The Regulatory Mechanism Of MDA5 In The Pathogenesis Of Vitiligo Under Virus Invasion

Background Vitiligo is a kind of autoimmune disease characterized by melanocytes destruction.The interaction between environment and heredity plays an important role in vitiligo.As an important environmental factor,virus has been identified to tightly corelated with vitiligo according to epidemical evidences but the specific mechanism is not explicit.Melanoma differentiation-associated gene 5(MDA5)is a kind of pattern recognition receptor sensing virus replication intermediate product double-stranded RNA(ds RNA)in cytoplasm.MDA5 contributes significantly to the anti-virus immunology by sensing and exterminating virus.Of note,the genome wide association study(GWAS)recently identifies IFIH1 encoding MDA5 as a susceptible gene in vitiligo.Nevertheless,the function and mechanism of MDA5 under the virus invasion are still elusive.Previous studies verified that CXCL10 and CXCL16 played an indispensable role in the process of melanocyte-targeting destruction by mediating the homing of CD8+ T cells to epidermis and blocking the production of chemokines could remarkably alleviate and reverse vitiligo.These chemokines are correlated with the disorder of epidermis immune microenvironment.The existing researches demonstrated that oxidative stress and inflammation stimulation promotes the chemokines secretion.However,whether virus infection could potentiate the secretion of chemokines and forwardly aggravate melanocyte death is yet to be determined.Virus infection could activate MDA5 and promote the secretion of IFN-β via MDA5-NF-κB pathway in keratinocytes.Besides,NF-κB has been already validated to play a kay part in the production of CXCL10 and CXCL16.So we hypothesized that MDA5 might participate in the pathogenesis of vitiligo by promoting the secretion of chemokines.Aims 1.To investigate the correlation between vitiligo and virus infection.2.To verify the impacts of virus infection on keratinocyte in vitiligo.3.To investigate the mechanism of how virus infection promotes the chemokines secretion in keratinocytes of vitiligo.Methods 1.The expression of anti-CMV Ig M in the serum of progressive vitiligo patients was detected by ELISA assay.The m RNA levels of CXCL10 and CXCL16 in the epidermis of progressive vitiligo patients was explored by qRT-PCR assay.The expression of MDA5 in the epidermis of progressive vitiligo patients was investigated by Western-blot assay.The expression of MDA5,IFN-β and the infiltration of CD8+ T cells in the skin was determined by immunofluorescence assay.2.Using the virus replication intermediate production double-stranded RNA(ds RNA)analog(Poly(I:C))to simulate the virus infection in keratinocytes in vitro.The safe and effective concentration of Poly(I:C)on NHKs was determined by IC50 and flow cytometry assay.The impacts of Poly(I:C)on the expression of MDA5 in NHKs in indicated time and concentrations were tested by qRT-PCR,Western-blot and immunofluorescence assay.3.Poly(I:C)was used to imitate the virus invasion in NHKs.The m RNA and protein levels of CXCL10 and CXCL16 in response to Poly(I:C)in NHKs was determined by qRT-PCR assay and ELISA assay.4.The impacts of Poly(I:C)on the MAVS aggregates formation were investigated by Western-blot assay and SDD-AGE assay.The colocalization of MDA5 and MAVS with the treatment of Poly(I:C)in NHKs and Ha Ca T cells was demonstrated by immunofluorescence assay.After silencing MDA5 and MAVS respectively using the corresponding si RNA,qRT-PCR and ELISA assay were used to detect the m RNA and protein levels of CXCL10 and CXCL16 in response to Poly(I:C).5.The expression of NF-κB P65,NF-κB p-P65,IRF3 and p-IRF3 was explored by Western-blot assay in response to Poly(I:C)in the indicated time.After silencing MDA5 and MAVS respectively using the corresponding si RNA,Western-blot assay was used to determine the expression alteration of NF-κB P65,p-P65,IRF3 and p-IRF3.After silencing NF-κB and IRF3 respectively using the corresponding si RNA,qRT-PCR and ELISA assay were used to detect the m RNA and protein levels of CXCL10 and CXCL16 in response to Poly(I:C).6.qRT-PCR and ELISA assay were used to probe the impacts of Poly(I:C)on the secretion of IFN-β via MDA5-MAVS-NF-κB/IRF3 pathway.The ELISA assay was used to explore the importance of IFN-β in the process of Poly(I:C)promoting the secretion of CXCL10 in keratinocytes.The contribution of JAK1-STAT1 pathway to the secretion of CXCL10 in response to Poly(I:C)was determined by Western-blot assay,qRT-PCR assay and ELISA assay.The direct binding between IRF3 and the CXCL16 promotor was probed by Ch IP assay.Results 1.The ELISA assay found that the positive rate and the antibody levels of anti-CMV Ig M were significantly higher in progressive vitiligo patients than in healthy controls.The m RNA levels of CXCL10 and CXCL16 were higher in the perilesional epidermis of progressive vitiligo patients with positive anti-CMV Ig M than that in the apparently healthy epidermis of patients with positive anti-CMV Ig M,in the perilesional epidermis of patients with negative anti-CMV Ig M and in healthy controls.The immunofluorescence assay showed the higher expression of MDA5,IFN-β in the perilesional epidermis of progressive vitiligo patients with positive anti-CMV Ig M than that in the apparently healthy epidermis of patients with positive anti-CMV Ig M,than that in the perilesional epidermis of patients with negative anti-CMV Ig M and in healthy controls.The infiltration quantity of CD8+ T cell in the skin showed the similar tendency,detected by the immunofluorescence assay.These data indicated virus infection correlates with some progressive vitiligo patients.2.The IC50 assay and flow cytometry assay exhibited that the safe and effective concentration of Poly(I:C)on keratinocytes was 0.6 μg/ml.Western-blot,qRT-PCR and immunofluorescence results manifested that Poly(I:C)promoted the expression of MDA5 in NHKs in time-and concentration-dependent manner.These results denoted virus infection promoted the expression of MDA5 in keratinocytes.3.At m RNA and protein levels,qRT-PCR and ELISA results demonstrated that Poly(I:C)could significantly potentiate the m RNA and secretion levels of CXCL10 and CXCL16 in a time-dependent way in NHKs.These results demonstrated that virus infection could potentiate the secretion of CXCL10 and CXCL16 in NHKs.4.SDD-AGE assay suggested that MDA5 mediated the formation of MAVS aggregates in Ha Ca T cells with the treatment of Poly(I:C).The immunofluorescence assay displayed that Poly(I:C)accentuated the colocalization of MDA5 and MAVS.The qRT-PCR and ELISA results denoted that the interfering MDA5 or MAVS using the corresponding si RNA could decrease the expression of CXCL10 and CXCL16 in m RNA and protein levels in response to Poly(I:C).These data illustrated that the secretion of CXCL10 and CXCL16 is mediated by the combination of MDA5 and MAVS in response to virus infection.5.The western-blot results exhibited that Poly(I:C)activated NF-κB and IRF3 in NHKs in a time-dependent manner,and with the pretreatment of si-MDA5 and si-MAVS,the upregulated expression of NF-κB p-P65 and p-IRF3 was disappear.After interfering NF-κB P65 or IRF3 using the corresponding si RNA,qRT-PCR and ELISA results manifested that the upregulated expression of CXCL10 and CXCL16 in response to Poly(I:C)was suppressed in m RNA and protein levels.These data manifested that NF-κB and IRF3 play a key role in the downstream of MAVS in Poly(I:C)promoting the secretion of CXCL10 and CXCL16.6.qRT-PCR and ELISA results suggested that Poly(I:C)potentiated the expression of IFN-β via MDA5-MAVS-NF-κB/IRF3 signaling pathway.Next,ELISA results displayed that the supernatant harvested from Poly(I:C)-treated Ha Ca T cells elevated the secretion of CXCL10 but not CXCL16 and neutralizing IFN-β disappeared the effect of promoting CXCL10.The Western-blot results revealed that IFN-β in the supernatant activated JAK1-STAT1 pathway and promoted the expression of MDA5 in Ha Ca T cells.To explore the importance of JAK1-STAT1 pathway on the secretion of CXCL10,qRT-PCR and ELISA results showed that rh IFN-β could increase the production of CXCL10,which could be inhibited by the pretreatment of tofacitinib in Ha Ca T cells.Last,the Ch IP assay validated that under the Poly(I:C)stimulation,IRF3 directly binds with the promotor of CXCL16 to regulate the secretion of CXCL16.These results suggested that MDA5-mediated IFN-β plays a key role in the process of Poly(I:C)promotes CXCL10 secretion,while the secretion of CXCL16 is regulated by IRF3.Conclusion In the present study,we initially manifested that the upregulated expression of MDA5 in some progressive vitiligo patients with positive anti-CMV Ig M.Then in vitro assays verified that virus infection,simulated by Poly(I:C),could promote the secretion of CXCL10 and CXCL16 which are the two vital chemokines in the pathogenesis of vitiligo.Sequentially,we demonstrated that IFN-β mediated by the MDA5-MAVS-NF-κB/IRF3 signaling pathway orchestrated the secretion of CXCL10 via the JAK1-STAT1 pathway and MDA5-meidiated IRF3 transcriptionally induced the production of CXCL16 in keratinocytes under virus invasion.