Surfactant Protein A Ameliorate Allergic Asthma Airway Inflammation By Modulating Tfh Subsets Differentiation In A DC-dependent Manner

Asthma is a common respiratory inflammatory disease characterized by airway hyperresponsiveness,which can be affected by people of all ages,bring a substantial economic burden to families and society to manage and cure asthma patients.The pathogenesis of asthma is very complicated,eosinophils and neutrophils infiltration,abnormal activation of lymphocytes,and synthesis of allergen-specific Ig E antibodies and so on are involved in the development of asthma.While significant progress has been reported in recent years,there are still no effective drugs that can control asthma,especially severe asthma.The clinical treatment of asthma is still highly dependent on drugs such as dexamethasone andβ2 receptor agonists,and these drugs not only have side effects,but it is still difficult to achieve good results in the treatment of patients with refractory or severe asthma.Therefore,there is still a long way to go to find new drugs and intervene more effectively in asthma.Surfactant protein A(SP-A)is produced by typeⅡalveolar epithelial cells,which belongs to the lectin family.Studies have proved that SP-A not only releases pulmonary tension,but also play a protective role in host defense and immune regulation,which is closely related to the occurrence of asthma.T follicular helper cells(Tfh)are newly recognized CD4~+T subsets located in Germinal Center and characterized by expression of CXCR5,which play a vital role in B cells somatic mutation,isotype class switching,and antibody affinity maturation.Mountains of studies have demonstrated that the abnormal activation of Tfh cells devotes to the development of asthma.Still,the mechanism of Tfh cells in the regulation of asthmatic airway inflammation remain unclear.In this study,we aimed to explore the role of Tfh cells in the development of asthma both in animal models and patients with asthma,and investigate whether SP-A can ameliorate asthmatic airway inflammation by regulating Tfh cell subsets in vivo and in vitro.It is hoped to provide a consult for clinicians and researchers in the selection of drugs and new targets.Part Ⅰ:Surfactant protein A ameliorate asthma airway inflammation by regulating Tfh subsets distribution6-8 weeks of female wild-type(WT)and SP-A knockout(Sp-a~-/~-)mouse on C57BL/6J background were immunized with ovalbumin(OVA),PBS control groups were established.HE staining was used to visualize the pathological changes in lung,ELISA was used to detect the level of Ig E,and Ig A antibodies and the level of IL-4,IL-6,and IL-17 in BALF,flow cytometry was used to analyze the distribution of Tfh cells and subsets in spleen and lymph nodes.It was found that a large number of inflammatory cells infiltrate and mucus exudate in the lung tissue of mice after OVA challenge,and Sp-a~-/~-mice showed more severe changes compared with WT mouse.Also,compared with PBS groups,the level of Ig E antibody in BALF increased significantly in the asthma model mouse,and the level of Ig E antibody increased in Sp-a~-/~-mouse while the level of Ig A antibody decreased,compared with WT mouse.These results suggested that SP-A plays a protective role in the development of asthma.In further study we have found that the distribution of Tfh cells skewed in WT and Sp-a~-/~-asthma mouse.The percentage of IL-4~+Tfh cells increased in Sp-a~-/~-mouse and was positively correlated with the level of Ig E.While the proportion of IFN-γ~+Tfh cells decreased in Sp-a~-/~-mouse,and was negatively correlated with the level of Ig E.And the ratio of IFN-γ~+Tfh/IL-4~+Tfh decreased significantly in Sp-a~-/~-mouse.In the further study we have found that Tfh derived from Sp-a~-/~-mouse have advantage on helping Ig E antibody secretion by B cells.These results indicated that the abnormal distribution of Tfh cell subsets was related to the development of asthma,and SP-A may alleviate the airway inflammation response by stabilizing the distribution of Tfh cell subsets.Part 2:Recombinant mouse SP-A modulate Tfh subsets differentiation in a BMDC dependent manner in vitroRecombinant pc DNA3.1?/His A-SP-A plasmid was transfected to CHO cells to construct a stable expression system of SP-A in vitro.Purified BMDCs and naive T cells were treated with SP-A or not;flow cytometry was used to analyze the distribution of Tfh subsets five days later.BMDCs were treated with LPS and SP-A in vitro;the expression of CD80/86 was measured by flow cytometry 24 hours later.And Q-PCR was used to estimate the relative level of IL-6,IL-4,and so on in WT and Sp-a~-/~-asthma mouse-derived DCs.Our results showed that SP-A significantly downregulates the ratio of IFN-γ~+Tfh/IL-4~+Tfh only in the situation of BMDCs existing in the coculture system.It indicated that SP-A participated in the differentiation of Tfh cells in a BMDC dependent manner in vitro.In a further experiment,we have found that SP-A significantly down-regulated the expression of CD86 on BMDC,which was activated by LPS,suggesting that SP-A inhibited the activation of BMDC.Besides,we have found that the relative level of IL-6 increased significantly in Sp-a~-/~-asthma mouse-derived DCs.Our data suggested that SP-A may participate in modulating Tfh subsets differentiation by inhibiting the activation of DC in vitro.Part 3:the distribution of circulating Tfh cells and subsets in asthmatic patientsTwenty-one asthma patients and nineteen gender-and age-matched healthy controls were enrolled;flow cytometry was used to analyze the distribution of circulating Tfh cells and subsets in asthmatic patients and healthy controls.We have found that there was no statistical difference in the proportion of CD4~+CD45RA~-CXCR5~+cells in peripheral blood between asthma patients and healthy controls.However,the distribution of Tfh subsets skewed in asthma patients,especially the propotion of Tfh2 cells increased significantly,while the percentage of Tfh1 cells and Tfh17cells showed no significant difference between asthma patients and healthy controls.Besides the proportion of Tfh2 cells in the group with elevated serum Ig E antibodies was significantly increased.These results suggested that the abnormal distribution of Tfh subsets,especially the abnormal activation of Tfh2 cells,may contribute to the development of asthma by affecting the synthesis of Ig E antibodies,which provided potential roles in allergic asthma treatment.In contrast,the mechanism of Tfh2 cells in the pathogenesis of asthma needs further researches.