Application of mesenchymal stem cell-derived small extracellular vesicles in treatment of neutrophil airway inflammation

Abstract

The invention provides application of mesenchymal stem cell-derived small extracellular vesicles in preparation of a product for preventing and / or treating airway inflammation mainly caused by neutrophil infiltration. Human cell in-vitro experiments and mouse in-vivo experiments find and prove that MSC-sEV can be directly taken by cells, activation of a cell JAK2-STAT3 pathway is inhibited, Th17 cell polarization is inhibited, neutrophil airway inflammation is remarkably relieved, and protein of MSC-sEV plays an important role in inhibition of Th17 cell polarization. The invention provides the application of the mesenchymal stem cell-derived small extracellular vesicles to prevention and treatment of airway inflammation mainly aiming at neutrophil infiltration, has the advantages of safety and effectiveness, and provides a new medicine for clinic.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically relates to the application of small extracellular vesicles derived from mesenchymal stem cells in the treatment of neutrophilic airway inflammation. Background Art

[0002] Both allergic rhinitis and allergic asthma are allergen-mediated respiratory inflammations. Allergen-mediated airway inflammation is a type of chronic respiratory inflammation induced by allergens, which causes airway inflammatory infiltration, airway hyperresponsiveness, and airway remodeling. According to the types of infiltrating inflammatory cells, allergen-mediated respiratory inflammation can be divided into inflammation mainly infiltrated by eosinophils and inflammation mainly infiltrated by neutrophils. Patients with eosinophilic inflammation are hormone-sensitive, while patients with neutrophilic inflammation are hormone-tolerant. Generally speaking, when the number of neutrophils in the induced sputum of a patient accounts for 40%-76% of the total number of inflammatory cells, the airway inflammation is mainly infiltrated by neutrophils. Previous studies have considered that allergen-mediated airway inflammation is mainly infiltrated by eosinophils. In recent years, the role of neutrophils in allergen-mediated airway inflammation has received extensive attention. Douwes et al. even believe that more than 50% of asthma cases have airway inflammation mainly infiltrated by neutrophils. A number of studies have found that in murine allergen-induced airway inflammation, neutrophils appear earlier than eosinophils. Neutrophils begin to increase 3-6 hours after allergen stimulation, while eosinophils do not begin to increase until 24 hours, indicating that neutrophils may be involved in the occurrence of airway inflammation earlier than eosinophils. Neutrophils activate airway inflammasomes by releasing pro-inflammatory factors tumor necrosis factor (TNF) and interleukin 1β (IL-1β), and by producing neutrophil extracellular traps (NETs). NETs also stimulate macrophages to induce pro-inflammatory factors, further promoting neutrophil infiltration and exacerbating airway injury.

[0003] Currently, the treatment methods for allergen-mediated respiratory inflammation mainly include drug treatment, specific immunotherapy, and biologic agent treatment. Drug treatment mainly uses glucocorticoids locally and systemically. This treatment method requires long-term medication, but the disease cannot be cured, and some patients with neutrophil infiltration as a characteristic develop hormone resistance, resulting in treatment effects that are not as expected. Specific immunotherapy can treat the cause by targeting allergens, but it has disadvantages such as a long desensitization period, poor patient compliance, high cost, few targeted treatment options, and only 50-70% of patients are effective.

[0004] In recent years, biotherapies such as monoclonal antibody therapies have shown good prospects in allergen-mediated airway inflammation. However, at present, the vast majority of monoclonal antibodies mainly target the treatment of airway inflammation dominated by eosinophil infiltration, and there is still no monoclonal antibody available for the treatment of airway inflammation dominated by neutrophil infiltration. In particular, how to effectively and safely treat airway inflammation with neutrophil infiltration remains a great challenge. Therefore, the development of new therapies for airway inflammation dominated by neutrophil infiltration has high medical application value and is also a technical difficulty.

[0005] Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) are rich in contents, carrying a variety of proteins, RNAs, and lipids, and their components vary depending on the source cells. Among them, the proteins and microRNAs (miRNAs) contained in MSC-sEVs are considered important components for exerting therapeutic functions. Both miRNAs and proteins in MSC-sEVs may play biological roles and affect the disease process, which also leads to uncertainties and complexities in the treatment of specific diseases. Moreover, the role and related mechanisms of MSC-sEVs in airway inflammation with neutrophil infiltration are still lacking in research. Summary of the Invention

[0006] Based on this, the purpose of the present invention is the new application of mesenchymal stem cell-derived small extracellular vesicles in medicine.

[0007] The technical solutions to achieve the above purpose include the following.

[0008] The application of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) in the preparation of products for preventing and / or treating airway inflammation dominated by neutrophil infiltration.

[0009] In some embodiments, the airway inflammation may also be airway inflammation dominated by neutrophil infiltration with combined infection and allergy.

[0010] In some embodiments, the above airway inflammation is Th17-dominated neutrophilic airway inflammation.

[0011] In some embodiments, the application includes that mesenchymal stem cell-derived small extracellular vesicles inhibit the polarization of human Th17 cells and inhibit the proliferation of Th17 cells. This application will not have a cytotoxic effect of promoting apoptosis.

[0012] In some embodiments, mesenchymal stem cell-derived small extracellular vesicles inhibit the activation of the JAK2-STAT3 pathway, thereby inhibiting the polarization of Th17 cells.

[0013] The small extracellular vesicles derived from mesenchymal stem cells mainly directly inhibit the polarization of Th17 cells, rather than delaying the inhibition of the immune balance of Th2 / Th17 or Th1 / Th17.

[0014] The small extracellular vesicles derived from mesenchymal stem cells can be prepared by a variety of conventional methods, such as ultracentrifugation, tangential flow filtration, etc., and also include methods that can obtain small extracellular vesicles derived from mesenchymal stem cells after various future biotechnological improvements.

[0015] In some of these embodiments, the preparation method includes the following:

[0016] Collect the cell culture supernatant after the passage and amplification of mesenchymal stem cells;

[0017] Prepare an eluate containing small extracellular vesicles derived from mesenchymal stem cells by anion exchange chromatography, and the supernatant liquid obtained by centrifugal ultrafiltration is MSC-sEV.

[0018] In some of these embodiments, the mesenchymal stem cells are derived from induced pluripotent stem cells, or from umbilical cord, bone marrow, adipose tissue, urine, or menstrual blood.

[0019] In some of these embodiments, the product is a drug or a biological agent.

[0020] In some of these embodiments, the drug or biological agent is an injection, such as an intravenous injection.

[0021] In some of these embodiments, the drug or biological agent is a nebulized inhalation agent, such as a dry powder inhalant or a nebulized solution.

[0022] The nebulized solution can be a finished product, i.e., a nebulized inhalation agent, or can be formed by diluting a lyophilized powder with a diluent to form a liquid and then nebulizing it.

[0023] In some of these embodiments, the drug or biological agent is an oral dosage form, such as an enteric-coated capsule or a nanoparticle embedding preparation.

[0024] In some of these embodiments, the drug or biological agent is a topical dosage form, such as a gel, cream, spray, or eye drops.

[0025] Neutrophilic airway inflammation is steroid-resistant, commonly seen in critically ill patients. Glucocorticoid therapy has poor efficacy, and new therapies such as monoclonal antibodies and antagonists also have unsatisfactory effects. Currently, there is no very effective treatment method. Through in vitro experiments with human cells and in vivo experiments with mice, the present invention has discovered and proven that MSC-sEV can significantly reduce neutrophilic airway inflammation by directly being taken up by cells, inhibiting the activation of the intracellular JAK2-STAT3 pathway, and inhibiting Th17 cell polarization. The protein of MSC-sEV plays an important role in inhibiting Th17 cell polarization. Based on this discovery, an application for preventing and treating airway inflammation mainly characterized by neutrophil infiltration through mesenchymal stem cell-derived small extracellular vesicles is obtained, and this application has the advantages of safety and effectiveness, and can provide new medications for clinical use. Brief Description of the Drawings

[0026] Figure 1 Identification of mesenchymal stem cell-derived small extracellular vesicles. Among them, (A) Nanosight was used to detect the diameter of small extracellular vesicles; (B) transmission electron microscopy was used to detect the structure and diameter of small extracellular vesicles; (C) Western Blot was used to detect exosome markers of mesenchymal stem cells and their small extracellular vesicles. MSC: mesenchymal stem cells, MSC-sEV: mesenchymal stem cell-derived small extracellular vesicles.

[0027] Figure 2 Schematic diagram for establishing a mouse airway inflammation model mainly characterized by neutrophil infiltration.

[0028] Figure 3 MSC-sEV alleviates allergic airway inflammation in mice. Among them, (B) H&E sections of the lungs show the degree of inflammatory cell infiltration in the trachea and blood vessels; (C) inflammatory scores of the trachea and blood vessels. *P<0.05, ***P<0.001. Br: trachea, Ve: blood vessel. Figure 4 MSC-sEV intervention reduces the number of inflammatory cells in the bronchoalveolar lavage fluid of mice. Among them, (A) flow cytometry gating strategy for inflammatory cells in the bronchoalveolar lavage fluid; (B) display diagram and statistical chart of the proportion of neutrophils in the bronchoalveolar lavage fluid; (C) display diagram and statistical chart of the proportion of eosinophils in the bronchoalveolar lavage fluid, *P<0.05, ****P<0.0001. Neu: neutrophils; Eos: eosinophils. Figure 5 MSC-sEV reduces the Th17 cell level in neutrophil-infiltrated airway inflammation in mice. Among them, (A) flow cytometry gating strategy for helper T cells in the lungs; (B) display diagram and statistical chart of the proportion of Th17 cells in the lungs; (E) statistical chart of the level of IL-17A in the bronchoalveolar lavage fluid, *P<0.05; ****P<0.0001.

[0029] Figure 6MSC-sEV inhibits the polarization of human Th17 cells. Among them, A. Flow cytometry analysis chart and statistical chart of Th17 cells co-cultured with MSC-sEV; B. Statistical chart of detecting the level of IL-17A in the co-culture supernatant by ELISA. *P<0.05; **P<0.01; ***P<0.001.

[0030] Figure 7 MSC-sEV significantly inhibits the proliferation of human Th17 cells but does not promote the apoptosis and necrosis of Th17 cells. Among them, A. Flow cytometry mean fluorescence intensity histogram and statistical chart of CFSE-stained Th17 cells; B. Flow cytometry chart and statistical chart of Th17 cell apoptosis and necrosis. *P<0.05; ***P<0.001; ns means no statistical significance.

[0031] Figure 8 Transcriptome sequencing of Th17 cells and Th17 cells treated with MSC-sEV. Among them, A. Heatmap of differentially expressed genes between naive Th17 cells and Th17 cells treated with MSC-sEV; B. Volcano plot of differentially expressed genes between naive Th17 cells and Th17 cells treated with MSC-sEV; C. Validation of the expression of IL22 mRNA in Th17 cells and Th17 cells treated with MSC-sEV by real-time quantitative PCR. **P<0.01.

[0032] Figure 9 Transcriptome sequencing analysis reveals that MSC-sEV downregulates the JAK-STAT pathway in Th17 cells. Among them, A. Display chart of differentially expressed gene enrichment pathways in Th17 cells co-cultured with MSC-sEV; B. Display chart of gene enrichment pathways in Th17 cells downregulated by MSC-sEV analyzed by GSEA; C. It is found by GSEA analysis that MSC-sEV downregulates the JAK-STAT pathway in Th17 cells. GO: Gene Ontology; GSEA: Gene Set Enrichment Analysis; NES: Normalized Enrichment Score. Figure 10 MSC-sEV inhibits Th17 cell polarization through the JAK2-STAT3 pathway. Among them, A. Detection of p-STAT3 level in mouse lung tissue by Western Blot; B. Detection of p-STAT3 and p-JAK2 levels in human Th17 cells by Western Blot; C. Detection of Th17 cell and IL-17A levels after co-culturing the STAT3 activator colivelin with MSC-sEV and Th17 cells by flow cytometry and ELISA respectively. *P<0.05; **P<0.01; ***P<0.001.

[0033] Figure 11Characterization of MSC-sEV after RNA removal. Among them, A. RNA content of MSC-sEV and MSC-sEV after RNA removal; B. Detection of exosome markers of MSC-sEV after RNA removal by Western Blot.

[0034] Figure 12 MSC-sEV after RNA removal inhibits Th17 cell polarization. Among them, A. Detection of Th17 cell level after co-culture of MSC-sEV after RNA removal and Th17 cells by flow cytometry; B. Detection of IL-17A level after co-culture of MSC-sEV after RNA removal and Th17 cells by ELISA. *P<0.05; **P<0.01; ns has no statistical significance.

[0035] Figure 13 Proteomic analysis of possible functional proteins of MSC-sEV. Among them, A. Venn diagram of the intersection of MSC-sEV proteins and the Vesiclepedia protein library; B. KEGG enrichment analysis of some pathways enriched by unique proteins of MSC-sEV; C. Venn diagram of the intersection of the pathways enriched by unique proteins of MSC-sEV and the pathways enriched by down-regulated genes in Th17 cells after treatment with MSC-sEV; D-F. Functional analysis of MSC-sEV proteins in terms of cellular component (D), molecular function (E), and biological process (F) through GO enrichment analysis of the pathways commonly enriched by unique proteins of MSC-sEV and down-regulated genes in Th17 cells after treatment with MSC-sEV. GSEA: Gene Set Enrichment Analysis. Detailed implementation manners

[0036] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0037] The experimental methods without specific conditions noted in the following examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturers. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0039] Definitions For the convenience of understanding the present technology, some terms and phrases are defined below.

[0040] MSC: Mesenchymal stem cell.

[0041] iPSCs: induced pluripotent stem cells, Induced pluripotent stem cells.

[0042] Small extracellular vesicels, sEV: Small extracellular vesicles.

[0043] MSC-sEV: Small extracellular vesicles derived from mesenchymal stem cells.

[0044] Allergen-mediated airway inflammation can be divided into eosinophil infiltration-dominated and neutrophil infiltration-dominated inflammation according to different infiltrating inflammatory cells. Eosinophil infiltration-dominated inflammation is generally caused by allergens in the external environment, such as dust mites and pollen, etc. After being inhaled into the respiratory tract by sensitive individuals, it induces an increase in Th2 cells, and then promotes eosinophil infiltration. This type is usually mild inflammation and has a good therapeutic effect with glucocorticoids. Neutrophil infiltration-dominated inflammation is caused by the combination of allergens stimulating the human body and infections with Gram-negative bacteria and viruses, etc., which induces an increase in Th17 cells, and then promotes neutrophil infiltration. This type is usually moderate to severe inflammation and has a poor therapeutic effect with glucocorticoids.

[0045] MSC-sEV has been proven to be able to inhibit type 2 inflammation and reduce airway inflammation with eosinophil infiltration by reducing the levels of Th2 cells and type 2 innate lymphoid cells (ILC2). However, neutrophil infiltration-dominated inflammation is an airway inflammation mainly characterized by an increase in Th17 cells. Th17 cells are the key pathogenic cells, and Th2 cells are not the key pathogenic cells and will not increase significantly in this type of inflammation. Therefore, whether MSC-sEV targeting type 2 inflammation can reduce Th17 cell-dominated airway inflammation remains unknown.

[0046] Through research, it has been found that in neutrophil-dominated airway inflammation, MSC-sEV does not affect the level of Th2 cells, but can reduce the level of Th17 cells and reduce airway inflammation with neutrophil infiltration, which is different from the previous treatment mechanism of MSC-sEV. The research shows that the treatment mechanism of MSC-sEV is different in different types of airway inflammation. In addition to treating allergic airway inflammation dominated by type 2 inflammation, MSC-sEV can also treat airway inflammation with neutrophil infiltration mainly caused by the combination of infection and allergy.

[0047] The present invention will be further described in detail below with reference to specific embodiments.

[0048] The main reagents involved in the experiment are as follows:

[0049]

[0050]

[0051]

[0052] The animals used in the experiments of the present invention were 4-6-week-old specific pathogen free (SPF)-grade female C57BL / 6 mice, purchased from Guangdong Medicilon Inc., and housed in the SPF-grade barrier environment of the Experimental Animal Center of Sun Yat-sen University (Area F, North Campus). All animal operations in this experiment were approved by the Animal Ethics Committee of Sun Yat-sen University and were carried out in accordance with the requirements of the welfare and ethics of experimental animals of Sun Yat-sen University to minimize the pain caused to animals by experimental operations as much as possible.

[0053] Example 1 Preparation of MSC-sEV

[0054] According to the existing and publicly disclosed methods, MSC-sEV was prepared. For example, the tangential flow method in CN116555006A is incorporated herein by reference in its entirety.

[0055] The mesenchymal stem cells described in the present invention are derived from induced pluripotent stem cells, and the preparation method of iPSC-MSC-sEV is exemplified as follows.

[0056] 1. Isolation and purification of iPSC-MSC-sEV

[0057] 1.1 Experimental procedures

[0058] (1) After resuscitating iPSC-MSC and expanding it to about 1.8×10 9 cells, remove the culture medium, wash 3 times with sterile phosphate buffered solution (PBS), and add 15 mL of serum-free conditioned medium to each dish and continue culturing for 6 h.

[0059] (2) After 6 h, remove the culture medium, add 30 mL of fresh serum-free conditioned medium to each dish, continue culturing for 42 h, collect the cell culture medium, centrifuge at 4°C and 2650 g for 20 minutes, and collect the supernatant.

[0060] (3) Use anion exchange chromatography to separate and purify sEV from the above supernatant.

[0061] 2. Identification of the characteristics of iPSC-MSC-sEV

[0062] 2.1 Experimental procedures

[0063] (1) The particle diameter and particle concentration of iPSC-MSC-sEV were detected using a Nanosight NS300 instrument. Each sample was detected 3 times, 30 seconds each time. The average particle diameter and particle concentration were obtained based on the data detected 3 times.

[0064] (2) The morphology of MSC-sEV was detected using a transmission electron microscope. The MSC-sEV sample was first fixed with 2% glutaraldehyde for 30 minutes. The treated sample was aspirated onto a copper grid for loading. The copper grid was placed with the Formvar side facing down on a droplet of 3% phosphotungstic acid (pH = 7) for 5 minutes, and then pure water was added dropwise for cleaning for 2 minutes. After air drying, it was photographed.

[0065] (3) The expression of sEV characteristic protein markers in iPSC-MSC-sEV was detected using Western Blot technology.

[0066] 2.2 Results

[0067] According to the Nanosight detection results, it was known that the diameter of the obtained MSC-sEV was mainly concentrated around 132 nm (see Figure 1 A). Through the transmission electron microscope, it was observed that MSC-sEV was a double-membrane circular vesicle with a diameter between 40 - 140 nm (see Figure 1 B). The results of Western Blot showed that the expression levels of the four exosome markers CD9, CD63, Alix, and TSG101 in MSC-sEV were significantly higher than those in cell lysates. The endoplasmic reticulum marker Alix was only present in cell lysates and not found in MSC-sEV.

[0068] In summary, the obtained MSC-sEV has typical exosome characteristics and meets the expected standards. The obtained iPSC-MSC-sEV was used in the experiments in the following examples.

[0069] Example 2: Establishment of a murine model of neutrophilic allergic airway inflammation and verification of the role of iPSC-MSC-sEV in this model

[0070] (I) Establishment of a murine model of neutrophilic allergic airway inflammation

[0071] 1 Experimental procedures

[0072] (1) Sensitization: Prepare the following sensitization solution according to the dosage per mouse: Mix 2.5 μl of ovalbumin (OVA, 40 μg / μl), 1 μl of lipopolysaccharide (LPS, 100 ng / μl) and 16.5 μL of PBS to a total of 20 μl. Sensitize on day 0 and day 7. Inject 1.2 ml of sodium pentobarbital into the right lower abdominal cavity of 4 - 6 week-old mice. Generally, 8 - 10 mice are injected each time. After the mice lie down and enter a state of deep anesthesia with slow and steady nodding, take out the mice, hang them vertically, pull out the tongue with forceps, and drip into the airway with a 20 μl pipette in coordination with the respiratory rhythm of the mice. The mice in the blank control group are dripped with an equal volume of PBS through the airway.

[0073] (2) Administration: The mice in the treatment group were intravenously injected with the iPSC-MSC-sEV described in Example 1 via the tail vein on day 12 and day 13, with a dosage of 100 μg / mouse / time. 4 hours before atomization on day 14, the exosomes were intravenously injected, with a dosage of 100 μg / mouse / time. The mice in the blank control group and the model group were intravenously injected with an equal volume of PBS via the tail vein.

[0074] (3) Challenge: Challenge the mice on day 14. On the day of challenge, prepare a 5% solution of OVA with PBS and filter it through a 0.2 μm filter to sterilize. Put the mice in the model group and the treatment group into the atomization box and close it. Add 8 ml of 5% OVA to the atomizer and atomize the mice for 40 minutes. The mice in the blank control group were atomized with an equal volume of PBS.

[0075] (4) Grouping: The blank control group is PBS / PBS / PBS; the model group is OVA / OVA / PBS; the treatment group is OVA / OVA / MSC-sEV.

[0076] For the technical roadmap of the experiment, see Figure 2 .

[0077] (2) Verify the role of iPSC-MSC-sEV in a murine model of neutrophilic allergic airway inflammation 1. Animal experiment material collection

[0078] (1) Exchange and treatment of broncho - alveolar lavage fluid (BALF): After anesthetizing the mice with isoflurane, trim their whiskers, draw blood from the eyeballs to sacrifice the mice, expose and free the trachea of the mice, pass a suture under the freed trachea for subsequent ligation of the trachea; connect a 1 mL syringe to the soft needle of an intravenous indwelling needle, aspirate 1 mL of pre - cooled PBS solution, and expel the air bubbles; make a small V - shaped incision under the throat of the mice with small scissors, insert the soft needle about 1 / 2 length, tie the soft needle to the trachea of the mice with the reserved suture, and fix it properly; slowly push and pull the syringe piston 3 times to wash the lungs of the mice with PBS, and about 0.8 mL of bronchial BALF can be obtained; centrifuge the BALF (400 g, 5 min, 4 °C), measure the level of IL - 17A in the supernatant by ELISA, and the precipitate after centrifugation is cells, which are used for flow cytometry staining analysis of inflammatory cells;

[0079] (2) Acquisition and treatment of lung tissue: Open the chest cavity of the mice, aspirate the Hank's balanced salt solution containing 1 mM EDTA aseptically with a 5 mL syringe, cut off the left atrial appendage, inject the solution into the right ventricle to wash the lungs, remove the residual blood in the lungs, cut off the left lung, soak it in 4% paraformaldehyde for tissue pathological staining, cut the right lung into pieces and soak them in 2 mL of collagenase digestion solution, digest in a 37 °C water bath for 1 hour, and terminate the digestion with 5 - fold volume of PBS. Use the head of the syringe piston to grind the lung tissue on a 75 μm filter until there is a small amount of white connective tissue remaining, centrifuge the ground cells (350 g, 5 min), discard the supernatant after centrifugation, and add red blood cell lysate to lyse red blood cells. Seed the obtained cells in a 24 - well plate at 2×10 6 / well with 1 mL of complete medium, stimulate with PMA (25 ng / mL) + ionomycin (1 μg / mL) + BFA (10 μg / mL) for 5 hours, collect the liquid, centrifuge (350 g, 5 min) to obtain the lower - layer cells, and detect lung Th cells by flow cytometry.

[0080] 2. H&E staining

[0081] Fix the lung tissue with 4% paraformaldehyde, take out the lung tissue after 1 day, dehydrate it with a gradient of alcohol density and permeabilize it with xylene (1 hour 75% alcohol → 1 hour 80% alcohol → 1 hour 90% alcohol → 40 minutes 100% alcohol → 40 minutes xylene I → 40 minutes xylene II), and then embed it in paraffin.

[0082] Embed the wax block into 5-μm sections, place them in an oven at 65 °C for 2 hours. Take out the sections and perform dewaxing and hydration (5 minutes in xylene I → 5 minutes in xylene II → 3 minutes in 95% ethanol I → 3 minutes in 95% ethanol II → 2 minutes in 80% ethanol → 2 minutes in 70% ethanol). Wash with distilled water for 20 seconds for hydration. Stain with hematoxylin for 5 minutes, then wash with distilled water to remove the floating color. Differentiate with 1% hydrochloric acid alcohol for 2 seconds, and rinse slowly with running water for 20 minutes. Stain with 0.5% eosin for 2 minutes, and wash with distilled water to remove the excess floating color. Dehydrate with an alcohol density gradient and permeabilize with xylene (2 seconds in 70% ethanol → 2 seconds in 80% ethanol → 2 seconds in 90% ethanol → 10 seconds in 100% ethanol I → 10 seconds in 100% ethanol II → 2 minutes in xylene I → 2 minutes in xylene II). Mount with neutral balsam, air-dry overnight, and observe under an optical microscope.

[0083] 3. Enzyme-Linked Immunosorbent Assay (ELISA)

[0084] The cytokine IL-17A level in the mouse BALF was detected according to the instructions of the Xinbosheng ELISA kit. The expression level of IL-17A in the co-culture supernatant of human Th17 cells and MSC-sEV was detected according to the instructions of the Linke ELISA kit.

[0085] 4. Flow cytometry

[0086] Use mouse CD45 FITC, CD11b APC-Cy7, CD64 PE, Ly-6G Alexa flour 700, siglec-F APC to label BALF cells; use CD4 Percp-Cy5.5, IFN-γ PE, IL-17A FITC, IL-4 PE-Cy7 to label mouse lung Th cells; use live / dead eFlour 506, CD4-PerCP-Cy5.5, IL-17A-FITC to label human Th17 cells.

[0087] The inflammatory cells in the BALF of mice were stained on the cell membrane surface, while the Th cells in the mouse lungs and human Th17 cells were stained intracellularly. For cell membrane staining, the cells were washed with 1 mL of PBS, centrifuged (350 g, 5 min) to remove the supernatant, resuspended with 50 μL of PBS, and the corresponding surface flow antibodies were added, followed by incubation at 4°C for 30 minutes and then washed with PBS. For intracellular staining: Cells were collected after being stimulated with PMA + ionomycin + BFA for 5 hours in advance. After cell membrane staining, the cells were resuspended with 100 μL of IC fixation solution and incubated at room temperature for 20 minutes. Then, 1 mL of 1× permeabilization solution was added and centrifuged (700 g, 5 minutes), and the supernatant was removed. The cells were resuspended with 50 μL of permeabilization solution, and the corresponding intracellular cytokine flow antibodies were added, followed by incubation at room temperature for 30 minutes. Then, 1 mL of permeabilization solution was added, centrifuged (700 g, 5 min), and the supernatant was discarded. The cells were resuspended with 400 μL of PBS and analyzed by flow cytometry using CytoExpert or FlowJo V10.

[0088] CFSE was dissolved in DMSO to a concentration of 10 mg / mL, and CD4 + T cells were resuspended with 1 mL of PBS, and 110 μL of PBS containing 0.5% FBS and 0.5 μL of CFSE were added. After covering the lid, the mixture was gently shaken in the hand for 10 minutes and then washed twice with ten-fold volume of complete culture medium (1000 r, 5 min). CFSE-labeled CD4 + T cells were used for Th17 cell induction.

[0089] After co-culturing Th17 cells and MSC-sEV for 4 days, the cell necrosis and apoptosis were detected according to the steps of the AnnexinV-FITC / PI cell apoptosis detection kit instruction manual.

[0090] 5. Inflammatory score of mouse lungs

[0091] The inflammatory score of mouse lungs was classified into 9 grades according to the number of bronchi with inflammatory cell infiltration and the number of layers of inflammatory cells in the lung H&E stained sections (see Table 2)

[0092] Table 2 Inflammatory score of mouse lung tissue

[0093]

[0094] 6. PBMC source

[0095] A total of 20 patients with allergic rhinitis caused by dust mite allergy were recruited in this part. After all volunteers signed the informed consent form, 10 mL of peripheral blood was drawn. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.

[0096] All recruited patients with allergic rhinitis due to dust mite allergy met the following inclusion criteria: (1) Onset or obvious symptoms in the past two weeks, meeting the diagnostic criteria for AR in the "Allergic Rhinitis and its Impact on Asthma (ARIA)"

[26] guidelines: two or more symptoms such as continuous sneezing, nasal congestion, nasal itching, and clear watery nasal discharge; (2) Positive dust mite-specific serum IgE test; (3) No use of nasal sprays, oral corticosteroids, antihistamines, or antibiotics in the past four weeks; (4) No upper respiratory tract infections such as common colds or influenza in the past four weeks; (5) Aged 18 - 50 years, regardless of gender; (6) No autoimmune diseases such as allergic dermatitis, systemic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, asthma, etc.; (7) Women are not in the menstrual period, pregnancy, or lactation period.

[0097] 7. Extraction of human peripheral blood mononuclear cells

[0098] PBMC extraction was performed within 1 hour after 10 mL of peripheral blood was drawn from each volunteer. The steps are as follows:

[0099] (1) Centrifuge the peripheral blood (600 g, 10 min), aspirate the upper plasma and store it in a -80°C refrigerator, and dilute the lower blood with the same volume of PBS;

[0100] (2) Gently and slowly add the diluted blood to a 15 mL centrifuge tube pre-added with the same volume of lymphocyte separation medium, taking care not to disrupt the layering;

[0101] (3) Use a centrifuge to centrifuge (2000 rpm, 20 min, acceleration 2, deceleration 0), and aspirate the middle white membrane layer with a Pasteur pipette (the first layer from top to bottom is the plasma platelet layer, the second layer is a thin milky white membrane layer, the third layer is a transparent lymphocyte separation medium layer, and the fourth layer is the red blood cell layer), and transfer it to a new centrifuge tube;

[0102] (4) Add an appropriate amount of PBS for washing, centrifuge with a centrifuge (350 g, 5 min), discard the supernatant, add 650 μL of 1× red blood cell lysate diluted with sterile distilled water to resuspend, and let it stand for 5 minutes;

[0103] (5) Add 5 times the volume of PBS to terminate, centrifuge with a centrifuge (350 g, 5 min), discard the supernatant, add complete medium (90% RPMI + 10% FBS + 1% double antibody) to resuspend for subsequent experiments.

[0104] 8. CD4 + T cell sorting

[0105] (1) Determine the number of PBMC cells using a cell counting chamber, centrifuge (350 g, 5 min), and discard the supernatant;

[0106] (2) For every 10 7Add 10 μL of CD4 magnetic bead antibody and 40 μL of MACS Buffer to the cells, resuspend, and incubate in a 4°C refrigerator for 20 minutes; (3) For every 10 7 Resuspend the cells in 500 μL of MACS Buffer, centrifuge (350 g, 5 min), and discard the supernatant;

[0107] (4) For every 10 8 Resuspend the cells in 500 μL of MACS Buffer and pass through an MS separation magnetic column. The cells that flow through are CD4-positive cells.

[0108] 9. Induce co-culture of Th17 cells and MSC-sEV

[0109] Induction of human Th17 cells:

[0110] (1) Prepare Th17 induction culture medium: Complete culture medium (90% RPMI 1640 + 10% FBS + 1% double antibody) + anti-CD3 (1 μg / mL) + anti-CD28 (5 μg / mL) + IL-1β (10 ng / mL) + IL-6 (20 ng / mL) + TGF-β1 (1 ng / mL) + IL-23 (100 ng / mL);

[0111] (2) After CD4 + T cell counting, in the control group, take 2 × 10 5 cells and add them to 200 μL of complete culture medium. In the Th17 induction group, take 2 × 10 5 cells and add them to 200 μL of Th17 induction culture medium. Seed them in 96-well flat bottom plates respectively, gently shake well. In the MSC-sEV treatment group, based on the Th17 induction group, add 30 μg / mL of MSC-sEV;

[0112] (3) On the second day after seeding, change the medium semi-quantitatively. On the fourth day, centrifuge (300 g, 5 min). The cells in the lower layer are used for subsequent flow cytometry, immunofluorescence, and RNAseq analysis, and the supernatant is used for ELISA detection of IL-17A.

[0113] 10. Statistical analysis

[0114] All data analysis in this part is performed using GraphPad 8.3.0. The results of each group of experimental data are expressed by the method of Mean ± SD. One-way ANOVA is used for analysis among the three groups in the mouse experiment, and paired one-way ANOVA is used for analysis among the three groups in the human cell experiment. When P < 0.05, the results are statistically different.

[0115] (III) Results

[0116] The results of H&E staining of lung tissue sections showed that there were multiple layers of inflammatory cell infiltrations around the trachea and blood vessels in the model group, and the inflammation score was relatively high. The intervention treatment with MSC-sEV significantly reduced the number of inflammatory cell infiltrations in the trachea and blood vessels in the treatment group and decreased the lung inflammation score( Figure 3 ), indicating that MSC-sEV significantly alleviated the degree of lung inflammation in mice.

[0117] Furthermore, alveolar lavage fluid cells were collected, and flow cytometry was used to detect the levels of inflammatory cells. Lymphocytes were defined as CD45+, neutrophils as CD45+CD11b+Ly-6G+, and eosinophils as CD45+CD11b+Ly-6G-SiglecF+CD64-. Scatter plots were used to define the "lung cell gate" to exclude dead cells and debris; CD45 labeling was used to identify all white blood cells; among CD45+ cells, double positivity for CD11b and Ly-6G was used to define neutrophils; among the remaining CD45+ cells, non-neutrophils were defined based on Ly-6G negativity. Non-neutrophils were plotted as macrophages and eosinophils according to CD64 positivity and negativity( Figure 4 A). The results of the detection showed that the proportion of neutrophils in the alveolar lavage fluid of this mouse model was significantly increased( Figure 4 B), but there was no significant difference in the proportion of eosinophils compared with the blank control group( Figure 4 C), further demonstrating that this model was an inflammation model mainly characterized by neutrophil infiltration. The intervention treatment with MSC-sEV significantly reduced the level of airway neutrophils and did not significantly change the level of eosinophils( Figure 4 ), indicating that MSC-sEV could reduce the level of neutrophil infiltration in the lungs and alleviate allergic inflammation in the lungs of mice.

[0118] Th17 cells are key immune cells in the occurrence and development of neutrophil-infiltrating airway inflammation. Flow cytometry analysis showed that( Figure 5 A) the level of Th17 cells in the lungs of mice in the model group was significantly increased, and the level of Th17 cells was significantly decreased after treatment with MSC-sEV( Figure 5 B). IL-17A is the most important functional factor of Th17. By detecting the level of IL-17A in the supernatant of alveolar lavage fluid using enzyme-linked immunosorbent assay (ELISA), it was found that the intervention treatment with MSC-sEV decreased the level of IL-17A in the alveolar lavage fluid of mice( Figure 5 E). These results suggest that MSC-sEV alleviates allergic airway inflammation by inhibiting the level of Th17 cells.

[0119] MSC-sEV can inhibit the polarization of human Th17 cells

[0120] Previous studies have demonstrated that MSC-sEV can alleviate neutrophilic airway inflammation in mice and reduce the level of Th17 cells in the lungs. However, it remains unclear whether MSC-sEV has a direct immunomodulatory effect on Th17 cells. Therefore, CD4 + T cells were isolated and purified from human peripheral blood and co-cultured with MSC-sEV in the presence of Th17 cell differentiation induction medium. First, lymphocyte populations were gated physically, and non-single cells were excluded using FSC-A and FSC-H. Dead cells stained with the eFlour506 dye were excluded from the single-cell population, and human Th17 cells were defined as CD4 + IL-17A + ( Figure 6 A). Flow cytometry analysis revealed that after adding the Th17 cell differentiation induction medium, CD4 + T cells polarized towards Th17 cells and the number of Th17 cells increased. However, after co-culture with MSC-sEV, MSC-sEV significantly inhibited the differentiation of human Th17 cells, which was consistent with the results of the above animal experiments( Figure 6 A). ELISA detection of IL-17A in the co-culture supernatant also confirmed that MSC-sEV inhibited the secretion of IL-17A in the co-culture supernatant( Figure 6 B). Therefore, MSC-sEV can directly inhibit the polarization of human Th17 cells and the secretion of the functional factor IL-17A by Th17 cells.

[0121] To further explore the mechanism by which MSC-sEV inhibits Th17 cell polarization, CD4 + T cells that induce Th17 cell polarization were stained with CFSE, and the proliferation of Th17 cells was detected by flow cytometry. Cells labeled with the CFSE dye will have a weakened CFSE fluorescence intensity as the number of generations increases. Therefore, the rightmost peak represents primary cells, and each peak to the left represents the next generation and the next next generation of proliferation. From the experimental results, it was found that after adding the Th17 cell differentiation induction medium, the proliferation of Th17 cells was significant, but after co-culture with MSC-sEV, the proliferation of Th17 cells was significantly inhibited( Figure 7 A). At the same time, MSC-sEV did not promote the apoptosis or necrosis of Th17 cells( Figure 7 B). These results indicate that MSC-sEV inhibits the polarization of Th17 cells by inhibiting the proliferation in the Th17 cell direction, rather than through a cytotoxic effect that promotes cell apoptosis.

[0122] Example 3: Transcriptome sequencing analysis of the mechanism of MSC-sEV acting on Th17 cells

[0123] Experimental method

[0124] This part of the experiment was commissioned by BGI Tech Solutions Co., Ltd. on a paid basis. After co-culturing human Th17 cells and MSC-sEV for 4 days, mRNA in human Th17 cells was extracted, and its concentration and purity were detected using NanoDrop and Agilent 2100 Bioanalyzer. Oligonucleotide (dT)-linked magnetic beads were used to ligate the fragmented mRNA and convert it into single-stranded circular DNA as the mRNA library. The sequencing data were filtered using SOAPnuke (v1.5.2) to obtain clean reads. HISAT2 (v2.0.4) was used to map the clean reads to the reference genome. Bowtie2 (v2.2.5) was applied to align the clean reads with the reference coding gene set. RSEM (v1.2.12) was used to calculate the gene expression levels. Pheatmap (v1.0.8) was applied to generate the heatmap. DESeq2 (v1.4.5) was used to analyze differential gene expression, with Q < 0.05 considered statistically significant. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software. DAVID (https: / / david.ncifcrf.gov / tools.jsp) was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis.

[0125] Results

[0126] Compared with the original Th17 cells, 27 upregulated genes and 32 downregulated genes were detected in Th17 cells after co-culture with MSC-sEV ( Figure 8 A), and the downregulated genes included ONECUT2, KIAA1217, TNFRSF19, IL22, and KCNA2, etc. ( Figure 8 B). IL-22 is one of the most important functional factors of Th17 cells. Among these downregulated genes, the significant decrease in the expression of IL22 mRNA in Th17 cells co-cultured with MSC-sEV was verified by real-time quantitative PCR, which was consistent with the transcriptome sequencing results, indicating the reliability of the mRNA sequencing results and also indicating that MSC-sEV can inhibit the function of Th17 cells. ( Figure 8 C).

[0127] Pathway enrichment analysis was performed on the differential genes obtained from the transcriptome sequencing of the original Th17 cells and Th17 cells after co-culture with MSC-sEV, and it was found that the differential genes changed in Th17 cells after MSC-sEV treatment were enriched in pathways closely related to the functions of Th17 cells, such as inflammatory response, immune response, lymphocyte chemotaxis, and chemokine-mediated signaling pathways.Figure 9 A). GSEA is a statistical method for analyzing the enrichment degree of gene sets, which is usually used to study biological pathways and disease states. Through GSEA analysis, it was found that the downregulated genes in Th17 cells co-cultured with MSC-sEV were mainly enriched in the JAK-STAT signaling pathway, the T cell receptor signaling pathway, and other pathways related to the function of Th17 cells ( Figure 9 B). The JAK-STAT signaling pathway has been proven to play a key role in Th17 cell polarization. Therefore, MSC-sEV may inhibit Th17 cell polarization by downregulating the JAK-STAT pathway. Figure 9 C).

[0128] MSC-sEV inhibits Th17 cell polarization by suppressing JAK-STAT pathway activation

[0129] It was speculated by transcriptome sequencing that MSC-sEV may act on Th17 cells by inhibiting the JAK-STAT pathway, and the JAK-STAT3 pathway is the most important pathway for Th17 cell differentiation. Therefore, attention was paid to the effect of MSC-sEV on the JAK-STAT3 pathway of Th17 cells. The phosphorylation level of STAT3 in the lung tissue of airway inflammatory mice with neutrophil infiltration was detected by Western Blot, and it was found that the level of phosphorylated-STAT3 (p-STAT3) in the lungs of model mice was significantly higher than that of the blank control group. After MSC-sEV treatment, the level of p-STAT3 in the lungs of model mice decreased significantly. Figure 10 A). The phosphorylation levels of STAT3 and its upstream molecule JAK2 in human Th17 cells treated with MSC-sEV were also detected, and it was found that the levels of p-STAT3 and p-JAK2 in Th17 cells increased significantly, and the levels of p-STAT3 and p-JAK2 decreased significantly after MSC-sEV treatment. Figure 10 B).

[0130] In addition, in order to further verify the mechanism of action of MSC-sEV, the STAT3 phosphorylation activator colivelin was added while MSC-sEV was co-cultured with Th17 cells. The Th17 cell level was detected by flow cytometry and the level of IL-17A in the culture supernatant was detected by ELISA. It was found that colivelin could reverse the inhibitory effect of MSC-sEV on Th17 cells, restore the Th17 cell level, and also had a certain effect on reversing the level of IL-17A in the supernatant, but there was no statistical difference. Figure 10 C). These results suggest that MSC-sEV inhibits Th17 cell polarization and alleviates lung inflammation in mice by inhibiting the JAK2-STAT3 pathway.

[0131] Transcriptome sequencing analysis found that the downregulated genes in Th17 cells treated with MSC-sEV were enriched in the JAK-STAT signaling pathway. By detecting the lung tissues of airway inflammation mice with neutrophil infiltration and human Th17 cells by Western Blot, it was found that the phosphorylation level of STAT3 was significantly decreased after MSC-sEV treatment, and the phosphorylation level of JAK2, the upstream molecule of STAT3, was also decreased. When the STAT3 phosphorylation activator colivelin was added during the co-culture of MSC-sEV and Th17 cells, it was found that colivelin could reverse the inhibitory effect of MSC-sEV on Th17 cells. These results indicate that MSC-sEV is directly taken up by CD4 + T cells, inhibits the activation of the JAK2-STAT3 pathway, and thus inhibits the polarization of Th17 cells and alleviates pulmonary inflammation in mice.

[0132] Example 4 Regulation of Th17 cell function by MSC-sEV protein components

[0133] I. Experimental methods

[0134] 1 Preparation of RNA-depleted MSC-sEV

[0135] (1) Prepare MSC-sEV prepared by anion exchange chromatography under sterile conditions;

[0136] (2) Add 0.1% Triton X-100 and incubate at 37 °C for 30 minutes;

[0137] (3) Add 200 μg / mL RNaseA without DNase and protease and incubate at 37 °C for 30 minutes;

[0138] (4) Add 4 volumes of PBS and ultrafilter with a 2650g 4 °C ultrafiltration tube for 8 minutes;

[0139] (5) Repeat step (4) twice to re-extract RNA-depleted MSC-sEV.

[0140] 2 Detection of MSC-sEV RNA content

[0141] Prepare 200 μL each of MSC-sEV and RNA-depleted MSC-sEV at a concentration of 450 μg / mL, extract RNA using the method in Section 3.3.8 of Part III, resuspend with 5 μL of ultra-pure enzyme-free water, and detect the RNA concentration with NanoDrop.

[0142] 3 Co-culture of RNA-depleted MSC-sEV with human Th17 cells

[0143] For the induction of human Th17 cells, refer to Section 2.3.17 in Part II. During the induction and differentiation process, 30 μg / mL of RNA-depleted MSC-sEV was added. After culturing for 4 days, the cells were harvested for flow cytometry analysis, and the level of IL-17A in the culture supernatant was detected by ELISA.

[0144] 4 Proteomics analysis

[0145] The proteomics data was sourced from previously measured proteomics data using nano liquid chromatography-tandem mass spectrometry. The extracellular vesicle protein database was derived from the Vesiclepedia database. DAVID ( https: / / david.ncifcrf.gov / tools.jsp ) was used for KEGG and GO enrichment analysis of the proteins.

[0146] 5 Statistical methods

[0147] All data analysis in this study was performed using GraphPad 8.3.0. The results of each group of experimental data were expressed as Mean±SD. Paired one-way ANOVA was used for analysis among the three groups in in vitro cell experiments. When P<0.05, the results were considered statistically significant.

[0148] II: Experimental results

[0149] 1. MSC-sEV can inhibit Th17 cell polarization through proteins

[0150] Proteins and miRNAs are the two major functional components of MSC-sEV. To determine the functional components of MSC-sEV that inhibit Th17 cells, MSC-sEV was perforated with 0.1% Triton X-100, and RNase A was added to remove all RNA from MSC-sEV. RNA-depleted MSC-sEV was prepared in this way. After extracting RNA with Trizol, the RNA content was detected using NanoDrop to confirm that this MSC-sEV contained no RNA components ( Figure 11 A), still expressed exosome markers CD9, CD63, Alix, TSG101, and did not express the cell marker Alix ( Figure 11 B), indicating that this batch of RNA-depleted MSC-sEV was qualified.

[0151] When this RNA-depleted MSC-sEV was co-cultured with Th17 cells, it was found that the RNA-depleted MSC-sEV could still inhibit Th17 cell polarization ( Figure 12 A), inhibit the secretion of IL-17A in the co-culture supernatant ( Figure 12 B), which was consistent with the effect of MSC-sEV, indicating that the proteins of MSC-sEV had the function of inhibiting Th17 cell differentiation and were the functional components of MSC-sEV.

[0152] 2. Potential functional proteins of MSC-sEV

[0153] The Vesiclepedia database is an extracellular vesicle database that contains protein, RNA, and lipid data of vesicles derived from immune cells, tumor cells, red blood cells, etc. published in previous literature. By reanalyzing the proteomic data measured in the previous research results of the research group, among the 2,655 proteins detected in MSC-sEV, 2,073 proteins can be found in the latest Vesiclepedia protein database. These proteins are mostly structural proteins or internal reference proteins of MSC-sEV, etc., and 582 are unique to MSC-sEV. Therefore, it is speculated that these 582 proteins may be the functional proteins of MSC-sEV( Figure 13 A). KEGG analysis showed that the functions of these 582 MSC-sEV-specific proteins were enriched in pathways such as the T cell receptor pathway and Th17 cell differentiation( Figure 13 B). In the third part of the transcriptome sequencing, the genes downregulated in Th17 cells after treatment with MSC-sEV were enriched in 57 pathways, and 17 of these pathways were also the pathways enriched with specific proteins in MSC-sEV( Figure 13 C), such as the T cell receptor signaling pathway, FcεRI signaling pathway, cell cycle, etc. Therefore, it is speculated that the proteins enriched in these 17 pathways in MSC-sEV may be the functional proteins that inhibit Th17 cell polarization.

[0154] There are 102 proteins in MSC-sEV in these 17 pathways (Appendix). To better study the functions of these 102 enriched proteins, DAVID( https: / / david.ncifcrf.gov / summary.jsp ) was used for GO enrichment analysis. First, in terms of cellular components, these proteins are closely related to the cytoplasm, extracellular exosomes, cell adhesion, etc., indicating that these proteins are specific proteins of exosomes( Figure 13 D); in terms of molecular functions, these protein molecules mainly have aspects such as nitric oxide synthase regulation, binding proteins, chromatin, etc.( Figure 13 E), to some extent indicating that these proteins can enter cells and bind to intracellular proteins to play a role; in terms of biological processes, these proteins are mainly involved in aspects such as positive regulation of protein kinases, signal transduction, mucosal innate immune responses, etc.( Figure 13 F). In summary, the proteins of MSC-sEV have a wide range of biological functions, and MSC-sEV has the effects of inhibiting Th17 cell polarization and treating airway inflammation mainly characterized by neutrophil infiltration.

[0155] After obtaining RNA-depleted MSC-sEV using Triton-100 and RNase A, it was found that the RNA-depleted MSC-sEV still had the function of inhibiting Th17 cell differentiation, indicating that proteins are important components for MSC-sEV to exert its functions. Through the proteomic data measured by combined nano-liquid chromatography-tandem mass spectrometry and the Vesiclepedia protein database, 582 proteins unique to MSC-sEV were identified. There were 17 pathways in the gene enrichment pathways downregulated in Th17 cells treated with MSC-sEV that were also the enrichment pathways of the unique proteins in MSC-sEV. Therefore, the functional proteins of MSC-sEV may be the proteins that stimulate the gene downregulation pathways in Th17 cells, and these proteins have a wide range of biological functions. MSC-sEV can inhibit the JAK-STAT pathway through proteins and inhibit Th17 cell polarization, thereby treating neutrophil infiltration-dominated and allergen-mediated airway inhibition.

[0156] In this study, through in vitro experiments with human cells and in vivo experiments with mice, it was discovered and proven that MSC-sEV can directly be taken up by cells, inhibit the activation of the intracellular JAK2-STAT3 pathway, inhibit Th17 cell polarization, and significantly alleviate neutrophilic airway inflammation. The proteins of MSC-sEV play an important role in inhibiting Th17 cell polarization.

[0157] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0158] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. Application of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) in the preparation of products for preventing and / or treating airway inflammation mainly characterized by neutrophil infiltration.

2. The application according to claim 1, characterized in that, The airway inflammation is airway inflammation mainly characterized by neutrophil infiltration with infection combined with allergy.

3. The application according to claim 1, characterized in that, The airway inflammation is Th17-dominated neutrophilic airway inflammation.

4. The application according to claim 1, characterized in that, The application includes that mesenchymal stem cell-derived small extracellular vesicles inhibit the polarization of human Th17 cells and inhibit the proliferation of Th17 cells.

5. The application according to claim 4, characterized in that, The application includes that mesenchymal stem cell-derived small extracellular vesicles inhibit the activation of the JAK2-STAT3 pathway, thereby inhibiting the polarization of Th17 cells.

6. The application according to claim 1, characterized in that, The preparation method of the mesenchymal stem cell-derived small extracellular vesicles includes the following: Collect the cell culture supernatant after the mesenchymal stem cells are passaged and amplified; Prepare an eluate containing mesenchymal stem cell-derived small extracellular vesicles by anion exchange chromatography or tangential flow method, and obtain the supernatant liquid by centrifugal ultrafiltration to obtain MSC-sEV.

7. The application according to claim 1, characterized in that, The mesenchymal stem cells are derived from induced pluripotent stem cells.

8. The application according to claim 1, characterized in that, The mesenchymal stem cells are derived from umbilical cord, bone marrow, adipose tissue, urine, and menstrual blood.

9. The application according to any one of claims 1-8, characterized in that, The product is a drug or a biological agent.

10. The application according to any one of claims 1-8, characterized in that, The drug or biological agent is one of an injection, an aerosol inhalant, an oral dosage form, and a topical dosage form.

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