The Role And Mechanism Of Nlrp3 Inflammasome In The Obliterative Bronchiolitis Lesion After Lung Transplantation

Object: Obliterative bronchiolitis(OB)remains the major complication limiting long-term survival of patients after lung transplantation.Nlrp3 inflammasome is an integral part of innate immunity,and it has been reported to participate in acute rejection after kidney and skin transplantation and graft verse host disease after allogeneic hematopoietic stem cell transplantation.However,to date,the effect and mechanism of the Nlrp3 inflammasome on OB after lung transplantation has not been clarified.Methods:(Part one)Mouse orthotopic tracheal transplant model was used to mimic the OB lesion after lung transplantation.RT-q PCR,Western blot,and ELISA were used to measure the expression and activation of Nlrp3 inflammasome after trachea transplantation.To study the effect of Nlrp3 inflammasome on OB,the specific inhibitor of Nlrp3 inflammasome,MCC950,were administrated to the allogeneic trachea transplant mice.HE staining,Masson's trichrome staining,and Hydroxyproline assay were performed to evaluate the degree of luminal occlusion and fibrosis of the trachea grafts.The infiltration of CD4+/CD8+ T lymphocytes and Ly6G+ neutrophils in the tracheal intima were detected by immunofluorescent or immunohistochemical staining.Apoptosis of tracheal epithelia,marked by CK19,was detected by TUNEL assay.(Part two)The influence of MCC950 on the differentiation and function of Th1/Th17/Treg cells were evaluated via Flow cytometry,ELISA,and RT-q PCR.The splenocytes from C57BL/6 mice(Responder)and BALB/c mice(Stimulator,which were pretreated with mitomycin C)were co-cultured for mixed lymphocyte reaction(MLR).The effect of Nlrp3 inflammasome and MCC950 on the differentiation and function of Th1/Th17/Treg cells were further tested in MLR system.To further explore the downstream molecular mechanism by which the Nlrp3 inflammasome contribute to OB,the recombinant mouse IL-1? and/or IL-18 were added to MCC950 treated experiments.Results:(Part one)This study indicated that compared with syngeneic trachea transplantation,the activation level of Nlrp3 inflammasome was significantly elevated after allogeneic trachea transplantation,and the transcription of Nlrp3 inflammasome components(Nlrp3,Caspase-1,and IL-1?),except IL-18,were also markedly upregulated.MCC950 treatment significantly inhibited the activation of Nlrp3 inflammasome after allogeneic tracheal transplantation,however,it had little effect on the transcription of the Nlrp3 inflammasome components.The suppression of Nlrp3 inflammasome by MCC950 markedly ameliorated the luminal occlusion,fibrosis of the subepithelial area,infiltration of Ly6G+ neutrophils and CD4+/CD8+ T lymphocytes in the intima,and apoptosis of epithelia after allogeneic trachea transplantation.(Part two)Mechanism study found that MCC950 treatment dramatically decreased the differentiation and function of Th1/Th17 cells after allogeneic trachea transplantation,while increased the Treg cell response.However,these effects were reversed by the addition of IL-1? and/or IL-18 both in vitro and in vivo.The alleviating effect of MCC950 on luminal occlusion,collagen deposition,epithelial apoptosis,and neutrophil infiltration were also abolished by the addition of IL-1? and/or IL-18.Conclusions: Nlrp3 inflammasome contribute to the development of OB after mouse orthotopic trachea transplantation;the potential mechanism may be mediating the production of IL-1? and IL-18,and further promoting the Th1/Th17 cell responses,while suppressing the Treg cell response.MCC950 can ameliorate OB lesion through inhibiting the production of IL-1? and IL-18 and regulating the balance between Th1/Th17 cells and Treg cells.Therefore,targeting the Nlrp3 inflammasome is a promising strategy for controlling OB after lung transplantation.