Application of alpha-melanocyte stimulant in treating asthma by inhibiting type II innate lymphocytes

Abstract

The invention provides an application of alpha-melanocyte stimulant in treating asthma by inhibiting type II innate lymphocytes. An IL-7R-STAT pathway in activated lung ILC2 is inhibited through an alpha-MSH-MC5R axis, it is found that the neuroendocrine hormone alpha-MSH can target ILC2 to limit lung inflammation, and screening of new drugs for treating lung inflammation is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of alpha-melanocyte stimulating hormone in treating asthma by inhibiting type II innate lymphocytes. Background Art

[0002] With the development of society, complex environmental factors have a profound impact on human health and disease. Whether immune diseases such as asthma are affected by environmental factors deserves our in-depth study. The risks and benefits of sun exposure can mostly be explained by the effects of solar ultraviolet (UV) radiation on human health. Studies have reported the regulatory effect of UVB (290-315nm) irradiation on experimental allergic diseases in mice, but the specific mechanism of UVB immunosuppression is still unclear.

[0003] Asthma has become a chronic inflammatory lung disease with strong heterogeneity, seriously affecting the lives of about 300 million people worldwide and causing enormous socioeconomic pressure.

[0004] Asthma has long been considered a type II immune disease, with high immunoglobulin E (IgE) levels and the main involvement of Th2 cells. In addition to CD4+Th2 cells, emerging evidence shows that type II innate lymphocytes (ILC2s) are also important and earlier sources of type II cytokines such as IL-5, IL-13, Areg, and IL-4. Under different initial triggers (such as protease allergens, fungi and viruses), lung epithelial cells secrete interleukin (IL) -33, IL-25 and thymic stromal lymphopoietin (TSLP), which activate ILC2 cells to induce large amounts of IL-5 and IL-13, thereby inducing type II immune responses. These cascades drive eosinophil infiltration, airway hyperresponsiveness (AHR) and mucus production.

[0005] The current mainstream treatment for asthma is a combination of inhaled corticosteroids (which suppress inflammation) and short-acting or long-acting β2-adrenergic agonists (which open the contracted bronchial smooth muscle), but approximately 5-10% of patients are ineffective against corticosteroids and are often hospitalized due to respiratory viral infection with rhinovirus. With a deeper understanding of the pathogenesis of asthma, more and more targeted therapies are being developed for type II inflammatory airway diseases.

[0006] Therefore, there is an urgent need in this field to conduct in-depth research on the pathogenesis of asthma and explore effective drugs for preventing and / or treating lung inflammatory-related diseases. Summary of the invention

[0007] The purpose of the present invention is to provide an application of alpha-melanocyte stimulating hormone in treating asthma by inhibiting type II innate lymphocytes.

[0008] In a first aspect of the present invention, there is provided a use of α-melanocyte stimulating hormone (α-MSH) or a promoter thereof for preparing a preparation or a kit, wherein the preparation or the kit is used for:

[0009] (1) Upregulate the expression of α-MSH receptor in ILC2 cells;

[0010] (2) Upregulation of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC);

[0011] (3) downregulate the number of ILC2 cells;

[0012] (4) downregulating the function of ILC2 cells to secrete cytokines;

[0013] (5) downregulating the expression of IL-5, IL-13, and / or eosinophils by ILC2 cells;

[0014] (6) Inhibit the immune response of ILC2 cells;

[0015] (7) inhibiting JAK / STAT and NF-κB signaling pathways; and / or

[0016] (8) Prepare drugs for treating and / or preventing ILC2-mediated pulmonary inflammatory diseases.

[0017] In another preferred embodiment, the subtype of the α-MSH receptor is MC1R, MC2R, MC3R, MC4R, MC5R; preferably MC5R.

[0018] In another preferred embodiment, the ILC2 is a lung ILC2 cell.

[0019] In another preferred embodiment, the ILC2-mediated pulmonary inflammatory disease includes type II respiratory inflammation, including but not limited to asthma and chronic obstructive pulmonary disease (COPD).

[0020] In another preferred embodiment, the ILC2-mediated pulmonary inflammatory disease is allergic pulmonary inflammation.

[0021] In another preferred embodiment, the promoter refers to a substance that can increase the content of α-MSH in vivo or in vitro; the substance can be an artificially synthesized or natural compound, protein, nucleotide, or immunomodulator.

[0022] In another preferred embodiment, the α-MSH promoter includes a substance that promotes the expression of α-MSH.

[0023] In another preferred embodiment, the α-MSH promoter is selected from the following group: small molecule compounds, vectors expressing α-MSH, immunomodulators or a combination thereof.

[0024] In another preferred embodiment, the immunomodulator comprises ultraviolet radiation (UVR).

[0025] In another preferred embodiment, the α-MSH is selected from the following group:

[0026] (1) a polypeptide whose amino acid sequence is shown in SEQ ID NO.1;

[0027] (2) an α-MSH polypeptide derivative or an active fragment thereof formed by replacing, deleting or adding one or more (usually 1-5, preferably 1-3, more preferably 1-2, and most preferably 1) amino acid residues of the amino acid sequence shown in SEQ ID NO.: 1;

[0028] (C) an α-MSH polypeptide derivative or an active fragment thereof having a sequence homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% to the amino acid sequence shown in SEQ ID NO.: 1.

[0029] In another preferred embodiment, the α-MSH polypeptide derivatives include modified α-MSH, polypeptide molecules having amino acid sequences homologous to natural α-MSH and having natural α-MSH activity, dimers or multimers of α-MSH polypeptides, and fusion proteins containing α-MSH polypeptide amino acid sequences.

[0030] In another preferred embodiment, the α-MSH polypeptide derivative is selected from the following group: (D-Phe7)-α-MSH, (Nle4)-α-MSH, Ac-[Nle4,D-Phe7]-α-MSH(4-10)-NH2, Melanotan I, Bremelanotide, CCZ01048.

[0031] In another preferred embodiment, the MC5R is the MC5R on the surface of ILC2 cells.

[0032] In a second aspect of the present invention, there is provided a use of an α-MSH receptor subtype MC5R or a promoter thereof for preparing a preparation or a kit, wherein the preparation or the kit is used for:

[0033] Downregulation of ILC2 cell numbers; and / or

[0034] Down-regulate the function of ILC2 cells to secrete cytokines;

[0035] Down-regulate the expression of IL-5, IL-13 and / or eosinophils by ILC2 cells;

[0036] Inhibit the immune response of ILC2 cells;

[0037] Inhibit JAK / STAT and NF-κB signaling pathways; and / or

[0038] Treating and / or preventing ILC2-mediated pulmonary inflammatory diseases.

[0039] In another preferred embodiment, the ILC2 is derived from a mammal, preferably from a human, a mouse, a rat, or a cynomolgus monkey.

[0040] In another preferred embodiment, the ILC2 is lung ILC2.

[0041] In another preferred embodiment, the α-MSH receptor subtype MC5R promoter is selected from the following group: polypeptides, small molecule compounds, vectors expressing MC5R, or a combination thereof.

[0042] In another preferred embodiment, the α-MSH receptor subtype MC5R promoter includes a substance that promotes MC5R expression.

[0043] In another preferred embodiment, the α-MSH receptor subtype MC5R promoter includes a MC5R protein promoter and / or a MC5R gene promoter.

[0044] In another preferred embodiment, the α-MSH receptor subtype MC5R promoter is α-MSH or a polypeptide derivative thereof.

[0045] In a third aspect of the present invention, a composition is provided, wherein the pharmaceutical composition comprises:

[0046] (i) a first active ingredient, wherein the first active ingredient is selected from the group consisting of α-melanocyte stimulating hormone (α-MSH) or a promoter polypeptide derivative thereof; or a combination thereof; and / or

[0047] (ii) a second active ingredient, the second active ingredient being selected from the group consisting of: an agonist of the α-MSH receptor subtype MC5R; or a combination thereof;

[0048] (iii) a pharmaceutically acceptable carrier or excipient.

[0049] In another preferred embodiment, the α-melanocyte stimulating hormone (α-MSH) includes a polypeptide derivative of α-MSH.

[0050] In another preferred embodiment, the composition is a pharmaceutical composition.

[0051] In another preferred embodiment, the composition is used to upregulate MC5R expression in ILC2 cells.

[0052] In another preferred embodiment, the composition further contains a pharmaceutically acceptable carrier.

[0053] In another preferred embodiment, the dosage form of the composition is selected from: solid preparations, liquid preparations, preferably in the form of dry powder or solution.

[0054] In another preferred embodiment, the composition is a pharmaceutical composition, a health product composition or a vaccine composition.

[0055] In another preferred embodiment, the composition or preparation can be used alone or in combination for the prevention and / or treatment of ILC2-mediated pulmonary inflammatory diseases.

[0056] In another preferred embodiment, the ILC2-mediated pulmonary inflammatory disease includes type II respiratory inflammation, including but not limited to asthma and chronic obstructive pulmonary disease (COPD).

[0057] In another preferred embodiment, the combined use includes: combined use with other methods for preventing and / or treating ILC2-mediated pulmonary inflammatory diseases.

[0058] In another preferred embodiment, the other drugs for preventing and / or treating ILC2-mediated pulmonary inflammatory diseases are selected from the following group: omalizumab (anti-IgE antibody), mepolizumab (anti-IL-5 antibody), reslizumab (anti-IL-5 antibody), benralizumab (anti-IL-5 receptor antibody), dupilumab (anti-IL-4Rα antibody) and tezepelumab (anti-TSLP antibody) or a combination thereof.

[0059] In a fourth aspect of the present invention, a method for inhibiting an IL2 cell immune response is provided, comprising the steps of:

[0060] (1) in the presence of α-MSH or a promoter thereof; and / or

[0061] (2) Cultivating stellate cells in the presence of the α-MSH receptor subtype MC5R gene, or its protein, or its promoter, thereby inhibiting the IL2 cell immune response.

[0062] In another preferred embodiment, the α-melanocyte stimulating hormone (α-MSH) includes a polypeptide derivative of α-MSH.

[0063] In another preferred embodiment, the inhibition of IL2 cell immune response comprises:

[0064] Down-regulate the expression of IL-5, IL-13 and / or eosinophils by ILC2 cells;

[0065] Inhibit the IL-7 / IL-7R signaling pathway of ILC2 cells.

[0066] In a fifth aspect of the present invention, a method for screening potential therapeutic agents for ILC2-mediated pulmonary inflammatory diseases is provided, comprising:

[0067] (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the MC5R gene for a period of time T1, and detecting the expression level E1 of the MC5R gene in the culture system of the test group;

[0068] And in a control group without the test compound and with other conditions being the same, detecting the expression level E2 of the MC5R gene in the culture system of the control group; and

[0069] (b) Comparing E1 and E2, if E1 is significantly higher than E2, it indicates that the test compound is a potential therapeutic agent for lung inflammatory diseases.

[0070] In another preferred embodiment, the “significantly higher than” refers to E1 / E2 ≥ 2, preferably ≥ 3, more preferably ≥ 4.

[0071] In another preferred embodiment, the cells include lung IL2 cells.

[0072] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0073] 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 the pulmonary inflammatory disease of the mammal.

[0074] In another preferred embodiment, the mammal includes a human or a non-human mammal.

[0075] In another preferred embodiment, the non-human mammals include rodents and primates, preferably, mice, rats, rabbits and monkeys.

[0076] In another preferred embodiment, the ILC2-mediated pulmonary inflammatory disease includes type II respiratory inflammation, including but not limited to asthma and chronic obstructive pulmonary disease (COPD).

[0077] 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

[0078] Figure 1The results show that the intensity of UVB in sunlight affects the frequency of acute visits to the doctor in asthma patients and inhibits ILC2s-mediated type II pneumonia. A. Papain-induced lung inflammation model and UVB radiation treatment mode. B. Flow cytometry detection of eosinophils (FVD - CD45 + CD11c - / lo Siglec + C. Statistical analysis of the proportion of eosinophils in bronchoalveolar lavage fluid (BALF) + The proportion and number of cells. D. HE and PAS staining of paraffin sections of lung tissue pathology (scale bar: 200 μm). E. ELISA detection of IL-5 (left) and IL-13 (right) concentrations in bronchoalveolar lavage fluid. F. Flow cytometry detection and calculation of the number of ILC2s (FVD-CD45.2+LIN-CD90.2+CD127+ST2+) in pulmonary monocytes. GH. Flow cytometry detection of the proportion of intracellular IL-5 and IL-13 in ILC2 after 4 hours of PMA, ionomycin and BFA stimulation of pulmonary monocytes. P values ​​were calculated by unpaired t test, Pearson's correlation coefficient analysis and two-tailed Student's t test, and errors are shown as means±SDs. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant; non-immunized group, n=3; immunized group, n=7-8; The experiment was repeated more than twice independently.

[0079] Figure 2 Figure 3 shows the contribution of ILC2s in the inhibitory effect of UVB irradiation on papain-induced lung inflammation. A. ILC2s were adoptively transferred into Rag - / - Il2rγ - / - Schematic diagram of mouse experiment. ILC2s cells were sorted from WT mice immunized with IL-33, and the expression of each Rag2 - / - Iq - / - 4×10^5 ILC2s were injected into the tail vein of recipient mice and irradiated with UVB (2.5 kJ / m 2 ), and finally IL-33 immunization (30 ng / mouse) was performed 3 days later to detect related indicators. - / - Iq - / -The recipient mice were used as controls (n=3 in the non-adoptive transfer group and n=7 in the adoptive transfer group). B. Flow cytometry was used to detect eosinophils in the bronchoalveolar lavage fluid of mice in the ILC2s adoptive transfer experiment; Statistical analysis of the proportion of eosinophils in the bronchoalveolar lavage fluid of mice was performed. + The proportion of cells. C. ELISA detected the concentrations of IL-5 (left) and IL-13 (right) in the alveolar lavage fluid of mice. D. Flow cytometry detected the number of eosinophils in mouse lung tissue (left), the proportion of ILC2s in pulmonary monocytes to CD45.2+ cells (middle), and the number of ILC2s in pulmonary monocytes (right). E. Schematic diagram of the hypothalamus-pituitary axis. F. Scatter plot showing the normalized mean counts of neural-related gene expression in lung ILC2s isolated from Naive mice (left). Normalized counts of Mc-r and opioid receptor Opr gene expression in lung ILC2s isolated from Naive mice and analyzed by RNA-seq, shown as a heat map (right). G. qRT-PCR analysis of the relative expression of Mc-r in sorted lung ILC2s. H. qRT-PCR analysis of the relative expression of Mc5r in other immune cell populations in the lung, Hprt as an internal reference and 2 -△△Ct Statistical analysis was performed after treatment with the method. P values ​​were calculated by unpaired t test and two-tailed Student's t test, and the errors are shown as means±SDs. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant; the experiment was repeated more than twice independently.

[0080] Figure 3 It shows that UVB eye irradiation leads to increased α-MSH levels and activation of hypothalamic CRH-POMC signaling. A. Schematic diagram of the mouse UVB irradiation experiment. WT C57BL / 6 female mice were anesthetized with sodium pentobarbital and divided into a skin irradiation group and an eye irradiation group. The skin irradiation group had their back skin shaved and exposed, and their eyes were shielded with tin foil for UVB (2.5 kJ / m 2 ) irradiation; the eyes of the eye irradiation group were exposed, and the back skin was shielded with tin foil to UVB (2.5 kJ / m 2 ) irradiation; sampling and testing after 12h / 24h. B. ELISA was used to detect the concentration of α-MSH in mouse serum 12h / 24h after UVB irradiation (n=6-7). C. qRT-PCR was used to detect the gene expression of Crh in the hypothalamus of mice 12h after UVB eye irradiation, with Hprt as the internal reference and 2 -△△CtStatistical analysis was performed after treatment. D. Immunofluorescence was used to detect the expression of CRH in the hypothalamus 24 hours after UVB eye irradiation (CRH: red; Dapi: blue; scale bar: 50μm). E. Western blot was used to detect the protein level of CRH in the hypothalamus 24 hours after UVB eye irradiation. F. Statistical analysis of the changes in CRH in Figures D and E was performed using ImageJ software for quantitative analysis. G. qRT-PCR was used to detect the gene expression of Pomc in the hypothalamus, skin, and pituitary of mice 12 hours after UVB eye irradiation. H. Schematic diagram of POMC shearing. I. qRT-PCR was used to detect the changes in the transcription levels of the shearing enzyme genes Pcsk1 and Pcsk2 in the pituitary of mice 12 hours after UVB eye irradiation. Hprt was used as an internal reference and 2 -△△Ct Statistical analysis was performed after treatment. J. Immunofluorescence was used to detect the expression of POMC in the pituitary region 24 hours after UVB eye irradiation (POMC: green; Dapi: blue; scale bar: 50μm). K. Western blot was used to detect the expression of POMC protein levels in the pituitary region 24 hours after UVB eye irradiation; the changes in POMC in Figure H were statistically analyzed, and ImageJ software was used for quantitative analysis. L. Flow cytometry was used to detect the proportion of intracellular IL-5 and IL-13 in ILC2s after 2 hours of PMA, ionomycin, and BFA stimulation; the proportion of intracellular IL-5 and IL-13 in ILC2s was statistically analyzed. M. ELISA was used to detect the concentrations of IL-5 and IL-13 in the culture supernatant of ILC2s and statistically analyzed. Error analysis was calculated by two-tailed Student's ttest, unpaired t test, and one-way ANOVA with Tukey's multiple comparisons test Student's t-test method, and the errors were displayed as means±SEMs. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

[0081] Figure 4 The results show that UVB eye radiation regulates ILC2s function through the α-MSH-MC5R axis. A. Papain-induced lung inflammation model and UVB radiation treatment pattern. B. Flow cytometry detection of eosinophils (FVD - CD45 + CD11c - / lo Siglec +) and statistically analyzed the number of eosinophils in the alveolar lavage fluid. C. HE and PAS staining of paraffin sections of lung tissue pathology (Scale bar: 200μm). D. ELISA detection of IL-5 (left) and IL-13 (right) concentrations in alveolar lavage fluid. E. Flow cytometry detection and calculation of the number of ILC2s (FVD-CD45.2+LIN-CD90.2+CD127+ST2+) in lung monocytes. F. Flow cytometry detection of the proportion of intracellular IL-5 and IL-13 in ILC2 after 4 hours of stimulation of lung monocytes with PMA, ionomycin and BFA, and statistically analyzed the proportion of intracellular IL-5 and IL-13 in ILC2. G. Papain-induced lung inflammation model and α-MSH bronchial treatment pattern. R5 / + With Mc5r fl / fl R5 / + The mice were treated with papain (4 μg per mouse) dripping into the bronchi for 5 days. The experimental group was treated with α-MSH (4 mg / kg) dripping into the bronchi every day, and the control group was treated with PBS. The relevant indicators were detected on the 6th day. H. Flow cytometry was used to detect eosinophils (FVD - CD45 + CD11c - / lo Siglec + ) ; Statistical analysis of eosinophils in bronchoalveolar lavage fluid (BALF) + I. HE and PAS staining of paraffin sections of lung tissue pathology (scale bar: 200 μm). J. ELISA detection of IL-5 (left) and IL-13 (right) concentrations in bronchoalveolar lavage fluid. K. Flow cytometry detection and calculation of the number of ILC2s (FVD-CD45.2+LIN-CD90.2+CD127+ST2+) in lung monocytes. LM. Flow cytometry detection and analysis of the proportion of intracellular IL-5 and IL-13 in ILC2s after 4 hours of stimulation of lung monocytes with PMA, ionomycin and BFA. N. Schematic diagram of the experiment. WT mice were treated with or without UVB eye irradiation (2.5 kJ / m 2 ), the pituitary glands were removed and ground, and the pituitary cells were cultured for about 48 hours. The concentration of α-MSH in the pituitary cell culture supernatant was detected by ELISA, and the pituitary cell culture supernatant was used to culture and sort the R5 cells immunized with IL-33. / + With Mc5r fl / fl R5 / +ILC2s from the lungs of mice (5000 cells per well), and the concentration of cytokines IL-5 / 13 in the supernatant of ILC2s culture was detected after three days of culture. O. ELISA detected the concentration of α-MSH in the supernatant of pituitary cells (left); ELISA detected the concentration of IL-5 / 13 in the supernatant of ILC2s culture (right). P values ​​were calculated by two-tailed Student's t test, and errors are shown as means±SDs. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant; more than 2 independent repeated experiments were performed.

[0082] Figure 5 The results show that the α-MSH-MC5R axis inhibits ILC2s function through the JAK-STAT and NF-κB signaling pathways. A. Gene enrichment analysis (GSEA) analysis of asthma-related signaling pathways. NES, enrichment score. B. Heat map showing the expression of asthma-related genes. A. Heat map showing R5 / + With Mc5r fl / fl R5 / + C. Volcano plot showing the expression of representative regulatory genes in ILC2s in the lungs of mice. / + With Mc5r fl / fl R5 / + Differentially expressed genes of ILC2s in the lungs of mice (log2(fold change)>1.0; p value<0.05); the main representative genes of ILC2s are marked and displayed in gray. E. Heat map showing R5 / + With Mc5r fl / fl R5 / + Upregulated genes in the JAK / STAT signaling pathway in ILC2s from the lungs of mice. F. Flow cytometry analysis of R5 / + With Mc5r fl / fl R5 / + The expression of CD127 in ILC2s from the lungs of mice after in vitro culture and α-MSH treatment was statistically analyzed. G. ELISA detected the concentrations of IL-5 and IL-13 in the culture supernatant of WT mouse ILC2 after 3 days of culture under different conditions. Mouse lung ILC2s sorted from IL-33-treated WT mice were cultured in vitro under the conditions of IL-2 (100U / ml), mIL-33 (1ng / ml) and IL-7 (0, 2pg / ml, 20pg / ml, 200pg / ml, 2ng / ml, 20ng / ml and 200ng / ml) in the presence or absence of α-MSH. H. Sorting R5 / + and Mc5rfl / fl R5 / + Mouse lung ILC2s were cultured in vitro with IL-2 (100 U / ml) and IL-7 (20 ng / ml) ± α-MSH (10 ng / ml) for 3 days, then cultured in the absence of cytokines for 24 hours, and Western blot was performed after adding IL-7 (20 ng / ml) for 0, 10, and 60 min of stimulation on the second day. The ratios of p-STAT3 / actin and p-STAT5 / actin in each group in A were statistically analyzed, and quantitative analysis was performed using ImageJ software. I. ELISA detected the concentrations of IL-5 and IL-13 in the culture supernatant of WT mouse ILC2 after 3 days of culture under the conditions of IL-2 (100 U / ml, mIL-33 (1 ng / ml) or IL-7 (20 ng / ml) alone ± α-MSH (10 ng / ml). J. ELISA detected the concentrations of IL-5 and IL-13 in the culture supernatant of WT mouse ILC2 under the conditions of IL-2 (100 U / ml + mIL-33 (1 ng / ml), IL-7 (20 ng / ml) + mIL-33 (1 ng / ml), IL-2 (100 U / ml + IL-7 (20 ng / ml) + mIL-33 (1 ng / ml), K. WT mouse lung ILC2s were sorted and cultured in vitro with IL-2 (100 U / ml) and IL-7 (20 ng / ml) ± α-MSH (10 ng / ml) for 3 days, then cultured in the absence of cytokines for 24 hours. The next day, IL-7 (20 ng / ml), mIL-33 (1 ng / ml), and IL-7 (20 ng / ml) + mIL-33 (1 ng / ml) were added for stimulation for 0, 10 min, and 60 min, respectively, and then Western blot was performed. Blot detection. The ratios of p-p65 / actin and p-STAT5 / actin in each group were statistically analyzed, and quantitative analysis was performed using ImageJ software. P values ​​were calculated by two-tailed Student's ttest, and the errors were shown as means±SDs. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant; the experiment was repeated more than twice independently.

[0083] Figure 6It shows that α-MSH inhibits the inflammatory response and immune response in the asthma model induced by house dust mites HDM. Among them, A. Schematic diagram of HDM immune modeling. WT mice were sensitized with high-dose allergen HDM (30ug / mouse) on days 1-3. After a 10-day break, mice were given a second sensitization with low-dose HDM (6ug / mouse) on days 14-17, in which α-MSH (4mg / kg) and HDM were simultaneously administered by bronchial instillation. B. ELISA was used to detect total IgE, HDM-specific IgE, and HDM-specific IgG1 in the serum of each group of mice. C. The lung function of each group of mice was detected. D. Flow cytometry was used to detect and analyze the proportion and number of eosinophils in the alveolar lavage fluid of mice. E. ELISA was used to detect the concentrations of IL-5 and IL-13 in the alveolar lavage fluid of each group of mice. F. HE and PAS staining of paraffin sections of lung tissue pathology (scale bar: 200μm). G. Flow cytometry was used to detect and calculate the number of ILC2s in pulmonary monocytes. H. Flow cytometry detection of intracellular IL-5 and IL-13 in ILC2 cells after 4 h of PMA, ionomycin and BFA stimulation of lung monocytes and statistical analysis of IL-5 + ILC2s and IL-5 + IL-13 + Number of ILC2s. Error analysis was calculated by unpaired t-test method. Errors are shown as means ± SEMs. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant. DETAILED DESCRIPTION

[0084] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that UVB promotes α-MSH to bind to MC5R on ILC2, downregulates IL-7R-JAK / STAT phosphorylation signals, and then inhibits ILC2s-mediated type II lung inflammation. Specifically, it was found in a mouse model that UVB radiation to the mouse eye significantly upregulated the expression of Pomc in the pituitary, increased the level of α-MSH in serum, and attenuated mouse allergic airway inflammation through the α-MSH receptor subtype Mc5r on the surface of ILC2 cells. Mechanistic studies have shown that the α-MSH-MC5R axis inhibits the IL-7R-STAT pathway in activated lung ILC2, which can be demonstrated by downregulation of IL-7R expression and STAT3 / 5 phosphorylation. In addition, the therapeutic effect of α-MSH was verified by clinical relevance and mouse models of chronic inflammation induced by HDM and OVA. These findings reveal the mechanism of the neuroendocrine hormone α-MSH in targeting ILC2 to limit lung inflammation, which is helpful for screening new drugs for the treatment of lung inflammation. The present invention was completed on this basis.

[0085] the term

[0086] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0087] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0088] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0089] Group 2 Innate Lymphocytes (ILC2s)

[0090] Similar to T cells, mature ILCs can be divided into cytotoxic (interleukin 7 receptor α-, IL-7Rα-) and non-cytotoxic (IL-7Rα+) ILCs. Natural killer (NK) cells are the oldest members of the ILC family and the only representatives of cytotoxic ILCs. Non-cytotoxic ILCs are divided into three different subtypes, namely ILC1, ILC2, and ILC3, according to the expression of lineage-specific transcription factors (TFs) (Spits et al., 2013).

[0091] Similar to T cells, ILC2s originate from bone marrow lymphoid progenitors (CLPs) and are widely distributed in the mesentery, intestine, lung, fat, and blood, usually expressing CD90.2 (Thy1), CD127 (IL7Rα), KLRG1, GATA3, ICOS, T1 / ST2 (IL33R), and CD25 (IL-2Rα), and contain Il13, Il5, IL4, Il9r, and Il17rb transcripts. ILC2s promote type II inflammation in the context of anti-helminth immunity, allergic responses, or tissue remodeling.

[0092] When the lung epithelium is stimulated by antigens or allergens, cytokines IL-25, IL-33, and TSLP are rapidly released and activate ILC2s. Classical Th2-related mediators, including lipids such as cysteinyl leukotrienes and prostaglandin D2, and tumor necrosis factor-like ligand 1A, can also activate ILC2s. Once activated, ILC2s rapidly secrete type II cytokines IL-5 and IL-13.

[0093] IL-5 is important for the growth, differentiation, recruitment and survival of eosinophils. IL-13 affects epithelial cells and airway smooth muscle cells, leading to bronchial hyperresponsiveness and promoting goblet cell mucus production and fibrosis. ILC2 can produce IL-4, which promotes CD4+Th2 cell differentiation and IgE antibody class switching (Rael and Lockey, 2011). In addition, ILC2 also secretes IL-9, which promotes mast cell accumulation, mucus secretion and the production of amphiregulin (epidermal growth factor receptor ligand), which can promote tissue repair after lung virus infection.

[0094] As used herein, "ILC2s-mediated type II lung inflammation" and "ILC2s-mediated allergic lung inflammation" can be used interchangeably to refer to ILC2-mediated type II inflammation of the respiratory tract.

[0095] α-MSH (alpha-melanocyte-stimulating hormone)

[0096] α-MSH (alpha-melanocyte-stimulating hormone) is an endogenous neuropeptide and an endogenous melanocortin receptor 4 (MC4R) agonist with anti-inflammatory and antipyretic effects. α-MSH is a post-translational derivative of pro-opiomelanocortin (POMC). As a member of MSH, α-MSH is an endogenous 13-peptide produced by the cleavage of the precursor hormone pro-opiomelanocortin (POMC) and is mainly detected in pituitary cells.

[0097] Structural formula: Ac-SYSMEHFRWGKPV-NH2 (SEQ ID NO.1)

[0098] In another preferred embodiment, the "α-MSH" also includes a polypeptide derivative selected from the following group:

[0099] (D-Phe7)-α-MSH (CAS No. 92937-43-4), (Nle4)-α-MSH (CAS No. 64887-70-3), Ac-[Nle4,D-Phe7]-α-MSH (4-10)-NH2 (CAS No. 82188-67-8), Melanotan I (CAS number 75921-69-6), Bremelanotide (CAS number 189691-06-3), CCZ01048.

[0100] As used herein, CCZ01048 is an α-MSH analog, and the structural formula is shown below:

[0101]

[0102] α-MSH receptor and its encoding nucleic acid

[0103] MC5R is one of five melanocortin receptors (MC1R-MC5R), and its ligand is melanocyte stimulating hormone (MSH).

[0104] The NCBI accession numbers of the MC5R of the present invention are: Mouse MC5R: NP_038624.3; human MC5R: NP_005904.1.

[0105] 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 MC5R sequence of the present invention.

[0106] 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.

[0107] The present invention also includes MC5R protein fragments and analogs having MC5R protein activity. As used herein, the terms "fragment" and "analog" refer to proteins that substantially retain the same biological function or activity as the natural MC5R protein of the present invention.

[0108] 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.

[0109] Table I

[0110]

[0111]

[0112] 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 MC5R protein 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 an amino acid with similar or similar properties is 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 difference between the natural MC5R protein analogs included in the present invention and the natural MC5R protein may be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. The 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.

[0113] 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.

[0114] The present invention also provides a polynucleotide sequence encoding the MC5R 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 the mature polypeptide includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include 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 it encodes.

[0115] 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.

[0116] 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%.

[0117] The proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] MC5R promoter

[0124] In the present invention, the MC5R promoter includes substances that can increase the activity and / or content of the MC5R gene or its protein in vivo or in vitro.

[0125] In the present invention, the MC5R promoter also includes substances that promote the formation of a complex between MC5R protein and integrin αv.

[0126] Among them, the expression level of MC5R can be increased by the following methods: the tissue itself secretes a large amount of MC5R protein or artificially overexpresses MC5R protein, or artificially delivers MC5R protein (for example, using a viral vector, such as an adeno-associated viral vector) or a MC5R promoter.

[0127] In the present invention, the MC5R promoter is not particularly limited, and any promoter that can promote the expression of MC5R or enhance the activity of MC5R protein is within the protection scope of the present invention.

[0128] In a preferred embodiment, the MC5R promoter comprises a small molecule compound.

[0129] Pharmaceutical compositions and methods of administration

[0130] The pharmaceutical composition of the present invention comprises the deubiquitinase or its derivatives, deubiquitinase agonists or antagonists (active ingredients) of the present invention within a safe and effective amount range and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 0.001-1000 mg of active ingredient / dose, preferably 0.05-300 mg of active ingredient / dose, and more preferably, 0.5-200 mg of active ingredient / dose.

[0131] The active ingredient of the present invention and its pharmacologically acceptable salt can be made into various preparations, which contain the active ingredient of the present invention or its pharmacologically acceptable salt within the safe and effective amount range and a pharmacologically acceptable excipient or carrier. The "safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the active ingredient is determined according to the specific circumstances such as the age, condition, and course of treatment of the subject.

[0132] "Pharmacologically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmacologically acceptable excipients or carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers and wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0133] The composition of the present invention may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), or topically.

[0134] The composition of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0135] The microcapsules containing the composition of the present invention can be used for sustained-release administration of the active ingredients of the present invention. The microcapsule sustained-release administration technology of recombinant proteins has been successfully applied to recombinant human growth hormone (rhGH), recombinant human interferon (rhIFN), interleukin-2 and MNrgp12.

[0136] The sustained-release formulation of the active ingredient of the present invention can be prepared with a lactic acid glycolic acid polymer (PLGA) with good biocompatibility and wide biodegradability. The degradation products of PLGA, lactic acid and glycolic acid, can be quickly removed by the human body. Moreover, the degradation capacity of the polymer can be extended from several months to several years depending on its molecular weight and composition.

[0137] When using the pharmaceutical composition, a safe and effective amount of the active ingredient of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the dosage per administration is usually 0.01 to 300 mg, preferably 0.5 to 100 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0138] The main advantages of the present invention include

[0139] (1) The present invention is the first to discover that α-MSH can inhibit a variety of allergic lung inflammations.

[0140] (2) The present invention first discovered that α-MSH inhibits the response of ILC2s through the α-MSH receptor subtype MC5R expressed on the surface of ILC2s, thereby alleviating lung inflammation.

[0141] (3) Ultraviolet radiation can act as a regulator of α-MSH, and UVB irradiation can inhibit ILC2s-mediated lung inflammation.

[0142] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0143] Example 1 UVB inhibits ILC2s-mediated type II lung inflammation

[0144] To explore whether UVB irradiation can inhibit ILC2s-mediated lung inflammation, the inventors constructed a classic papain model. Within 24 hours before modeling, the experimental group of mice were irradiated with whole-body UVB (2.5 KJ / m 2 , about one minimum edema dose), and then C57 / B6 (Wild Type) mice were treated with papain (4 μg / mouse) for 5 consecutive days by intrabronchial immunization ( Figure 1 A). The non-immunized group was treated with solvent PBS for 5 consecutive days under the same experimental conditions. On the 6th day, the mice were treated and bronchoalveolar lavage fluid, lung tissue and lung mononuclear cells were obtained.

[0145] In order to explore the effect of UVB irradiation on lung inflammation, the inventors performed flow cytometry staining on cells in bronchoalveolar lavage fluid to detect eosinophils (Eos; FVD - CD45 + CD11c - / lo Siglec + The inventors found that under non-immunized conditions, the overall level of eosinophils was low, and UVB irradiation had almost no effect on eosinophil infiltration. However, in the papain immunized group, UVB irradiation significantly reduced the proportion and number of eosinophils ( Figure 1 BC). At the same time, HE and PAS staining sections of lung tissue showed that UVB irradiation reduced the infiltration of inflammatory cells in the immune group ( Figure 1 D) and mucus production ( Figure 1 D below).

[0146] The inventors used ELISA to detect and found that UVB irradiation significantly reduced the concentrations of type II cytokines IL-5 and IL-13 in the bronchoalveolar lavage fluid of the immune group ( Figure 1 E). The above experimental indicators show that UVB irradiation inhibits papain-induced lung inflammation. In order to further explore whether UVB irradiation affects the immune response of ILC2s, the inventors isolated the lung monocytes of mice after modeling, and performed surface and intracellular flow staining on them to detect the number and function of ILC2s. The inventors found that the number of ILC2s in the non-immunized group was small, while the number of ILC2s increased significantly after immunization. After UVB irradiation treatment, the number of ILC2s decreased significantly ( Figure 1 F). At the same time, in order to detect the effect of UVB irradiation on the function of ILC2s in secreting cytokines, the inventors used the stimulator PMA (phorbol 12-myristate 13-acetate), Ionomycin and the Golgi transport inhibitor BFA to stimulate the isolated lung monocytes. After 4 hours, the cells were collected and flow cytometry staining was performed to detect the level of IL-5 / IL-13 expressed by ILC2 ( Figure 1 G) The inventors found that compared with the control group, the expression of IL-5 in ILC2s of mice irradiated with UVB + IL-13 + IL-5 + and IL-13 + The proportion of Figure 1 H). This indicates that UVB irradiation can significantly inhibit the number of ILC2 and its function of secreting type II cytokines IL-5 and IL-13 in the case of lung inflammation.

[0147] Example 2 Contribution of ILC2s in the process of alleviating asthma by UVB irradiation

[0148] The mechanism of UVB's effect on immune diseases is complex and diverse, involving many immune cells. The inventors adopted ILC2s into Rag cells lacking T, B, NKs and ILCs. - / - Il2rγ - / - In female mice, it was convenient for the inventors to further study the role of ILC2s in UVB irradiation-alleviated asthma in a cleaner immune background.

[0149] First, the inventors immunized WT female mice with IL-33 (50 ng / mouse) intrabronchially for 3 days, and then sorted the primary ILC2s in the lungs of mice after 3 days of rest. The sorted ILC2s were expanded and cultured in vitro in the presence of cytokines for 6 days and then injected into Rag- / -Il2rγ- / - female mice through the tail vein. - / - Il2rγ - / - Mice were irradiated with UVB (2.5KJ / m 2 ), and then all mice were immunized with IL-33 bronchi for 3 days. On the 4th day, the mice were euthanized and the corresponding tissues were taken to detect the corresponding inflammatory indicators ( Figure 2 A) The experimental results show that in the absence of ILC2s adoptive Rag - / - Il2rγ - / - In mice with IL-33 immunization, there was almost no eosinophil infiltration in the cells of the bronchoalveolar lavage fluid (BALF), whereas in the Rag - / - Il2rγ - / - In mice irradiated with UVB, the proportion of eosinophils in BALF reached about 60%, but decreased significantly after UVB irradiation ( Figure 2 B). At the same time, in the BALF supernatant, type II cytokines IL-5 and IL-13 were significantly decreased after UVB radiation ( Figure 2 C). In addition to BALF, the inventors also digested lung tissue and isolated lung monocytes. Consistent with the previous experimental phenomenon in BALF, the number of eosinophils in mouse lung tissue decreased significantly after UVB irradiation ( Figure 2 D, left); the proportion of ILC2s in lung monocytes ( Figure 2 D, middle) and quantity ( Figure 2 D, right) also decreased significantly. This experiment adoptively transferred ILC2s into a background mouse lacking an immune system, demonstrating that ILC2s play an important role in the inhibitory effect of UVB on papain-mediated lung inflammation.

[0150] The immune regulation process of UVB for the body is very complex, and the endocrine system plays an important regulatory role in it. Corticotropin-releasing hormone (CRH) and precursor pro-opiomelanocortin (POMC)-derived peptides are one of the important components of the body's response to UVB (Fell et al., 2014; Parikh et al., 2022). When the body is stimulated by stress such as UVB, CRH in the hypothalamus will be secreted into the pituitary gland to promote the expression of POMC. As an important precursor, POMC will be cleaved by the cleavage enzymes in the environment into a variety of short peptides, such as melanocyte stimulating hormone (MSH) or β-endorphin. These short peptides will quickly enter the circulatory system and interact with cells expressing related receptors (Hiramoto et al., 2003) to play corresponding functions ( Figure 2 E).

[0151] In order to explore the mechanism of UVB's inhibitory effect on ILC2s, the inventors detected the expression of nerve or neuroendocrine-related receptors on ILC2s. By performing RNA-seq analysis on resting lung ILC2s, the inventors found that ILC2s highly expressed some nerve or neuroendocrine-related receptors. In addition to the reported related receptors, the inventors found that the expression level of Mc5r, a subtype of α-MSH receptor, was relatively high ( Figure 2 F, left). Compared with the Mc-R of the same family (Mc1r-Mc4r), Mc5r has the highest expression, while the β-endorphin receptor Op-r is almost not expressed ( Figure 2 F, right). This result was also verified by fluorescence quantitative PCR experiment: Mc5r had the highest expression level in ILC2s compared with other receptors of the same family ( Figure 2 G); Compared with other immune cells in the lungs, such as T cells, B cells, DC cells, granulocytes, etc., ST2 + The Mc5r expression level of ILC2s cells is higher ( Figure 2 H).

[0152] The inventors found that Mc5r was highly expressed on ILC2s, suggesting that the Mc5r receptor may play a role in the response process of ILC2s.

[0153] Example 3 UVB irradiation upregulates α-MSH levels and activates the hypothalamus-pituitary pathway in mice

[0154] MC5R is one of the five melanocortin receptors (called MC1-5R), and its ligand is melanocyte stimulating hormone (MSH). α-MSH, as a member of MSH, is an endogenous tridecapeptide produced by cleavage of the precursor hormone proopiomelanocortin (POMC) and is mainly detected in pituitary cells (Slominski et al., 1996). Skin and eyes are two tissues that are chronically exposed to UV radiation (Volatier et al., 2022), and this exposure will induce systemic α-MSH production (Cui et al., 2007; Hiramoto et al., 2003). In order to compare the contribution of these two tissues to UVB-induced systemic α-MSH production, the inventors used 2.5 kJ / m 2 UVB rays were irradiated on shaved back hair of C57BL / 6 mice (covering the eyes) and exposed eyes of C57BL / 6 mice (covering the back skin) ( Figure 3 A). After 12h and 24h, the α-MSH levels in the serum of the control group and the experimental group were detected by ELISA. The inventors found that the α-MSH concentration in the serum of the mice exposed to UVB eye radiation was higher ( Figure 3 B). In order to explore whether the increase in α-MSH levels depends on hypothalamic-pituitary neuroendocrine-related signals, the inventors removed mouse hypothalamic tissue and found that the mRNA level of corticotropin-releasing hormone Crh increased significantly about 12 hours after UVB eye radiation ( Figure 3 C). Consistent with the results of qRT-PCR, immunofluorescence experiments ( Figure 3 D) and western blot ( Figure 3 E) The experimental results showed that after UVB eye radiation, the protein level of CRH in the hypothalamus of mice also increased significantly ( Figure 3 F).

[0155] The above experimental results show that UVB irradiation to the eyes can activate the expression of CRH in the hypothalamus, and the production of POMC in the pituitary gland will be regulated by CRH (Skobowiat and Slominski, 2015). After UVB eye irradiation of mice, the mRNA level of Pomc in the pituitary gland increased compared with the non-irradiated control group, while the transcription level of Pomc in the hypothalamus and skin did not change significantly ( Figure 3G). The pituitary gland is an important and complex endocrine gland in the body that can produce a variety of hormones and participate in regulating the body's physiological processes (Schiller et al., 2021). PC1 / 3 (Pcsk1) cleaves POMC to produce β-lipotropin (β-LPH) and ACTH, while PC2 (Pcsk2) can further cleave ACTH and β-LPH to produce α-MSH and β-endorphin ( Figure 3 H)(Fell et al., 2014; Parikh et al., 2022). However, after UVB irradiation of mouse eyes, the transcription of Pcsk2 but not Pcsk1 increased ( Figure 3 I). This is also consistent with the increase in serum α-MSH concentration mentioned above. At the same time, it is consistent with the results of qRT-PCR. Immunofluorescence experiment ( Figure 3 J) and western blot( Figure 3 K) The experimental results all showed that 24 hours after UVB eye radiation, the protein level of POMC in the hypothalamus area of ​​mice also increased significantly.

[0156] In the above experiments, the inventors found that UVB irradiation can enhance CRH-POMC signals in the neuroendocrine system and increase the concentration of α-MSH in the circulatory system. Combined with the inventors' previous findings, UVB inhibits ILC2s-mediated lung inflammation experiments.

[0157] In order to further verify whether UVB may cause changes in ILC2s function by promoting the secretion of α-MSH, the inventors conducted an in vitro culture experiment of ILC2s. The inventors performed bronchoalveolar immunization of WT mice with IL-33 for three consecutive days, and sorted them on the seventh day to obtain primary ILC2s in the mouse lungs. During the culture of ILC2s, cytokines IL-2, IL-7 and IL-33 were added, and α-MSH or β-endorphin (β-endorphin) was added for treatment. Three days later, the function of ILC2s to secrete type II cytokines was detected by flow cytometry and ELISA. The flow cytometry results showed that in vitro treatment with α-MSH can reduce IL-5 + and IL-13 + The proportion of ILC2s ( Figure 3 L). The ELISA results were consistent with the flow cytometry results. After in vitro treatment with α-MSH, the IL-5 and IL-13 in the culture supernatant of ILC2s decreased ( Figure 3 M). However, β-endorphin treatment in vitro did not affect ILC2s, which is consistent with the conclusion that ILC2s hardly express β-endorphin receptors. The above experiments show that α-MSH can inhibit the function of ILC2s in vitro.

[0158] Example 4 UVB ocular radiation regulates ILC2s function via the α-MSH-MC5R axis

[0159] In the above experimental results, the inventors found that ILC2s highly expressed a subtype of α-MSH receptor Mc5r, and α-MSH may play an important role in UVB inhibiting ILC2s-mediated lung inflammation. The inventors speculated that α-MSH may exert its inhibitory effect through the receptor MC5R on the surface of ILC2s. To verify this hypothesis, the inventors made mice with conditional knockout of the receptor Mc5r in ILC2. fl / fl R5 / + R5 / + Tool mice are currently commonly used ILC2s conditional knockout mice, and the CRE enzyme is controlled by the Il-5 site. / + Mouse Mc5r gene with loxP sites inserted before and after fl / fl Mice were mated and reproduced to obtain IL-5 + Mice with Mc5r knockout in cells. / + Insertion of mice affects the function of IL-5+ cells, and all subsequent mice are R5 / + Heterozygous background; control mice were R5 / + , the experimental group was Mc5r fl / fl R5 / + .

[0160] To verify whether the inhibitory effect of UVB ocular radiation on ILC2s in mouse lungs depends on its surface receptor MC5R, the inventors / + With Mc5r fl / fl R5 / + UVB eye irradiation and papain modeling were performed. Figure 4 A). The inventors found that, consistent with the above experimental results, UVB eye irradiation can inhibit the R5 / + Lung inflammation in mice, but the experimental group Mc5r fl / fl R5 / + Lung inflammation in mice. Related indicators include the proportion and number of eosinophils in bronchoalveolar lavage fluid ( Figure 4 B), Pathological HE and PAS staining sections of lungs ( Figure 4 C), and the levels of IL-5 and IL-13 in bronchoalveolar lavage fluid ( Figure 4 D). At the same time, the inventors also detected the changes in ILC2s in lung monocytes. Consistent with the changes in the aforementioned inflammatory indicators, UVB eye irradiation can reduce the R5 / + The number of ILC2s in mouse lung monocytes is reduced but Mc5r fl / fl R5 / + ILC2s in mice Figure 4 E). Pulmonary monocytes were stimulated with PMA / Ionomycin / BFA for four hours, and the ability of ILC2s to release cytokines was detected by flow cytometry. The inventors found that UVB eye irradiation could attenuate the R5 / + IL-5 in the lungs + and IL-13 + This phenomenon was not observed in Mc5r fl / fl R5 / + Observed in mice ( Figure 4 F). This indicates that UVB ocular radiation inhibits the immune response of lung ILC2s in a manner dependent on its surface receptor Mc5r.

[0161] As an important product of UVB irradiation and an important ligand of MC5R, α-MSH was found to inhibit the function of ILC2s in the in vitro experiments mentioned above. Therefore, the inventors speculated that α-MSH may have an effect on ILC2s-mediated lung inflammation in mouse lungs in vivo, and this process may depend on the receptor MC5R on the surface of ILC2s. To verify this hypothesis, the inventors / + With Mc5r fl / fl R5 / + The papain immunomodulation model was established for 5 days, and α-MSH was dripped into the bronchi. On the 6th day, the lung inflammation indexes and ILC2s immune response were detected ( Figure 4 G) Flow cytometry analysis of eosinophils in lung lavage fluid revealed that α-MSH treatment significantly inhibited R5 / + Eosinophil infiltration in mice but unable to suppress Mc5r fl / fl R5 / + The acidophilus ratio ( Figure 4 H) HE and PAS staining sections also showed that α-MSH treatment could significantly inhibit R5 / + Infiltration of immune cells and mucus production around the lung airways of mice, while Mc5r fl / fl R5 / + However, no similar changes were observed ( Figure 4 I). At the same time, the ELISA test results of IL-5 and IL-13 in the alveolar lavage fluid also showed that α-MSH could inhibit the expression of R5 in the inflammatory model. / + Type II cytokine production in mice, but Mc5r fl / fl R5 / + There was no significant change in mice ( Figure 4 J).

[0162] To further explore whether the inhibition of α-MSH on ILC2s in mouse lungs depends on its surface receptor MC5R, the inventors detected the expression of R5 / + With Mc5r fl / fl R5 / + The number and function of ILC2s in the lungs of mice were significantly reduced by α-MSH treatment. / + The number of ILC2s in mouse lungs, but not in Mc5r fl / fl R5 / + This change was not observed in mice ( Figure 4 K). At the same time, PMA / Ionomycin / BFA was used to stimulate mouse lung monocytes and flow cytometry was used to detect the ability of ILC2s to secrete type II cytokines. The inventors found that similar to the results of UVB eye irradiation mentioned above, α-MSH bronchial treatment could inhibit R5 / + IL-5 in ILC2s in mouse lungs + IL-13 + ratio, but could not inhibit Mc5r fl / fl R5 / + Function of mouse lung ILC2s in secreting IL-5 and IL-13 ( Figure 4 LM). This is similar to the results of the UVB-irradiated knockout mouse experiment mentioned above. α-MSH inhibits ILC2s-mediated lung inflammation and related immune responses in a manner dependent on its surface receptor MC5R.

[0163] In the above experiments, the inventors found that UVB eye irradiation can enhance the expression of POMC in the pituitary and lead to an increase in the concentration of α-MSH in mouse serum. In order to study whether pituitary-derived α-MSH can inhibit the function of ILC2s, the inventors cultured the mouse pituitary cells after UVB irradiation. Two days later, the inventors detected the concentration of α-MSH in the pituitary cell culture supernatant ( Figure 4 N), and found that the α-MSH concentration in the UVB irradiation group increased ( Figure 4 O, left). The inventors took the culture supernatant and cultured the separated R5 / + With Mc5r fl / fl R5 / + The inventors found that in R5 / + In the group, the culture supernatant of pituitary cells irradiated by UVB could inhibit the secretion of cytokines by ILC2s, but could not inhibit the function of ILC2s with Mc5r gene deletion ( Figure 4 O, right).

[0164] These results suggest that UVB ocular irradiation may inhibit the function of ILC2s by increasing the concentration of α-MSH in serum and acting on the α-MSH receptor subtype MC5R on ILC2s.

[0165] Example 5 α-MSH-MC5R axis inhibits ILC2s function via JAK-STAT and NF-κB signaling pathways

[0166] Through the above animal experiments, the inventors proved that α-MSH can inhibit the function of ILC2s through the receptor MC5R on the surface of ILC2s. Then the inventors need to explore the mechanism of α-MSH-MC5R downstream. The inventors used flow cytometry to sort R5 in the papain model. / + With Mc5r fl / fl R5 / + ILC2s in the lungs of mice with Mc5r deficiency were identified and RNA-seq analysis was performed to compare the transcriptomes of the two. Gene enrichment analysis (GSEA) showed that asthma-related signaling pathways were enriched in Mc5r-deficient ILC2s (NES=1.5512, p value<0.0307) ( Figure 5 A), including increased expression of IL-13, IL-4, and IL-5 ( Figure 5 B). This is consistent with the phenomenon of the inventor's previous animal experiments. The loss of Mc5r will lead to increased expression of asthma-related genes. ILC2s, as a type of innate immune cell that is relatively old in evolution but has only been widely reported in the past decade, is regulated by a variety of regulatory factors. By analyzing the representative regulatory genes of ILC2s that have been reported, Mc5r deficiency will lead to increased expression of important regulatory genes of ILC2s, such as Il7r, Tnfrsf4, Klrg1, Ramp1, Pdcd1, Ccl9, Gata3, Arg1, etc. ( Figure 5 C). Among them, the most significantly upregulated gene is Il7r ( Figure 5 D). The signals activated downstream of IL-7R mainly include the three pathways of JAK / STAT, PI3K and MAPK (Leonard, 2001). The results of GSEA analysis showed that the JAK / STAT signaling pathway was more enriched than the other two pathways, and the representative genes in the JAK / STAT signaling pathway were significantly upregulated ( Figure 5 E). In order to verify the changes in the protein level of IL-7R (CD127), the inventors performed R5 / + With Mc5r fl / fl R5 / + Mice were immunized with IL-33 for 3 days, and ILC2s in the lungs were sorted by flow cytometry on the 7th day. They were cultured under the conditions of IL-2, IL-7 and α-MSH for 3 days, and cells were collected on the 4th day for flow cytometry detection of CD127. The results showed that in the R5 of the non-deleted Mc5r / + In the Mc5r group, α-MSH treatment could reduce the expression of CD127 in ILC2s. fl / fl R5 / + group, α-MSH treatment could not reduce the expression of CD127 ( Figure 5 F). This indicates that α-MSH may regulate the expression of Il7r / CD127 through Mc5r. This also suggests to the inventors that the inhibitory effect of α-MSH on ILC2s may be dependent on the IL-7 / IL-7R signaling pathway. To verify this hypothesis, the inventors immunized WT mice with IL-33 for 3 days, and then sorted and cultured lung ILC2s on the seventh day. The inventors added the same concentrations of IL-2 and IL-33 to the culture system, and set a gradient concentration of IL-7 (0, 2pg / ml, 20pg / ml, 200pg / ml, 2ng / ml, 20ng / ml and 200ng / ml), and detected the inhibitory effect of α-MSH on ILC2s after 3 days of culture. The experimental results show that when the concentration of IL-7 is higher, the stronger the inhibitory effect of α-MSH on ILC2s, while in the absence of IL-7 or at a lower concentration, α-MSH has almost no inhibitory effect ( Figure 5 G). This indicates that the inhibitory effect of α-MSH on ILC2s may depend on IL-7 / IL-7R signaling. JAK / STAT is an important signaling pathway downstream of IL-7R, which is crucial for the activation and proliferation of immune cells. In order to explore whether α-MSH affects the activation of JAK / STAT signals downstream of IL-7 / IL-7R, the inventors used western blot to detect ILC2s intracellular related signals. First, the inventors sorted R5 cells immunized with IL-33. / + With Mc5r fl / fl R5 / +Mouse ILC2s, and the cells were divided into α-MSH untreated group and treatment group, and cultured for 3 days under IL-2, IL-7, IL-33 and with or without α-MSH. In order to eliminate the influence of various cytokines in the culture system on JAK / STAT signals, the inventors removed the culture supernatant of each group of ILC2s on the 4th day, and then cultured for 24 hours under culture conditions without any cytokines. After 24h, IL-7 (20ng / ml) was added to the culture system again to stimulate 0, 10min, and 60min. At the corresponding time points, ILC2s of each group were collected, and protein lysates were prepared, and the signals of p-JAK1 / 3 and p-STAT3 / 5 were detected by western blot method. Through the results, the inventors found that after culturing ILC2s overnight without cytokines, the signals of p-JAK1 / 3 and p-STAT3 / 5 of the cells were almost completely absent, and 10min after re-adding IL-7h, the signals of p-JAK1 / 3 and p-STAT3 / 5 were activated. This shows that the inventors can use this system to detect JAK / STAT signals downstream of IL-7 in ILC2s. Using this method, the inventors found that in R5 / + In the same group, the p-JAK1 / 3 and p-STAT3 / 5 signals in ILC2s cells were weakened after α-MSH treatment, while in Mc5r fl / fl R5 / + group, α-MSH treatment or not did not affect the p-JAK1 / 3 and p-STAT3 / 5 signals in ILC2s cells ( Figure 5 H). Statistical analysis showed that p-STAT3 / 5 of ILC2s without Mc5r deletion was significantly decreased after α-MSH treatment, while there was no corresponding change in ILC2s with Mc5r deletion ( Figure 5 H), which indicates that α-MSH may affect the JAK / STAT signaling downstream of intracellular IL-7 in ILC2s through MC5R.

[0167] In addition to the IL-7 / IL-7R signaling pathway, IL-33 also plays a very important role in the activation of ILC2s. It is of great significance to explore the role of α-MSH in the cytokine activation pathway of ILC2s. The inventors cultured ILC2 under different cytokine conditions under the background of α-MSH treatment. Under the conditions of IL-2, IL-7 or IL-33 cultured separately, α-MSH treatment did not significantly inhibit the function of ILC2 to release cytokines ( Figure 5 I). However, the inhibitory function of α-MSH was effective under the condition of IL-7 plus IL-33, but was ineffective under the condition of IL-2 plus IL-33 ( Figure 5 J). In the presence of all three cytokines, the inhibitory function of a-MSH on ILC2s was amplified ( Figure 5 J). Meanwhile, under the condition of IL-7 plus IL-33, α-MSH treatment downregulated phosphorylated p65 (active form) in the NF-κB pathway ( Figure 5 K). These findings suggest that α-MSH acts as a potent inhibitory signal of the JAK / STAT and NF-κB pathways to regulate type II cytokine production in lung ILC2s.

[0168] Example 6 α-MSH can inhibit allergic lung inflammation in mice

[0169] In order to further explore the effect of α-MSH on effector cells in allergic bronchi, the inventors constructed a house dust mite HDM model that is more relevant to clinical asthma. HDM is a very common type of allergen protein in clinical practice, which has protease activity. After entering the respiratory tract, it will damage lung epithelial cells and cause lung inflammation. The inventors sensitized mice with high doses of allergen HDM (30ug / mouse) for 3 consecutive days through bronchial immunization. After stopping for 10 days, they gave mice a second sensitization with low doses of HDM (6ug / mouse) for 4 consecutive days starting from the 14th day. α-MSH (4mg / kg) and HDM were administered simultaneously by bronchial instillation, and inflammatory response and related immune cell response detection were performed on the 18th day ( Figure 6 A).

[0170] The detection of antibodies in mouse serum revealed that the serum total IgE, HDM-specific IgE, and HDM-specific IgG1 levels of mice in the α-MSH treatment group were significantly reduced ( Figure 6 B); Lung function of mice decreased ( Figure 6 C); the proportion and number of eosinophils in bronchoalveolar lavage fluid decreased ( Figure 6 D), while the concentrations of type II cytokines IL-5 and IL-13 decreased ( Figure 6 E). The results of lung tissue pathology showed that after α-MSH treatment, the levels of inflammatory cell infiltration and goblet cell mucus secretion in the mouse lungs also decreased ( Figure 6 F) At the same time, lung ILC2s and IL-5 + IL-13 + The number of ILC2s was also suppressed ( Figure 6 G, H). The above experiments show that α-MSH can inhibit the immune response mainly based on ILC2s in allergic asthma.

[0171] discuss

[0172] The interactions and mutual influences between various systems in the body affect the body's various life reactions to varying degrees. In the study of lung inflammation, the nervous system and neuroendocrine system have always been inextricably linked to type II inflammation or type II inflammatory cells. Due to the complexity of the lung microenvironment, the regulatory mechanism of immune effector cells needs further study. Changes in the environment cause the body to take corresponding countermeasures to maintain the body's homeostasis, and UVB, as a type of seasonal environmental factor, has complex and diverse regulation of the body. In addition to being mutagenic, moderate UVB irradiation has certain immunomodulatory properties, but because the UVB reaction involves a variety of systemic and local immune cells, its mechanism is not yet clear. In order to explore the relationship between UVB and lung inflammation such as asthma, the inventors conducted a series of clinical observation experiments and mouse model experiments.

[0173] In this study, the inventors discovered the potential mechanism of lung ILC2 in the context of lung inflammation such as allergic asthma, and proposed the possibility of sunlight-assisted asthma treatment. Given the importance of ILC2s in the pathogenesis of asthma, the inventors found in a mouse model that UVB can attenuate lung inflammation mediated by papain-induced ILC2s cells. Correspondingly, after detecting the relevant surface receptors of ILC2s cells, the inventors found that ILC2s specifically and highly expressed a class of α-MSH receptors MC5R. This suggests the inventors' potential function of α-MSH on ILC2. As an important product driven by UVB radiation, α-MSH is produced more after eye irradiation with UVB, and the expression of Pomc and Pcsk2 in the pituitary gland increases, thereby promoting the production of α-MSH. α-MSH in the circulation system inhibits ILC2s response through MC5R expressed on the surface of ILC2s, thereby reducing lung inflammation. In terms of mechanism, the inventors observed that the α-MSH-MC5R axis led to the downregulation of IL-7R-JAK / STAT phosphorylation signals. As an endogenous neuroendocrine hormone, α-MSH can effectively inhibit lung inflammation induced by fungi, house dust mites, ovalbumin, etc., demonstrating the potential clinical application value of α-MSH. These observations indicate that the UVB radiation-driven neuroendocrine hormone α-MSH is a new type of lung ILC2s regulator and opens up a promising therapeutic avenue for the treatment of lung inflammation.

[0174] 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.

Claims

1. A use of α-melanocyte stimulating hormone (α-MSH) or a promoter thereof, characterized in that: For use in preparing a preparation or a kit for: (1) Upregulate the expression of α-MSH receptor in ILC2 cells; (2) Upregulation of corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC); (3) downregulate the number of ILC2 cells; (4) downregulating the function of ILC2 cells to secrete cytokines; (5) downregulating the expression of IL-5, IL-13, and / or eosinophils by ILC2 cells; (6) Inhibit the immune response of ILC2 cells; (7) inhibiting JAK / STAT and NF-κB signaling pathways; and / or (8) Prepare drugs for treating and / or preventing ILC2-mediated pulmonary inflammatory diseases.

2. The use according to claim 1, characterized in that The subtypes of the α-MSH receptor are MC1R, MC2R, MC3R, MC4R, and MC5R; preferably MC5R.

3. A use of α-MSH receptor subtype MC5R or a promoter thereof, characterized in that: For use in preparing a preparation or a kit for: Downregulation of ILC2 cell numbers; and / or Down-regulate the function of ILC2 cells to secrete cytokines; Down-regulate the expression of IL-5, IL-13 and / or eosinophils by ILC2 cells; Inhibit the immune response of ILC2 cells; Inhibit JAK / STAT and NF-κB signaling pathways; and / or Treating and / or preventing ILC2-mediated pulmonary inflammatory diseases.

4. The use according to claim 1, characterized in that The ILC2 is derived from a mammal, preferably from a human, a mouse, a rat, or a cynomolgus monkey.

5. A composition, characterized in that The pharmaceutical composition comprises: (i) a first active ingredient, wherein the first active ingredient is selected from the group consisting of α-melanocyte stimulating hormone (α-MSH) or a promoter polypeptide derivative thereof; or a combination thereof; and / or (ii) a second active ingredient, the second active ingredient being selected from the group consisting of: an agonist of the α-MSH receptor subtype MC5R; or a combination thereof; (iii) a pharmaceutically acceptable carrier or excipient.

6. The composition according to claim 5, characterized in that The α-melanocyte stimulating hormone (α-MSH) includes polypeptide derivatives of α-MSH.

7. The composition according to claim 5, characterized in that The composition or preparation can be used alone or in combination in the prevention and / or treatment of ILC2-mediated pulmonary inflammatory diseases.

8. The composition according to claim 7, characterized in that The ILC2-mediated lung inflammatory disease applications include type II inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD).

9. A method for inhibiting IL2 cell immune response, comprising the steps of: (1) in the presence of α-MSH or a promoter thereof; and / or (2) Cultivating stellate cells in the presence of the α-MSH receptor subtype MC5R gene, or its protein, or its promoter, thereby inhibiting the IL2 cell immune response.

10. A method for screening potential therapeutic agents for ILC2-mediated pulmonary inflammatory diseases, comprising: (a) in a test group, in a culture system, in the presence of a test compound, culturing cells expressing the MC5R gene for a period of time T1, and detecting the expression level E1 of the MC5R gene 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 level E2 of the MC5R gene 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 lung inflammatory diseases.

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