Study On The Role And Activation Mechanisms Of ILC2s In RSV-induced Airway Inflammation

Objective: Asthma is a respiratory disease.The main characteristic of asthma is airway hyperresponsiveness and airway inflammation.There are about 23500 to 334 million asthmatic patients world wide,and about 250 thousand people die from asthma each year.In China,the total incidence of asthma is about 1.24%,and in recent years it has a significant upward trend.The innate immune cells,including eosinophils and mast cells,as well as adaptive immune cells,such as Th2 cells,are involved in the development of asthma.These immune cells and their secreted cytokines play an important role in inducing asthma formation and aggravating respiratory inflammation.The study found that abnormal expression of inflammatory mediators and cytokines in patients with asthma,such as thymic stromal lymphopoietin(TSLP thymic,stromal lymphopoietin)and interleukin-33(IL-33,interleukin-33),IL-31,IL-4 etc.it is possible to Ig E level in serum and lung tissue of eosinophil infiltration in airway inflammation and airway hyperresponsiveness.There are reports of thymic stromal lymphopoietin can activate dendritic cells and activated Th2 immune response,especially in the early stage of asthma inflammation and inflammation play a role;interleukin-33 activation and recruitment of macrophages,induced eosinophil maturation and survival,and promote mast cells to secrete high levels of IL-13 and Th2;cytokine IL-4 known as the classic is mainly induced by cytokines produced by Ig E;in recent years,the study confirmed that IL-31 is also an important factor in asthma.But so far,the expression of cytokines including TSLP,IL-33,IL-31 and other cytokines in patients with asthma,rhinitis and asthma and rhinitis is not enough.How are the expressions of IL-4,IL-31,IL-33,and TSLP associated with asthma,rhinitis,and asthma combined with rhinitis? Is there a difference in the levels of IL-4,IL-31,IL-33,and TSLP in the serum of allergic and non allergic patients? These problems should be studied and discussed in depth.Among the many inducements that induce asthma formation and attack,virus infection,especially respiratory syncytial virus(RSV)infection is particularly important.Although studies show that RSV infection could induce Th2 cells to secrete more Th2 type cytokines and induce or aggravate asthma,but the conventional drug treatment of asthma in the treatment of poor,suggesting that in asthma induced by RSV infection in the occurrence and development of some pathogenic mechanism or pathway different from Th2 cells.Group 2 innate lymphiod cells(ILC2s)is a recently discovered non T non B type 2 natural lymphocyte.Its main biological function is to participate in the development of allergic diseases by secreting Th2 cytokines.It has been found that influenza virus infection can lead to a significant increase in airway responsiveness in experimental rats.The increased airway responsiveness is independent of adaptive response,but depends on natural helper cells and their resulting cytokines,especially IL-13.It is found that the natural auxiliary cells in the lung tissue are important sources of IL-5 and IL-13 in the ovalbumin induced allergic asthma model,suggesting the key role of natural helper cells in the pathogenesis of asthma.However,it is not clear whether ILC2s are involved in acute asthma induced by respiratory syncytial virus(RSV),and what is its biological activity and mechanism of activation.Studies have shown that IL-33 plays a key role in inducing the activation of ILC2s.Then,during RSV-induced airway inflammation,whether ILC2s are activated by the stimulation of IL-33,and then secrete the cytokines associated with asthma is not clear.On the other hand,whether the levels of IL-4,IL-31,IL-33 and TSLP are related to the pathogenesis of asthma or rhinitis,or which allergies are more likely to induce the expression of these cytokines,have not yet been reported.In the present study,the functional role of lung ILC2s in RSV-induced airway inflammation as well as the underlying molecular mechanisms relative to IL-33 was investigated.Furthermore,the role of IL-4,IL-31,IL-33 and TSLP in the pathogenesis of asthma and rhinitis in the patients with asthma,rhinitis and patients with both asthma and rhinitis were investugated.Methods: 1.Patients.All patients were treated in the Department of allergy in the General Hospital of Shenyang Military Command from October 2015 to May 2016.Of 64 patients with asthma,32 patients(s Ig E[+])and 32 patients(s Ig E[-]).In 64 patients with rhinitis,32 patients(s Ig E[+]),and 32 patients(s Ig E[-]).Of 64 patients with asthma combined with rhinitis,32 patients(s Ig E[+])and 32 patients(s Ig E[-]).Patients excluded include: autoimmune disease,tumor or other serious systemic diseases,other allergic diseases,and allergic family history.In addition,the patients need not to take antihistamine before examination,or do not use glucocorticoid within 1 months.In the control group,32 cases of the normal control group were all the healthy subjects in the physical health center of the physical examination in our hospital.The control group had no systemic disease,endocrine system and heart,lung,liver,kidney and other diseases.In the control group,there was no local or systemic infection in the near future,and no allergic dermatitis,such as allergic dermatitis.Our research agreement has been approved by the medical ethics and the human clinical trial committee.2.Specimen collection.All patients collected venous blood 5 m L in the early morning,2000 rpm centrifugation 10 min at room temperature,after centrifugation,the upper serum was transferred to Ep tube(Eppendorf tubes),then stored at-20℃ refrigerator.3.ELISA: using IL-31,IL-4,IL-33 and TSLP detection reagents,IL-4,IL-31,IL-33 and TSLP concentrations in serum were measured by ELISA.The operation method is in strict conformity with the standard requirements.The German European Union testing system is used.The measuring unit is pg/ml.4.Real-time RT-PCR.The total ribonucleic acid(RNA)was extracted by the Trizol method.The reverse transcriptional kit was used to reverse the 1μg total RNA to complementary deoxyribonucleic acid(c DNA).Complete the real-time PCR experiment according to the instructions.All the experiments were repeated 3 times,using the housekeeping protein actin(β-actin)as reference.5.Western blot.Extract protein from 1 × 106 cells by using lysis solution.After protein quantification,take 60 μg proteins and carry out sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).After electrophoresis,after transferring to the membrane,the skim milk powder diluted with Tris Physiological Buffered Saline plus Twain 20(TBST)was closed up with antibody and then electrochemiluminescence(ECL).6.Animal model.The C57BL/6 mice were randomly divided into control group,dust mite group and mugwort group.Dust mite group and mugwort group respectively use dust mites and mugwort allergen,in experiment 0,7 and 14 days of mice were intraperitoneally injected with phosphate as Physiological Buffered Saline group.The twenty-eighth day,dust mite group and mugwort group mice for 7 days to accept the dust mite and mugwort extract stimulation,while the control group was given phosphate buffered saline stimulation.The symptoms of asthma in each group were observed and recorded.Finally,the serum and lung tissue were taken for experiment.7.RSV infection model.Mice were infected intranasally with RSV at a dose of 2 × 106 TCID50 per mouse.For blocking the effect of IL-33,some mice were treated intraperitoneally with 200 μg of neutralizing anti-IL-33 or isotype-matched control antibody within 2 h before RSV infection.8.Airway inflammation.Lungs were washed with 1 ml of phosphate-buffered saline(PBS)and bronchoalveolar lavage fluids were collected at the intervals.After centrifugation,the pellet was cytospun on to glass slides and stained with Wright-Giemsa.Two hundred cells were counted on each slide and the proportions of different leukocyte subtypes were counted.9.Preparation of lung single cell suspension.Lungs were minced and incubated at 37?C for 60 min on a rocker with 200 μg/ml collagenase D and 40 μg/ml DNase I.The enzyme-digested lung tissue was passed through a stainless steel mesh.Red blood cells in lung cells were lysed using osmotic lysis buffer.After centrifugation,single cell suspensions were resuspended in RPMI 1640 medium.10.Isolation and identification of ILC2s in lung.After blocking CD16/32,the lung cells were incubated with the following specific antibodies: CD45,ST2,mixed antibody: CD3、CD4、CD5、CD8、CD11b、GR-1、CD19、B220、DX5(or NK1.1)and(TCRδ).A total of 500,000 stained lung cells were analyzed by flow cytometry and the pulmonary ILC2s were identified as CD45+Lin-ST2+ cells.11.Adoptive transfer of pulmonary ILC2s.To gain enough number of NH cells for adoptive transfer experiments,lung cells from ten mice,which were infected with RSV on day 3,were pooled and purified by using a Mo Flo high-speed cell sorter.Then,2 ×104 of the purified NH cells(purity was >90%)in 200 μl of PBS were injected via the tail vein into mouse 2 h before RSV infection.12.Determination of cytokine.The levels of IL-4,IL-5,IL-10 and IFN-γ in alveolar lavage fluids were measured by using the mouse cytokine ELISA detection kit.13.Determination of the expression of cytokine gene.The gene expression levels of IL-4,IL-5,IL-10 and IFN-γ in the alveolar lavage fluid was measured by real-time PCR.14.Statistical analysis.Results are expressed as mean ± SE.Statistical analyses were performed using Prism 6.One-way ANOVA as well as χ 2 analysis was used.p-values < 0.05 were considered statistically significant.Results: 1.The levels of IL-4,IL-31,IL-33 and TSLP in the serum of patients with asthma,rhinitis,asthma and rhinitis were significantly higher than those of the healthy individuals.The levels of IL-4,IL-31,IL-33 and TSLP in the serum of patients with asthma,rhinitis,asthma and rhinitis were significantly higher than those of normal people.However,the levels of IL-4,IL-31,IL-33 and TSLP in the serum of patients with s Ig E[+] and s Ig E[-] were almost no different.This means that IL-4,IL-31,IL-33 and TSLP can be used to determine whether a patient is suffering from asthma or rhinitis,but is not sure whether the patient has an allergy.2.Mite and mugwort may more likely to arise IL-4,IL-31,IL-33 & TSLP in patients with asthma and rhinitis.By analysing the cytokine levels in the serum of patients with asthma and rhinitis,we found that dust mites and mugwort can promote more production of IL-4,IL-31,IL-33 and TSLP in the serum of patients with asthma and rhinitis.3.In vivo experiments confirmed that dust mites and mugwort can result in elevated cytokines.To study whether mites and mugwort are responsible for allergy in patients,a mice model was constructed.Results showed that IL-4,IL-31,IL-33 and TSLP significantly increased in the serum of allergic mice.4.ILC2s are a source of type 2 cytokines during RSV infection.Rreal-time RT-PCR was used to detect the changes in the expression levels of Th1/Th2-type cytokines in the lung tissue.The results showed that the expression level of IL-4,IL-5 and IL-10 in ILC2s cells increased significantly after RSV infection,especially in the early stage of infection.However,in the course of viral infection,did not find the upregulated expression of IFN-γ in ILC2s,suggesting that infection with RSV may predispose ILC2s to elicit Th2-dominate immune responses via a dominate production of Th2-type cytokines.5.ILC2s act as a promoter for RSV-induced airway inflammation.Adoptive retransfusion experiments showed that ILC2s could significantly aggravate local inflammatory response in RSV infected mice.The number of eosinophils in the lung tissue was significantly increased.In addition,adoptive transfer of pulmonary ILC2s augmented the amounts of typical Th2-type cytokines in BAL fluids,in parallel with an increased expression of m RNAs for IL-4,IL-5 and IL-10 in the lungs of transferred mice.In contrast,the amounts of IFN-γ in BAL fluids of all tested were undetectable.6.ILC2s are responsive to an in vivo source of IL-33.In order to verify whether lung ILC2s produce type 2 cytokines in an IL-33-dependent manner,we neutralized the IL-33 in vivo by Mc Ab.We found that blockade of IL-33 diminished not only the number of lung ILC2s,but also the expression of m RNAs for type 2 cytokines,particularly IL-5 and IL-13 m RNA in ILC2s,suggesting that IL-33 is necessary,either directly or indirectly,to induce Th2-type ILC2s.Conclusion: 1.The levels of IL-4,IL-31,IL-33 and TSLP in the srum of patients with asthma and rhinitis were significantly higher than those in healthy individuals.Mite and mugwort may more likely to arise type 2 cytokin production in patients with asthma and rhinitis.2.Pulmonary ILC2s are an important cellular source of type 2 cytokines during RSV infection.Th2-inducing cytokine IL-33 is a critical factor for producing type 2 cytokines by ILC2s.