The Role Of Regulatory T Cell In Non-typeable Haemophilus Influenza Induced Acute Exacerbation Of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease(COPD)is a chronic inflammatory disease characterized by incompletely reversible airflow obstruction and progressive inflammation.Currently,COPD becomes the third leading cause of death in the world and its mechanism is still unknown.Studies show that cigarette smoke(CS)is the major cause of COPD.Some believe that due to the infiltration and accumulation of inflammatory cells caused by harmful particles from CS,lungs might generate abnormal immune responses.CS could activate immune responses of bronchial epithelial cells and reduce cilia movement,which influences the repairing of mucus consequently.Additionally,sustained activation of alveolar macrophages by CS could further strengthen the airway inflammatory response.Acute exacerbations of chronic obstructive pulmonary disease(AECOPD)is the main cause of high morbidity and mortality rates of COPD.Researches show that Non-typeable Haemophilus influenza(NTHi)is the most important cause of AECOPD,but the mechanism has not been completely elucidated.By the research on NTHi and regulatory T cells(Tregs),it is confirmed that the induction of AECOPD by NTHi infection was closely related to adaptive immune response mediated by Tregs,however the mechanism needs further investigation.Hence,focusing on Tregs,illuminating the immunological mechanisms of the induction of AECOPD by NTHi infection becomes meaningful for explaining the pathogenesis of COPD andAECOPD.In the present study COPD model was built by passive smoking on BALB/c mice with whole body exposure.Animals were divided into the following groups:normal control group(Normal feeding for 17 weeks,NC group),smoking group(Smoking for 17 weeks,CS group),NTHi group(After normal feeding for 16 weeks,tracheally challenged with NTHi for 1 week,NTHi group)and NTHi-CS group(After smoking for 16 weeks,tracheally challenged with NTHi for 1 week,NTHi+CS).After model constructing,tests on mice lung function was performed by a spirometer(Buxco,PFT Controller).HE staining was used on lung tissue after immobilization.Peripheral blood serum was collected in mice to measure the levels of TGF-?1 and IL-17 in serum via enzyme-linked immune sorbent assay(ELISA).Cell suspension of spleen was obtained for flow cytometry(FCM)to test the changes of Tregs in spleen.Immunohistochemistry was applied to measure the protien expression of FOXP3?ROR?t and IL-17.The m RNA expression of FOXP3 and ROR?t in lung tissue was assessed by real-time PCRThe results of lung function test indicated that the COPD murine model by whole body exposure to CS was feasible.Compared with NC group,the levels of Forced Expiratory Volume in 100ms(FEV0.1)decreased significantly in experimental groups,especially in CS and NTHi+CS groups(p<0.001,p<0.001);the level of Functional Residual Capacity(FRC)was increased in experimental groups,especially in CS and NTHi+CS groups(p<0.001,p<0.001);the level of lung resistance(RI)in CS and NTHi+CS groups was increased significantly(p < 0.001,p < 0.001).HE staining results revealed that compared with NC group,pathological changes at different levels were observed in lung tissues of mice from experimental groups.Inflammatory cell infiltration was detected in CS and NTHi groups,and there wasmild loss and drop in lung and bronchial epithelium tissue and some alveolar space expanded and fused into bullae.This phenomenon was more serious in NTHi+CS group.The results of FCM showed that CD4+CD25+Fox P3+Tregs from spleen decreased obviously in CS,NTHi and NTHi+CS groups compared with NC group,while the number of cells in NTHi+CS group increased relatively and there was no significant difference in total number compared with NC group(p > 0.05).The ELISA results showed that the expressions of TGF-?1?IL-17 in CS and NTHi+CS groups significantly increased(p<0.05,p<0.01),and the concentration of IL-17 increased significantly in NTHi group(p<0.001).Besides there was no significant difference in the expression of TGF-?1 in these two groups compared with NC group(p>0.05).The results of immunohistochemistry and real-time PCR indicated that the protein and m RNA expression of Foxp3 decreased in NTHi+CS group compared with this in NC group(p<0.05),while there was no significant difference in CS and NTHi groups(p > 0.05).Besides both the expression of ROR?t and IL-17 increased significantly in each experimental group(p<0.001).In summary.through the above research,we preliminarily concluded that CS could induce COPD,and inflammation in COPD subjects was closely related to TGF-?1 secretion by CS-induced Tregs activation,and that AECOPD was induced by stable COPD infected with NTHi through down-regulating Foxp3 and up-regulating ROR?t.