Oxidative Stress-induced TRPM2 Activates NLRP3 Inflammasome In Autoimmune Vitiligo

Background: Vitiligo is an autoimmune disease of the skin and characterized by epidermal melanocytes loss due to CD8⁺T cells-mediated immune response.Previous studies and our recent findings have demonstrated that oxidative stress plays a critical role in the initiation of autoimmunity in patients with vitiligo.And we have further confirmed that the keratinocyte-derived chemokines including CXCL10 and CXCL16,by interaction with their corresponding receptors CXCR3 or CXCR6,mediates the skin trafficking of CD8⁺T cell in response to oxidative stress and thus contributes to melanocytes loss in patients with vitiligo.However,the detailed mechanism of chemokines production in stressed keratinocytes is not well understood,and the effect of oxidative stress on human keratinocyte and its capacity to regulate CD8⁺T-cell immune response is still unknown.The NLRP3 inflammasome is a multiprotein complex consisting of three kinds of proteins,NLRP3,ASC,and pro-caspase-1,and plays a role in sensing pathogens and danger signals in the innate immune system.Inflammasome assembly results in caspase-1 activation,which in turn drives maturation and secretion of the pro-inflammatory cytokines interleukin（IL）-1β and IL-18.Incresing evidences have indicated that NLRP3 inflammasome played a critical role in inducing T cells migration through regulating chemotaxis-related proteins.In addition,NLRP3 inflammasome could regulate immune cells proliferation,activation,the specific antigens recognition,as well as the inflmmatory cytokins production,thus initiating various autoimmune diseases.Recently,the NLRP3 inflammasome is thought to be involved in the development of vitiligo.However,the mechanism by which the NLRP3 inflammasome induces vitiligo is not clear.The NLRP3 inflammasome is activated in response to the widest array of stimuli,among of which,the reactive oxygen species（ROS）-generating mitochondria is necessary and required for NLRP3 inflammasome activation.Further studies elucidate a critical role for Ca²⁺ mobilization in promoting mitochondrial damage as well as the assembly and activation of the NLRP3 inflammasome by multiple stimuli.Interestingly,the redox-sensitive cation channel transient receptor potential melastatin 2（TRPM2）has been reported to produce mitochondria ROS generation via mediating Ca²⁺ influx.More importantly,activation of TRPM2 could play an important role in the modulation of cytokine/chemokine expression under oxidative stress and that such changes contribute to initiation and amplification of immune cells migration and autoimmune signals.Based on these researches,we hypothesised that TRPM2-induced Ca²⁺ influx may function as a key player that links oxidative stress to the NLRP3 inflammasome activation,which lead to chemokines production,CD8⁺T cells skin accumulation,and thereby contribute to melanocyte aotoimmune destruction in vitiligo.Objectives: 1.To explicate the detailed mecanism of NLRP3 inflammasome activation under ooxidative stress in keratinocytes in patients with vitiligo.2.To explicate the effecs of ROS-induced NLRP3 inflammasome activation on vitiligo CD8⁺T cells skin migration and other immune functions.Methods: 1.Firstly,we detected NLRP1,NLRP3,NLRC4,and AIM expression in vitiligo skin tissues by using immunohistochemistry;and then we detected the IL-1β expression in peripheral blood samples of patients with progressive vitiligo by using ELISA;Further,the IL-1β m RNA level and its associations with H₂O₂ accumulation,CXCL10 or CXCL16 expression in skin lesions of patients with vitiligo were detected and analyzed.2.To investigate a possible implication of mitochondria in NLRP3 inflammasome activation,we artificially induced oxidative stress in primary human keratinocytes by H₂O₂,and then we detected the mitochondrial membrane potential and mitochondrial ROS production by using three types of mitochondria-specific labels.Next,the real-time PCR,Western blot,immunofluorescence,and ELISA were used to detect NLRP3 expression,localization as well as activation.3.To investigate the possible mechanism of NLRP3 inflammasome activation,we first detected TRPM2 expression and Ca²⁺ influx by using Western blot and using Ca²⁺-specific label Fluo-4 in H₂O₂-treated primary human keratinocytes;and then we knockdown the TRPM2 expression in Ha Ca T cells by transfection with TRPM2 Si RNA,and subsequently detected the Ca²⁺ influx,mitochondrial damage and NLRP3 activation.4.To investigate the possible influences of TRPM2-mediated NLRP3 inflammasome activation on chemokines production,we analyzed the secretion of CXCL10 and CXCL16 by using ELISA in Ha Ca T cells knocking down either TRPM2 or NLRP3,and also in untransfected Ha Ca T cells treated with either IL-1β neutralization antibody or IL-1R antagonist.5.To investigate the possible influences of TRPM2-mediated NLRP3 inflammasome activation on chemokine receptors expression in CD8⁺T cells,we sorted peripheral blood CD8⁺T cells from patients with progressive vitiligo,and then cultured these T cells in supernatants from Ha Ca T cells transfected with either TRPM2 or NLRP3 Si RNA,or that treated with IL-1β neutralization antibody.We also block the activity of IL-1R on CD8⁺T cells by using IL-1R antagonist.Finally,the flow cytometry were used to analyze the expression of CXCR3 and CXCR6 in CD8⁺T cells.6.To confirm the effects of TRPM2-mediated NLRP3 inflammasome activation on vitiligo CD8⁺T cells migration,we performed transwell assay by using blood-derived CD8⁺T cells from patients with progressive vitiligo and supernatants from Ha Ca T cells transfected with either TRPM2 or NLRP3.Next,we added recombined human CXCL10（rh CXCL10）and rh CXCL16 in above mentioned transfected Ha Ca T supernatants.In addition,CXCR3-CD8⁺T cells and CXCR6-CD8⁺T cells derived from patients with vitiligo were also used for the migration assay.After 3 hours of trafficking,the migrated cells in the lower chamber were counted by means of flow cytometric analysis acquisition.7.To investigate the possible influences of TRPM2-mediated NLRP3 inflammasome activation on proinflammatory cytokine IFN-γ expression in CD8⁺T cells,we sorted CD8⁺T cells and then cultured these T cells in supernatants from Ha Ca T cells transfected with either TRPM2 or NLRP3 Si RNA,the flow cytometry were used to analyze the expression of IFN-γ in CD8⁺T cells.Results: 1.The immunohistochemistry results confirmed that among the four inflammasome markers examined,NLRP3 was highly expressed in epidermal keratinocytes.According to the ELISA results,IL-1β expression in peripheral blood of patients with progressive vitiligo were 5.617±0.174 pg/m L（n=30）,slightly decresed than in healthy dornors（6.821±0.367 pg/m L,n=18,P < 0.05）,there was no significant differences between the petients before treatment and after treatment（n=30,P = 0.7154）.Notewoorthy,the IL-1β m RNA levels were markedly upregulated in skin lesions of patients with vitiligo than healthy dornors（with 2.04±0.28 folds upregulated,n=16,P < 0.01）;And the increased IL-1β level was significantly correlated with epidermal H₂O₂ accumulation（r=0.6588,P = 0.0055）,increased CXCL10（r=0.6118,P = 0.0118）and CXCL16（r=0.6118,P = 0.0118）m RNA levels.2.In primary human keratinocytes,addition of H₂O₂ resulted in the loss of mitochondrial membrane potential and robust ROS production.In addition,H₂O₂ significantly promoted NLRP3 expression and translocalization in mitochondria,increased Caspase-1 activation and IL-1β secretion.These data suggested that H₂O₂ could induce mitochondrial damage and thus activate NLRP3 inflammasome in keratinocytes.3.In primary human keratinocytes,H₂O₂ significantly induced TRPM2 expression and Ca²⁺ influx.Importantly,knocking down TRPM2 in Ha Ca T cells attenuated Ca²⁺ influx,mitochondrial damage induced by H₂O₂.In accordance with these,the H₂O₂-induced NLRP3/ASC mitochondrial ranslocation and Caspase-1,IL-1β activation were also attenuated.These data suggested that TRPM2 is required for ROS-induced NLRP3 inflammasome activation in keratinocytes.4.We found knocking down either TRPM2 or NLRP3 attenuated CXCL10 and CXCL16 expression induced by H₂O₂ in Ha Ca T cells,more prominently in groups with TRPM2 knockdown.In addition,bloking IL-1β/IL-1R signal by using IL-1β neutralization antibody or IL-1R antagonist displayed the similar results.These data suggested that TRPM2-induced NLRP3 inflammasome activation involve in chemokine CXCL10 and CXCL16 expression under oxidative stress in keratinocytes,at least partially via IL-1β/IL-R1.5.According to the flow cytometry results,knocking down either TRPM2 or NLRP3 in stressed Ha Ca T cells,or neutralization IL-1β in untransfected stressed Ha Ca T cells could attenuate CXCR3 and CXCR6 expression in CD8⁺T cells derived from patients with vitiligo.Further we found blocking the activity of IL-1R in CD8⁺T cells could significantly reduce the CXCR3 and CXCR6 expression.These data suggested that TRPM2-induced NLRP3 inflammasome activation in keratinocytes could regulate CXCR3 and CXCR6 expression in vitiligo CD8⁺T cells via IL-1β/IL-R1 signal.6.Chemotaxis experiment in vitro confirmed that TRPM2-induced NLRP3 inflammasome activation in keratinocytes play a key role in attracting vitiligo CD8⁺T cells migration via at least two chemokine pairs,CXCL10-CXCR3 and CXCL16-CXCR6.7.We found that knocking down either TRPM2 or NLRP3 in stressed Ha Ca T cells could attenuate IFN-γ expression in CD8⁺T cells derived from patients with vitiligo,suggesting TRPM2-induced NLRP3 inflammasome activation in keratinocytes could promote IFN-γ expression in vitiligo CD8⁺T cells.Conclusion:In this study,we first demonstrate that TRPM2 play a critical role in activating NLRP3 inflammasome through mediating Ca²⁺ influx and mitochondrial damage in keratinocytes under oxidative stress.And further we discovered for the first time that TRPM2-induced NLRP3 inflammasome activation could not only significantly upregulated the expression of chemokines such as CXCL10 and CXCL16 in keratinocytes,but also promote the chemkine receptors like CXCR3 and CXCR6 expression in CD8⁺T cells derived from patients with vitiligo via IL-1β/IL-R1 signal,thus facilitating the migration of CD8⁺T cells to skin.In addition,we also found TRPM2-induced NLRP3 inflammasome activation could increase the IFN-γ expression in vitiligo CD8⁺T cells.Our study provides insight into the exact mechanism of TRPM2 induces NLRP3 inflammasome activation under oxidative stress in keratinocytes,and more importantly,this study suggests a strong connection between the NLRP3 inflammasome and immune cell migration and function through induction of chemotaxis-related proteins and proinflammatory cytokines in patients with vitiligo.Therefore,targeting TRPM2 may be effective for the treatment of NLRP3 inflamamsomeassociated autoimmune disorders like vitiligo.