Effect Of IL-17A On Airway Eosinophilic Inflammation In Asthma And Its Immunomodulatory Mechanism

BackgroundBronchial asthma(Hereinafter referred to as "asthma")is a highly heterogeneous chronic airway inflammatory disease.Asthma usually has reversible airflow limitation,airway hyperresponsiveness and chronic airway inflammation and other clinical features.According to the different clinical features,asthma can be divided into different clinical phenotypes,such as allergic and non-allergic asthma and so on.Different clinical phenotypes of asthma have different pathogenesis,that is,existing different endotypes.Different endotypes may explain the pathophysiological processes of different asthma "phenotypes".At present,mostly mentioned asthma type,such as“Th2 high”asthma,showed exceedingly Th2 immune response,with enhanced activation of Th2 cells and production of associated cytokines IL-4,IL-5,while the airway was mainly infiltrated with eosinophils.Allergic asthma is a common“Th2 high”type of asthma,usually induced by inhaled allergens,with a significant Th2 immune response,accompanied by eosinophils,mast cells and lymphocytes aggregation,airway smooth muscle hyperplasia,as well as elevated serum IgE.IL-17 A is a cytokine secreted by Th17 cells.Th17 cells are distinctly different from Th1 and Th2 cells,and their differentiation is negatively regulated by IFN-? and IL-4.IL-17 A is involved in resistance to pathogens or inducing rheumatism diseases by causing aggregation and activation of neutrophils in the body.IL-17 A is a very important cytokine involved in neutrophilic inflammation.In recent years,we had found that the concentration of IL-17 A in the blood and lungs of asthmatic patients is increased and is associated with the severity of asthma.IL-17 A may be involved in the progression of chronic airway inflammation in asthma,especially in “Neutrophilic asthma ”.A number of studies at home and abroad found that IL-17 A has a proinflammatory effect and a certain anti-inflammatory effect,that is,IL-17 A may have dual effects,and the effect of inhibition of inflammation may be caused by low concentrations of IL-17 A.Our previous study found that OVA-induced asthma model accompanied by giving LPS into the airway established with IL-17 A knockout mice has increased airway eosinophilic inflammation and splenic Th2 cell differentiation than wild mice.Therefore,we hypothesized that IL-17 A can reduce airway eosinophil inflammation in asthmatic mice by directly inhibiting Th2 cell differentiation.In this study,mice were sensitized and challenged with OVA to establish a asthmatic model with increased airway eosinophil inflammation,and IL-17 A was infused into the airway to observe the effect of IL-17 A on airway eosinophilic inflammation and Th2 type immunization in asthmatic mice and explore its mechanism.Research purposesTo investigate the role of IL-17 A in pathogenesis related changes of airway inflammation in asthmatic mice,including the characteristics of airway inflammatory cell classification,the infiltration of peribronchial and perivascular inflammatory cells,and the changes of inflammatory cytokines in bronchoalveolar lavage fluid.To investigate the role of IL-17 A in bronchial lymph nodes and splenic Th2 cell differentiation in asthmatic mice and the effect on the differentiation of naive CD4 + T cells into Th2 cells in vitro.Research methodChapter I Effects of IL-17 A on pathology of airway inflammation1.Establishment of OVA-induced Asthma Mouse Model and Airway InterventionMice were divided into three groups: control group,asthma model group,IL-17 A treatment group.On day 1 and 7,OVA was injected intraperitoneally into the mice of model group and the IL-17 A group,and were challenged with 1% OVA by aerosol inhalation for 1 h from day 14 to 18,and mice in the control group were sensitized and challenged with the same amount of normal saline.For the mice in IL-17 A treatment group,100 ng of IL-17 A was dropped into the airway of mice 1 hour before each challenge,and mice in the control group and model group were given the same amount of saline into the airway.2.The acquisition and detection of BALF and lung tissue samples in miceThe total number of cells in BALF solution was counted.After centrifugation,the concentration of IL-4?IL-5?INF-? and IL-17 A in the supernate were detected by ELISA,and the cell pellet obtained by centrifugation were smeared and stained with Wright-Giemsa dye for cytology.The left lung tissue was fixed and stained with HE and PAS and graded semi-quantitatively to value the pathological change.Chapter II Effect of IL-17 A on the differentiation of Th2 cells in asthma1.Flow cytometry was used to detect Th cell differentiationSingle cell suspension of bronchial lymph nodes and spleen were prepared,and cells were stimulated and cultured.After stained,cells were detected by FCM for Th1/2/17 differentiation ratio.2.Effect of IL-17 A on the differentiation of immature CD4 + T cells into Th2 cells in vitroIsolate spleen and prepare single cell suspension.Naive CD4+T cells were sorted by MACS,and cell number was counted,then cultured with a completed medium containing polarizing factors(with 30 ng IL-17 A or not).After 48 h,the cells were washed and cultured with completed medium containing IL-2 to 96 h.After cultured for 24 hours and 96 hours,the cells were detected by FCM for Th differentiation.The cells cultured for 24 hours were detected by Annexin V / PI for the degree of apoptosis.The CCK8 reagent was used for measuring the cell proliferative activity at 24 h under the same culture condition.Research ResultsChapter I Effects of IL-17 A on pathology of airway inflammationComparison of the total number of cells and differential count in BALFTotal number of cells were significantly higher in the model group than the control group(P <0.01)and IL-17 A treatment group(P <0.05);The number of eosinophils(P <0.05)and its ratio(P < 0.01)in IL-17 A treatment group were significantly higher than the control group but lower than the model group.The number and ratio of neutrophils and lymphocytes in the model group did not show significantly different from IL-17 A treatment group(P > 0.05).Comparison of peribronchial and perivascular inflammatory changes in miceIn the control group,no infiltration of inflammatory cells was observed in the lungs;Compared with the control group,the model group mice had more inflammatory cells infiltration around the bronchus and the blood vessel,and higher semi-quantitative score(P <0.01).And IL-17 A treatment group had lower peribronchial and perivascular infiltration than the model group(P <0.05),but the score was still significant higher than the control group(P <0.01).3.Comparison of metaplasia degree and semi-quantitative score of airway goblet cells in miceThere was no obvious goblet cell metaplasia in the airway of the control group;the number of PAS-positive cells in the airway of the model group was significantly higher than that in the control group(P <0.01);Compared with the model group,the degree of metaplasia of goblet cells in the airway of IL-17 A treatment group was significantly decreased(P <0.01).4.Comparison of Th cells-related cytokines in the BALF.Compared with the control group,the concentration of Th1-related factor IFN-? in the BALF of model group was significantly lower(P <0.01),while the levels of IL-4 and IL-5 were significantly higher(P <0.01),and IL-17 A concentration was also higher(P <0.01);Compared with the model group,the levels of IL-4 and IL-5 in BALF were significantly decreased(P <0.01),while the concentration of IL-17 A was significantly increased in IL-17 A treatment group(P <0.01),but IFN-? concentration was not significantly different(P> 0.05);The concentration of IL-4(P<0.01)and IL-5(P<0.05)are much higher in the IL-17 A treatment group than in the control group.Chapter II Effect of IL-17 A on the differentiation of Th2 cells in asthma1.Comparison of Th cell differentiation in bronchial lymph nodesCompared with the control group,the ratio of Th1 cells in the lymph nodes of the model group was lower(P <0.01),the ratio of Th2 cells was higher(P <0.05),but the ratio of Th17 cells did not change significantly;Compared with the model group,IL-17 A treatment group had lower lymph node Th2 cell ratio(P <0.05),while Th1 and Th17 cells had no significant difference;The ratio of Th2 cells in IL-17 A treatment group was still higher(P <0.05),and Th1 cell ratio was lower(P <0.01)than the control group.2.Comparison of differentiation of splenic Th cells in miceCompared with the control group,the ratio of splenic Th1 cells was significantly lower(P <0.01),while the ratio of Th2 cells was significantly higher(P <0.01)in the model group,but the ratio of Th17 cells was almost the same;Compared with the model group,after giving IL-17 A treatment,the percentage of Th2 cells in the spleen of the mice was significantly decreased(P <0.01),but the ratio of Th1 cells did not change(P> 0.05);Compared with the control group,the ratio of Th1 cells in the spleen of the IL-17 A treatment group was still lower(P <0.01),but the ratio of Th17 cells was not significantly different.3.Effect of IL-17 A on the differentiation of immature CD4 + T cells into Th2 cells in vitroThe differentiation ratio of Th2 cells in Th2+IL-17 A polarized group seemed to decrease at 24 h(P> 0.05),and IL-17 A significantly inhibited Th2 cell differentiation(P < 0.01)at 96 h.There was no significant difference in apoptosis and proliferation between Th2 polarized group and Th2 + IL-17 A polarized group(P> 0.05)at 24 h.Research conclusions1.OVA-induced asthmatic model had significant airway eosinophilic inflammation and increased levels of Th2-related cytokines IL-4 and IL-5 in the BALF.Airway infusion of IL-17 A can reduce the concentration of Th2-related factors IL-4 and IL-5 and eosinophils in the BALF of asthmatic mice.2.The ratio of Th2 cells in bronchial lymph nodes and spleen of asthmatic mice was significantly increased,and the ratio of Th2 cells in spleen and lymph nodes was decreased after instillation of IL-17 A into the airway,but no significant effect on Th1 cells was observed.IL-17 A can inhibit the differentiation of naive CD4 + T cells into Th2 cells in vitro,but has no significant effect on proliferation and apoptosis.Research SignificanceThe opinion of "hygiene hypothesis" in asthma mentioned that,exposure to bacterial endotoxin in childhood can stimulate Th1 cell differentiation to inhibit the differentiation of Th2,ameliorating the balance of Th2/Th1,thereby reducing the incidence of asthma.This study confirmed that,giving IL-17 A through the airway,can also inhibit the differentiation of Th2,ameliorate airway eosinophilic inflammation.Because bacterial endotoxin is an important reason to promote Th17 differentiation of the body.Therefore,the results of this article enriched the understanding of the immune regulation mechanism of "hygiene hypothesis".Our previous researches have shown that too strong endotoxin stimulation can lead to excessive Th17 cell response,converting asthma airway eosinophilic inflammation into neutrophilic inflammation,leading to the difficulties of asthma treatment.While relatively lower doses of IL-17 A were used in our research and did not cause significant neutrophilic inflammation.