The Immune Modulation And Mechanism Of InterleuKin-17A And Osteopontin During The Antituberculosis Therapy

Backgrouds:Tuberculosis(TB),a chronic lung disease caused by Mycobacterium tuberculosis(M.TB),ranks among the top ten deadliest communicable diseases in low and middle-income countries.The occurrence and development of TB are closely related to the immune function,especially the cellular immune function,in which the main effector cells are T Lymphocytes.CD4 naive cells can differentiate into Thl,Th2 and Thl7 subgroups,and IL-17 is the major cytokine secreted by Th17 cells.It has been found that IL-17 mRNA expression can be detected in tuberculous pleural effusion,pulmonary tuberculosis bronchial lavage fluid and peripheral blood,suggesting that Th17 cells and IL-17 are involved in the pathogenesis of tuberculosis.Regulatory T(Treg)cell is an important cell for modulation of the immune responses,which plays a crucial role in immune tolerance and dynamic balance.However,the immune tolerance is broken when the number or the function of Treg cells is impaired.In many chronic infections,including the tuberculosis infection,IL-17 and IFN-y can cause pathological damage.Full length OPN(OPN-FL)is a matricellular soluble protein and a matrix-bound phosphoglycoprotein.Some recent studies found that OPN,as a cytokine,take part in the occurrence and development of the rheumatoid arthritis,hepatitis,liver cancer and other diseases.OPN can increase the pathological damage of the lung after Mycobacterium tuberculosis infection,by increasing the secretion of IL-12 and IFN-y.In vitro culture of macrophage cells,which were infected with Mycobacterium tuberculosis,the level of OPN in the culture supernatant increased significantly.These data indicate that OPN is involved in the development of TB.Th17 cells,Treg cells,and OPN are important for the occurrence,development and prognosis of tuberculosis.However,there are few reports on the dynamic changes during the course of TB treatment.In our study,the levels of IL-17,IL-23,IL-6,IFN-y,IL-10,OPN and IP-10 in patients with pulmonary tuberculosis who were positive for Acid Fast Bacilli-smear(AFB),were dected by ELISA,and the dynamic changes of Treg cells during the course of treatment were dected by flow cytometry.The purposes are to investigate the changes of immunological characteristics during tuberculosis treatment and the mechanisms of these changes,and the reliable indicators for the treatment of tuberculosis.Methods:A total of 20 AFB-positive pulmonary TB patients and 20 age-and gender-matched volunteers were included in our study.The percentage of T lymphocytes and Treg cells in peripheral blood mononuclear cells(PBMCs)were measured by flow cytometry,and the IL-17-producing cells were measured by Enzyme-linked Immunospot Assay(ELISPOT).Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of cytokines(IL-17A,IL-23,IL-6,IFN-y and IL-10)and OPN in the plasma of patients and healthy control.The levels of cytokines and Lactate Dehydrogenase(LDH)in culture supernatant of alveolar epithelial cell lines(A549)were detected by ELISA,with the stimuli of BCG,IL-17A,IL-10,OPN.The purpose is to investigate the function and mechanism of IL-17A,IL-10,OPN in the tuberculosis.The levels of cytokines in culture supernatant of PBMC in healthy volunteers and pulmonary tuberculosis patients were detected by ELISA after 24 hours of stimulation with BCG and OPN.Results:Part1The level of IL-17A in AFB-positive TB patients was significantly higher than that in healthy controls,while the level of IL-17A was significantly decreased after anti-tuberculosis treatment when the AFB was negative,but still higher than that of healthy controls.The levels of IL-23,IL-6 and IFN-y in AFB-positive patients were also significantly higher than that of healthy controls.In AFB-positive patients,except for classical T cells(CD3+CD56-),CD3+CD56+ NKT like cells are another major source of IL-17A,detected by flow cytometry.Treg cells and their secreted cytokines IL-10 can inhibit Th17 cells secreting IL-17A.IL-17A was positively correlated with C-reactive protein(CRP)and Erythrocyte Sedimentation Rate(ESR),but negatively correlated with IL-10.In vitro experiments showed that IL-17A could induce the apoptosis of alveolar epithelial cells and increase the damage of alveolar epithelial cells induced by BCG,while IL-10 could inhibit the damage of alveolar epithelial cells by IL-17A.Part2The level of OPN in AFB-positive TB patients was significantly higher than that in healthy controls.And the level of OPN in AFB-positive patients with tuberculous pleural effusion(TPE)was significantly higher than that in AFB-positive patients without TPE.And OPN was positively correlated with CRP.OPN could induce the expression of IFN-y-induced protein 10(IP-10),and the expression of IP-10 was also significantly increased in sputum smear-positive patients.OPN and IP-10 decreased only in the sputum smear negative.At the same time,BCG can promote the secretion of OPN in alveolar epithelial cells,and OPN can promote the secretion of IP-10.OPN also can promote the production of IP-10,IL-12 and IFN-y in PBMCs of healthy people and pulmonary tuberculosis.But after neutralized by the IFN-y-antibody,the ability of PBMC to secrete IP-10 and IL-12 was significantly reduced.Conclusions:In summary,in the M.TB infection,especially in AFB positive TB patients,IL-17A can induce lung injury.And CD3+CD56+ NKT like cells may be one of the IL-17A-producing cells in AFB positive TB patients.In the development of tuberculosis,Treg cells and IL-10 can inhibit IL-17A-induced lung injury.OPN can induce lung damage caused by M.TB.OPN and IP-10 may be a new indicator which can be used to detect the effect of tuberculosis treatment.