Regulatory Molecular Mechanism Of NLRP3 Inflammasome/Caspase-1/IL-1β Axis In Polymyositis

Polymyositis(PM)is an idiopathic inflammatory myopathy,which is induced by autoimmune response.Up to date,the mechanism of PM remains unclear.The NOD-like receptor family,pyrin domain containing 3(NLRP3)is implicated in infectious and autoimmune diseases.Recent studies have demonstrated that the NLRP3 inflammasome not only plays a central role in innate immune response,but also is very important for adaptive immune response through regulating the differentiation and function of the immunocytes.NLRP3 inflammasome can trigger the activation of caspase-1 and the cleavage of IL-1β(Interleukin-β,IL-1β.It was reported NLRP3 inflammasome/caspase-1/IL-1β axis was over expressed in PM patients,indicating this axis may be associated with PM.However,the role of NLRP3 inflammasome/caspase-1/IL-1β in modulating PM has not been elucidated.Therefore,we planned to investigate the effect of NLRP3 inflammasome/caspase-1/IL-1β on PM and the underlying cellular immunological mechanisms through which NLRP3 inflammasome/caspase-1/IL-1β axis modulates PM in PM patients,experimental rat polymyositis model and co-culture of Raw 264.7 macrophages with C2C12 myoblast,respectively.Additionally,we use NLRP3 inflammasome inhibitor on the polymyositis rat model and use siRNA to knock down NLRP3 and IL-1β to show the effect of NLRP3 inflammasome/caspase-1/IL-1β axis on PM.Our study may provide evidences for a better understanding of the immunopathological mechanism and possible therapeutic intervention of PM.PART ONEIncreased expression of NLRP3 inflammasome/caspase-1/IL-1βaxis in patients with polymyositisAims:We aim to examine the expression of NLRP3 inflammasome/caspase-1/IL-1β axis in PM patients.Methods:In this part,24 PM patients and 10 controls were recruited.The clinical data was collected.The histologic inflammation grade was evaluated by H&E stain.And expression of NLRP3 inflammasome,caspase-1,IL-1β and CD68+ macrophages were detected by immunochemistry.These markers were compared between the two groups.We also analyzed the correlation between NLRP3 inflammasome,caspase-1,IL-1β and histologic inflammation grade,CD68 expression and global activity score.Results:Histological grade score of muscle tissue was much higher in PM groups than control(3.0±0.9 vs 0.2±0.4;p<0.01).Percentage of NLRP3 inflammasome positive cells were higher in PM than control(8.8±3.5 vs 0.5±0.7;p<0.01).Percentage of NLRP3 inflammasome,caspase-1 and IL-1β positive cells was higher in PM than control(17.2±9.5 vs 1.2±1.3;p<0.01)、(15.2±8.4 vs 0.3±0.5;p<0.01)and(14.5±6.2 vs 0.2±0.4;p<0.01).NLRP3 inflammasome,caspase-1 and IL-1β expression correlated with histologic grade(r=0.588,p<0.05).(r=0.546,p<0.01)and(r=0.559,p<0.01),CD68+ macrophages infiltration(r=0.672,p<0.01)、(r=0.409,p<0.05)and(r=0.443,p<0.05)and Global visual analogue scale(V AS)(r=0.627,p<0.01)、(r=0.407,p<0.05)and(r=0.435,p<0.05).Conclusion:The NLRP3 inflammasome/caspase-1/IL-1β axis may play an active role in pathogenesis of PM and may be used as a marker of disease activity.PART TWOThe role of NLRP3 inflammasome/caspase-1/IL-1β axis in pathogenesis of PM in a myosin-induced rat modelAims:This study is designed to investigate the role of NLRP3 inflammasome/caspase-1/IL-1β axis in pathogenesis of PM in a myosin-induced rat model.Methods:Myosin-induced PM rat models were established.Rats were divieded into three groups:Model group(myosin,10mg/kg,5 times),Model + glibenclamide(Glyb)(myosin of the same dose accompanied with 5mg/kg Glyb daily)group and the control group(normal saline).The histologic inflammation grade was evaluated by HE stain.Double-labeling immunofluorescence analysis was used for localization of macrophages and NLRP3 inflammasome.qRT-PCR and Western Blot were used to test the expression of NLRP3 inflammasome,caspase-1 and IL-1β in three groups.Results:In the Model group(n=5),scattered muscle fibers were degenerated and necrotic,surrounded by numerous mononuclear cells.Inflammation involved fewer fibers with infiltration of inflammatory cells in Model+Glyb group(n=5).Morphology and structure of muscle fibers were normal,no obvious infiltration of inflammatory cells could be observed in the control group(n=5).Histological score of muscle tissue sections was much higher in the Model group than in the other two(p<0.01).Histological score in Model+Glyb group was higher than the control(p<0.05).CD68 and NLRP3 inflammasome were both expressed on infiltrated inflammatory cells detected by double-labeling immunofluorescence.The transcription level of NLRP3 mRNA in Model+Glyb group was lower than the Model group(p<0.05),but had no statistical difference from the control.And the similar results could be found in protein expression of NLRP3 inflammasome,caspase-1 and IL-1β by Western Blot.Conclusion:NLRP3 inflammasome can induce muscle inflammation in rat PM model,which.is involved with caspase-1 activity and IL-1β maturation.PART THREENLRP3 inflammasome/caspase-1/IL-1β axis activation in Raw 264.7 macrophages can up-regulate MHC-1 expression of C2C12 myoblastsAims:We aim to investigate the effect of NLRP3 inflammasome/caspase-1/IL-1β axis activation of Raw 264.7 macrophages on C2C12 myoblasts MHC-1 expression in vitro through transwell co-culture.Methods:In this study,we used LPS combined with ATP to stimulate Raw 264.7 macrophages in different condition.ELISA,qRT-PCR and Western Blot were used to test the expression of NLRP3 inflammasome,caspase-1 and IL-1β.In addition,we used a transwell co-culture system for Raw 264.7 macrophages and C2C12 myoblasts.We used LPS combined with ATP to stimulate Raw 264.7 macrophages and tested the MHC-1 mRNA transcription and protein expression.Then we used siRNA to knock down NLRP3 followed by LPS/ATP stimulation and examined MHC-1 mRNA transcription and protein expression.Futhermore,we used murine IL-1β cytokine to stimulate C2C12 myoblasts and detected the MHC-1 mRNA transcription and protein expression.Then we used siRNA to knock down IL-1β followed by LPS/ATP stimulation and examined MHC-1 mRNA transcription and protein expression.Results:1.We used LPS combined with ATP to stimulate Raw 264.7 macrophages,which showed mRNA and protein expression of NLRP3 was elevated,especially when LPS concentration was 200ng/ml at 24h(p<0.05).2.We used LPS combined with ATP to stimulate Raw 264.7 macrophages in a co-culture system and found MHC-1 mRNA transcription and protein expression of C2C12 myoblasts were significantly up-regulated,especially at 72h(p<0.01).Then we used siRNA to knock down NLRP3 followed by LPS/ATP and the MHC-1 mRNA and protein expression was significantly down-regulated compared to group without siRNA transfection(p<0.01).3.We used murine IL-1β cytokine to stimulate C2C12 myoblasts and detected that their MHC-1 mRNA and protein expression were elevated,especially when IL-1βconcentration was lOng/ml and stimulation time was 48h(p<0.01).Then we used siRNA to knock down IL-1β followed by LPS/ATP stimulation and the MHC-1 mRNA and protein expression was not statistically different from NS group.4.We found caspase-1 maturation increased after LPS/ATP stimulation in Raw 264.7 macrophages(p<0.01)and the effect could be significantly weakened when NLPR3 was knocked down(p>0.05)Conclusion:Our results suggest that the NLRP3 inflammasome/caspase-1/IL-1β axis activation in Raw 264.7 macrophages can up-regulate MHC-1 expression of C2C12 myoblasts in vitro,which is a marker of polymyositis.