc tggaatccag agacttctga 180 gactattagt tactcgggag agtccggccc ccacgcagcc agttggacag cgacaaagtg 240 aaatggacag cgcgagacag atattacagc catctctcca gcacccagag atttggaaga 300 tgctttcagc ccaactacag tttcctagct ggagcctctg agcgctcagg acccgcggga 360 gccaagcaag agagcgccgg tgagtgctcc tctcagatgc cttcgccaga gcaagtaaga 420 acttcccgct ccagtctcta gatcccctcc cttcgaaact ccccagaaag gaaggaagac 480 cctaggggga ggggcgaggg agatcccctt gcatttcttt ttccctccct tccccccacc 540 ccagcgggag tgcaggcagg catggatatg ttcagtcttg gccagggcaa caacaccaca 600 acgtccctgg agcccttcgg gacaggcggc aacgatactg gcctctccaa cgtgaccttc 660 agctaccaag tgatcacctc tcttttgctg gggacgctca ttttctgcgc ggtgctcggc 720 aatgcctgcg tggttgctgc catcgccctg gagcgctccc ttcagaatgt tgccaactat 780 ctcatcggct ccttggcggt caccgatctc atggtgtcag tgctggtgct gcccatggct 840 gctctgtacc aggtgctcaa caagtggact ctgggtcagg tcacctgtga cctgtttatc 900 gccctggatg tgctgtgctg cacttcgtcc atcctgcacc tgtgcgccat cgcgctagac 960 aggtactggg caatcaccga ccctatagac tacgtgaaca agaggacgcc ccggcgcgcc 1020 gctgcgctga tctcgctcac ttggctcatt ggctttctca tctccatccc gcctatgctg 1080 ggctggcgca ccccggaaga ccgctcgaac cccaacgagt gcaccatcag caaggaccac 1140 gggtacacca tctactccac tttcggcgct ttctatattc cgctgctgct catgctggtc 1200 ctctatgggc gcatcttcag agccgcgcgc ttccgaatcc gcaagacggt caagaaggtg 1260 gaaaagaagg gagcgggcac cagcttcgga acatcgtcgg ccccgccccc caagaagagc 1320 ctgaatggtc agccaggtag tggggactgc aggcgcagtg ctgagaacag ggcggtgggg 1380 actccgtgcg ctaatggggc ggtgagacag ggtgaggacg acgccaccct ggaggtgatc 1440 gaggtgcatc gagtgggcaa ctccaaaggg caccttcctc tgcccagcga atcaggagct 1500 acctcctatg tccccgcctg cttggagaga aaaaatgagc gcactgctga ggcaaagcgc 1560 aagatggcct tggcccgtga gaggaagaca gtgaagacac tgggcatcat catgggcacc 1620 ttcatcctct gctggctgcc ctttttcatt gtggccctgg tcctaccttt ctgtgagagc 1680 agttgccaca tgcctgagtt gttgggtgcc ataattaact ggctaggcta ctccaactcc 1740 ctgctcaacc cagttattta tgcttatttc aacaaagact ttcaaaacgc ttttaagaag 1800 atcatcaagt gcaagttctg ccgctgatga tgatgatggt ggtggtagtg gtggtaatag 1860 tggtggtgat ggagaaggaa gaagtggagg aagagtgtag ggcttacagg acccttcctg 1920 ttcactcaat atccccgccc tggaagcaac acctaagaaa acttgctttt cctagaacac 1980 gcagcttctt agcccatggc tttcagatct tctctctcta tctcatttta aggggacagt 2040 gctttttgca gaaatcttag agaaccccta gcatctgttt gtaaaattat tggctcggtc 2100 tttggcccta ggatcagttt tgatagttgc aataacctcc tctctattcg aaatcccaat 2160 tcttcacgat ggaagtttaa acctcggcga ttcaaggaag aaaacccaaa cagaaaatat 2220 aaactgtatg gacatctacc atgcccacta ggactgccac cctctgccct atatctggaa 2280 ctttgaggtt caagactgct ctgtctctcc ctccctcttg ctctagaaac atctgttccc 2340 tttgggtgtc aatgtacact gctgattctg gtctccatgc actcacggct ggtctgggaa 2400 agttggaact actttgggtt atggtgccct ggaattgttt gccttgctga atagcaaaag 2460 atccctttcc ccaatgcaca gcacccactt tctgtatttt gtttgtttgt ttgttttaac 2520 tcaaaatcaa gtgacaaaga tgtctgctgg agtggactta taagcaagaa gccaggccca 2580 aaaggaagag ccaagaaggc ctgcggatgt tttcctgtcc tggtgtcagc actcaccaaa 2640 gaaattgaca atcagactat ttggaaggaa gagttttatt cctgtagttt gttggctttt 2700 ctgtaagttc tggaaaggcc ttgtggactg cagaaggcgc ccactgctgg ccagggaggg 2760 ctcattgagg gtgctgagat gccgggatg gctcacctca ccctcagttt cttttctagg 2820 agtgagctat gccagagcag ctcaccttga gtttgcagcc tccgggttcc tccactcacc 2880 tctcacagta tccaggagct agtagagaag tttgagtttg tgcaagaggt tggagactta 2940 gaatttctaa ggaagtgtgg gtgtgaggca cgtagaggag gtaaggagca aaagggctcc 3000 taagtgcttc caactcctgc ggcgtgccca gggggtgggg gtgacctaaa ggggcaattt 3060 taagcatagg atgcaaagtt tctcaatcta ccccttcccc ctttcaagga acagcattgt 3120 gtagcatttc ctaaaaatgc ccggatttat tctccgggac attctgtgct cacacaaatt 3180 tcgcagatct ctgggctctc attgtgaagg gtatcaacac ctcaccatta cctacctagg 3240 atcttgatct cagagaccta aaagagagag cgatctctgt ggagtcccag ccaagtcagc 3300 ccccaaattc tgagggtaaa accagttttg tgtcctctca atttttaaag cattttctc 3360 cgctaggggt tcttttcctg ctgttattt tgctgtccca cccccacctc agagaagaaa 3420 attatttagg tgcttccttt gaatgtacat ttctagctaa gtgagagggc atcaaatggt 3480 tttgcgttat atctgaactt cgaattttct gttgaataat ggtttccttt tcaagcaatt 3540 atacggagat ttttttcacc ccccatcccc ttcagctgta tctttccaat gtctgcacct 3600 gctgttttga aaacccgtgc tgcattttt tttaaattgc caaagttgct ggtgagatct 3660 cccccttgtg tttaataaaca gggagaaagg actgccctgc cacatgaagc cattgtttt 3720 atttgttaaa ctgagcaaag tttcttttgc tctactctaa agtgggaatg agtattacag 3780 tcccttgcaa cagagccatt tcagaatgta agcctgtagg gtgtccacac ctgagagact 3840 gacaggggt tctgtggaga agacgcactg ggctggggctt ctccagtaca aacagtactt 3900 gtttgtaaat ttagcttgct ttattgttt gtttgtttat ttatttatca caaaaatgaa 3960 gtagaactat atggccaaat aatattttc tgtatgcttt actattttta gaccagatca 4020 tttacttgaa ataaagatca tttacttgaa ataaatcaat ccctaaattt cagatgttaa 4080 attggaaacc aagacctcta acttccaggt ctctgtattt ttttttttcc aattaggaat 4140 gtttgaccgt gatgggggag gggcattgct cttgttactt tgagaaagag actttaaaca 4200 agactggaaa gggggcaccc agctggaata taattgccaa gtatagagc atcagagaa 4260 ctaagtgcaa catgccgcac agctctaaag aacccctgat gcttgtttta ttttaaaata 4320 cttgtattac agtaaaatgt taatgcatca aaattggcct tcactttcc ttgtgataat 4380 aaaggacttt agtaatggca atgctgtaat aaactaagac cagtaacta tatgtacctt 4440 p 4441 <210> 12 <211> 2568 <212> DNA <213> Homo sapiens <400> 12 aaacctgcga tcgccacggt ccttccgccc tctccttcgt ccgctccatg cccaagagct 60 gcgctccgga gctggggcga ggagagccat ggaggaccg ggtgctcagt gcgctccacc 120 gccgcccgcg ggctccgaga cctgggttcc tcaagccaac ttacctcg ctccctccca 180 aaactgcagc gccaaggact accattacca ggactccatc tcctaccct ggaaagtact 240 gctggttatg ctattggcgc tcacctt ggccaccacg ctctccaatg cctttgtgat 300 tgccacagtg taccggaccc ggaaactgca caccccggct aactacctga tcgcctct 360 ggcggtcacc gacctgcttg tgtccatcct ggtgatgccc atcagcacca tgtacactgt 420 caccggccgc tggacactgg gccaggtggt ctgtgacttc tggctgtcgt cggacatcac 480 ttgttgcact gcctccatcc tgcacctctg tgtcatcgcc ctggaccgct actgggccat 540 cacggacgcc gtggagtact cagctaaaag gactcccaag agggcggcgg tcatgatcgc 600 gctggtgtgg gtcttctcca tctctatctc gctgccgccc ttcttctggc gtcaggctaa 660 ggccgaagag gaggtgtcgg aatgcgtggt gaacaccgac cacatcctct acacggtcta 720 ctccacggtg ggtgctttct acttccccac cctgctcctc atcgccctct atggccgcat 780 ctacgtagaa gcccgctccc ggattttgaa acagacgccc aacaggaccg gcaagcgctt 840 gacccgagcc cagctgataa ccgactcccc cgggtccacg tcctcggtca cctctattaa 900 ctcgcgggtt cccgacgtgc ccagcgaatc cggatctcct gtgtatgtga accaagtcaa 960 agtgcgagtc tccgacgccc tgctggaaaa gaagaactc atggccgcta gggagcgcaa 1020 agccaccaag accctaggga tcattttgg agcctttat gtgtgttggc tacccttctt 1080 catcatctcc ctagtgatgc ctatctgcaa agatgcctgc tggttccacc tagccatctt 1140 tgacttcttc acatggctgg gctatctcaa ctccctcatc aaccccataa tctataccat 1200 gtccaatgag gactttaaac aagcattcca taaactgata cgttttaagt gcacaagttg 1260 acttgccgtt tgcagtgggg tcgcctaagc gacctttggg gaccaagttg tgtctggttc 1320 cacaggtagg tcgaatcttc tttcgcggtt tctgggtccc agcgaggctc tctctcctgg 1380 gcaagggcaa tggatcctga gaagccagaa tagtcctgag agagagctct gaaaggagaa 1440 gtgttgaaac taaatgtaga gcttccctgc ccaggaggag gctcacttcc tcccctcaag 1500 ccccgggctc agcactgacc ctgcggcagc caatcccaaa gggggttgca acttttaaaa 1560 attgataatg gaagggaatc cctgccctgc tttggtatcg tggataatgc ccactagaag 1620 cagtgtactt gtaattgttg tctgaagcct gtctgagaca gatctacata cagcctggca 1680 gtacttgaac tagacgctta atgccctgtg tttttggggg gagaactttg tgttacagct 1740 taatttaaga acagttactt tggcatcatt cagtcttcac tttttgtcta tttaaacttg 1800 gttggagaaa cttgtggatt tggtgcttca aaccctatgt gtggcttgga tggcgcagag 1860 aaaccttgaa gagttaacag caaaattctg atgctgagat ctctattttt attatacttg 1920 aaactatatg ggggtgggtg ggtgggaatg ggagatgagg agtgttaaac tgagaatcaa 1980 cacctatgat tgtttgtttt ctgcagattt acaattttgt aattcctgtt tagcgattgt 2040 caagccacaa ctctaacaaa caaaccatta tgtgtgctag tgccgaagtc tgcagactgc 2100 tttatttttt ctcttaattt catgtacctg tcactttaca catttaaatc cccataaatg 2160 aagggtatga tgggtgactc agcccacact gctgctatat ttcttactaa tgcaattggt 2220 aaaaccgatt agtattggaa atatactgtt tcttaacaag aaaagtgtct ttatttctta 2280 tccaatttag tgagatgtga aggagactga tgacatgggg atagttctta cacaattgag 2340 gaatggggtg ggggcaatag gaggatgtat attttgactt gtaaaaaaat cttaaaatgc 2400 atgaaacttt tatctgatag tcatttgcac tctccttccc atctgtgatt ccttgtgtgc 2460 taacatataa agaaaccaag agaactatct tccttctcca gaaaccttaa aaatacagtt 2520 aagggcccta aaaacgatat tgaaaagaaa ataaacttgt ttctttt 2568

Claims

1. A method for screening potential therapeutic agents for ILC2 cell-mediated immune diseases, characterized in that: include: (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing serotonin receptors for a period of time T1, and detecting the expression level E1 of serotonin receptors in the culture system of the test group; And in a control group without the test compound and with other conditions being the same, detecting the expression amount E2 of the serotonin receptor in the culture system of the control group; and (b) comparing E1 and E2, if E1 is significantly higher than E2, it indicates that the test compound is a potential therapeutic agent for ILC2 cell-mediated immune diseases; And the serotonin receptor is 5-HTR2A; The cells are ILC2 cells, and the cells are cells cultured in vitro.

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