Nucleotide oligomerization domain?like receptor protein 1 inflammasome and vitiligo：progress in understanding the relationship

Ren-He Chen and Ru-Zhi Zhang*

Department of Dermatology,the Third Affiliated Hospital of Soochow University,Changzhou,Jiangsu 213003,China.

Introduction

Vitiligo is a depigmentation disease of the skin and/or mucous membranes caused by the interactions of various susceptibility genes and acquired factors.A variety of theories have been proposed to explain the occurrence of vitiligo:autoimmunity,genetics,viruses,oxidative stress,and others[1].Although the pathogenesis of vitiligo is not completely clear,autoimmune responses,which target functional melanocytes,are generally believed to play a major role[2].

A genome-wide association study(GWAS)revealed that the vast majority of vitiligo-associated genes have immunoregulatory functions and overlap with other autoimmune disease-related genes.Another evidence supporting this theory is that vitiligo responds to immunosuppressive therapy.Innate immune cells in the skin of patients with vitiligo are abnormally active,for example the infiltration of T cells,including CD4+and CD8+T cell populations,is usually seen at the edge of vitiligo lesions.Antigen-presenting cells in the skin,such as Langerhans cells,may migrate from the skin to the lymph nodes,present melanocyte antigens to T cells and activate them,thereby contributing to cellular stress pathways and T cell responses[3].The recruitment of natural killer cells and inflammatory dendritic cells is enhanced.All above evidences indicate that the activation of innate immunity plays a role in the pathogenesis of vitiligo.

Gene association analysis has found that nucleotide oligomerization domain-like receptor protein 1(NLRP1)is a susceptibility gene for vitiligo.The NLRP1 is assembled into NLRP1 inflammasome after activation,which in turn promotes the maturation and release of inflammatory factors such as interleukin(IL)-1β and IL-18[4].IL-1β can induce and enhance autoimmunity against melanocytes,which suggests that it may be a key link in converting the activation of NLRP1 inflammasome into an adaptive immune response.This article reviews recent progress in the association between NLRP1 inflammasome and the development of vitiligo,to improve the understanding of the disease pathogenesis and provide potential new targets for treatment of vitiligo.

NLRP1 inflammasome

Inflammasomes,first described in 2002,are multiprotein complexes composed of three elements:an AIM-like receptor(ALR)or NOD-like receptor(NLR),an adaptor protein,apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC),and an effector protein,caspase-1.Two receptor types have been identified—ALR and NLR.Multiple stimuli,including pattern recognition molecules and danger associated molecular patterns,can impinge on the ALR or NLR,which interacts with the ASC adaptor molecule.ASC then activates caspase-1.Activated caspase-1 cleaves pro-IL-1β and pro-IL-18,producing active IL-1β and IL-18,which finally motivate the inflammatory response.Activation of capase-1 through the inflammasome pathway also induces a type of inflammatory cell death—pyroptosis[5].Inflammasomes are named after their intracellular receptors.Many inflammasomes have been described:NLRP1,NLRP3,NLRP6,NLRP12,NLRC4/IPAF,andAIM-2[6].

NLRP1 was the first one to be discovered,and it consists of a pyrin domain,a caspase recruitment domain,a nucleotide-binding oligomerization domain(a NOD/NACHT domain),a leucine repeat domain,and a“function unknown”domain[7].A variety of exogenous stimuli,such as bacterial infections,and endogenous stimuli,such as tumorigenesis,can activate NLRP1.The activated NLRP1 then associates with ASC and caspase 1 or caspase 5 to form a complex termed the NLRP1 inflammasome.NLRP1 inflammasome promote caspase 1-dependent pro-cessing of IL-1β,which eventually leads to the secretion of IL-1β and downstream inflammatory responses.It is generally accepted that the adaptor protein,ASC only plays an auxiliary role in the activation of caspase-1 mediated by NLRP1 inflammasome.However,it has been reported that NLRP1 cannot induce the activation of caspase-1 and release IL-1β without ASC,indicating that ASC is indispensable for the activation of NLRP1 inflammasome[8].Multiple proteins can interact with the various domains of NLRP1 to inhibit the activation of NLRP1 inflammasome[9-10].

NLRP1 mutations increase susceptibility to vitiligo

A GWAS study on patients with vitiligo has identified multiple genes associated with innate immunity,includingNLRP1.Jinet al.[11]conducted extensive studies on NLRP1 variations in patients with vitiligo in the United States,the United Kingdom,and Romania.Their studies have shown that specific single nucleotide polymorphisms(SNPs)ofNLRP1increased susceptibility of subject to vitiligo including rs6502867/A,rs961826/A,rs925598/A,rs878329/G,rs3926687/T,rs7223628/G,rs12150220/A,rs2670660/C,rs8182352/G, rs11078575/C, rs1877658/T, rs2733359/G,rs4790796/A, rs35658367/ATGA, rs4790797/T,rs2716914/C,rs8182354/A.NLRP1mutations are genetically related to generalized vitiligo(GV).In Romanian vitiligo patients,there are at least two independent risk signals in NLRP1,one was labeled as SNP,rs6502867,and the other by SNPs,rs2670660 and rs8182352.Individuals carrying the high-risk allele of both rs6502867 and rs2670660 had a 4.2-fold increased risk of vitiligo compared with individuals carrying only one allele.Alkhateebet al.[12]studied patients with vitiligo in Jordan and reported that the SNPs,rs2670660 and rs1008588 in the promoter region ofNLRP1,had significant associations with vitiligo.Dwivediet al.[13]performed a similar study in Gujarat,and their results showed that theNLRP1SNPs,rs2670660(A/G)and rs6502867(T/C)were significantly associated with susceptibility to GV.However,GV was associated with the minor allele,“C”of rs6502867 in their study,whereas previous studies showed that GV is associated with the major allele,“T”in Caucasians.This difference suggests a recombination event that is closely linked to racial differences and indicates that the SNP itself is not a causal factor for GV.Earlier studies reported that three signals marked by the SNPs,rs4744411 on 9q22,rs734930 on 7q11 and rs6960920 on 7p13 were also significantly associated with GV.The NLRP1 SNP,rs6502867 affected vitiligo through gene-gene interactions of these other loci[14].

Recently,Liet al.[15]conducted a meta-analysis to assess the effects of the rs12150220,rs2670660,and rs6502867 polymorphisms of NLRP1 on vitiligo and vitiligo-associated autoimmune diseases.Based on data of 19 eligible case-control studies,they did not observe a strong association between NLRP1 rs12150220,rs2670660 or rs6502867 and the risk of vitiligo in the overall population.Similarly,the rs2670660 and rs6502867 polymorphisms of NLRP1 and the rs2670660/rs12150220 haplotype(A-A,G-T,G-A,AT)appeared to have no effect on the risk of vitiligoassociated autoimmune diseases.However,they found that there may be a genetic relationship between the rs12150220 and decreased susceptibility to autoimmune diseases that are closely associated with vitiligo,such as Edison’s disease,in Caucasian populations.So far,studies on the association of NLRP1 mutations with susceptibility to vitiligo have been limited,and there are differences on SNPs among the various studies.Further researches involving gene function analysis are required to figure out the critical NLRP1 mutations affecting susceptibility to vitiligo.

Expression of NLRP1 inflammasome in patients with vitiligo

NLRP1 inflammasome expressed in the peripheral blood of patients with vitiligo

Deoet al.[16]investigated the expression ofNLRP1gene in the peripheral blood cells of vitiligo patients and healthy individuals,and the results showed that,compared with healthy individuals,the expression ofNLRP1mRNA was decreased in patients with either generalized or segmental vitiligo,while it is worth noting that this study included only 28 subjects.Another study including 297 subjects reached the opposite conclusion using similar methods:the expression ofNLRP1mRNA in patients with GV was higher(7.6-fold)than that in healthy individuals;the expression ofNLRP1mRNA in active GV patients was also increased(5.3-fold)compared with stable GV patients[13].Expression of IL-1β in the peripheral blood of patients with active GV also increased,suggesting the involvment of activated NLRP1 inflammasome.These results indicate that NLRP1 inflammasome influence the progression of GV.In addition,women with GV had an earlier onset and their expression ofNLRP1mRNA was 6.0 times higher than that of men with GV.The authors described that this result corresponds to the fact that women are more susceptible to GV.However,most studies show that there is no gender difference in the incidence of vitiligo between sexes.The expression ofNLRP1mRNA was also increased in early-onset vitiligo patients compared with patients who developed the disease later,indicating that NLRP1 inflammasome play an important role in the early stages of the disease.Dwivediet al.[13]concluded that NLRP1 inflammasome had effects on the onset,development,and gender bias of GV.

NLRP1 inflammasome expressed in the lesion of vitiligo patient

Immunohistochemical staining of NLRP1 in biopsies of 20 vitiligo patients(without staging)showed that there were more NLRP1-positive cells at the edge of lesions than within the lesions and in non-lesional skin[17].They also found high levels of IL-1 β expression at the lesion edges,suggesting the presence of activated NLRP1 at this site.In addition,they also found that NLRP1 and CD207/Langerin are colocalized in the epidermal region at the edge of vitiligo lesions by dual immunofluorescence staining of NLRP1 and CD207/Langerin(Langerhans cell markers), suggesting the presence of NLRP1 inflammasome in Langerhans cells.

Maireet al.[18]collected samples from 14 patients with non-segmental vitiligo(eight cases in the active stage,one in the stable stage,and five in the regressive stage)and performed immunohistochemical staining.They found that staining for NLRP1 and IL-1β was negative in lesional specimens of all stages.However,at the edges of vitiligo lesions,NLRP1 was negative and IL-1β was positive in the stable and regressive stages,while both NLRP1 and IL-1β were positive in the active stage.These results suggest that NLRP1 inflammasome affect the progression of vitiligo.

NLRP1 is not only present in Langerhans cells,but also in both keratinocytes and melanocytes at the edges of active vitiligo lesions.Of even greater interest,the authors also observed NLRP1-positive staining in the nucleus,which is consistent with the findings of Kummeret al.in other tissues[19].The authors speculated that,in the development of vitiligo,NLRP1 drives two pathways:one is the inflammasome pathway located in the cytoplasm,and the other is an unknown pathway located in the nucleus,which needs further study.In addition,lymphocytic infiltration at the edges of lesions has been a good immunohistochemical proxy to assess vitiligo disease activity.Maireet al.[18]found that there was a significant correlation between NLRP1 and IL-1β expression at the edges of lesions and disease activity.If these markers were synergistically analyzed with lymphocyte infiltration,this could be expected to better assess disease activity and provide better guidance for clinical treatment.

NLRP1 inflammasome affect the development of vitiligo via IL?1β

Levandowskiaet al.[20]identified common NLRP1 haplotypes by high-throughput sequencing,at least two of which were associated with susceptibility to vitiligo and other autoimmune diseases.They found that the processing of IL-1β in mononuclear cells from high-risk individuals was 1.8 times greater compared with low-risk individuals.However,there was no significant change in the expression and stability of immature IL-1β,NLRP1transcription and NLRP1 protein expression.This result suggests that the highrisk haplotype increases the processing of immature IL-1β by enhancing the activation of NLRP1 inflammasome.In the study performed by Dwivediet al.[13]the expression ofNLRP1mRNA was increased in GV patients with the susceptible genotypes,GG(SNPrs2670660)and CC(SNPrs6502867).However,this did not contradict the report of Levandowskiaet al.[20]since the haplotypes they studied involved coding sequence variations,while the haplotypes studied by Dwivediet al[13].involved promoter and intron sequence variations.

Lachmannet al.[21]reported that in patients with cryopyrin-associated periodic syndromes(CAPS),nonsynonymous substitutions in theNACHTdomain of NLRP3 led to an approximately 5-fold increase in the production and secretion of IL-1β.The increased IL-1β leads to severe local or systemic inflammation even renal amyloidosis and death of patients.Recently,studies have found that a similar severe autoinflammatory phenotype is associated with anNlrp1mutation in mice[22].The common high-risk nonsynonymous mutation ofNLRP1haplotype is located outside theNACHTdomain,resulting in a 1.8-fold increase in IL-1 β in monocytes,and is only associated with an increased risk of vitiligo and part autoimmune diseases.Bhardwajet al.[23]reported that in Gujarati people,the IL-1β was significantly increased in patients with active non-segmental vitiligo,but not in the ones with stable vitiligo,which suggests that IL-1β has an association with disease activity of vitiligo.Therefore,IL-1β blockers may help treat or even prevent vitiligo,which needs clincial trails to prove.The NLRP1 inflammasome pathway is expected to become a new target for vitiligo treatment.

Nevertheless,vitiligo is a multi-gene-related autoimmune disease,and variation in a single gene such asNLRP1is obviously not sufficient to cause the disease.Levandowskiaet al.[17]speculated that increasing the immature IL-1β may contribute to the autoimmune aspects of vitiligo.IL-1β may act as an“adjuvant”and promote the presentation of autoantigens that may trigger or provide the specificity of the autoimmune response.This is consistent with the previous finding of NLRP1 inflammasome presentation in Langerhans cells.In fact,IL-1β can act on lymphocytes,upregulate the expression of IL-2 receptor,prolong the survival of T cells,enhance antibody production by B cells,and increase proliferations of B cells[24].IL-1β also plays a key role in promoting differentiation and proliferation of Th17 and Th1 cells,and may be a key link in converting the activation of NLRP1 inflammasome into an adaptive immune response.IL-18 is also a downstream cytokine of NLRP1 inflammasome,and has been previously found to increase in the peripheral blood of vitiligo patients.However,it has not been investigated in studies of the association with vitiligo.

NLRP1 inflammasome and melanocyte destruction

It is generally believed that autoimmune responses targeting functional melanocytes play a major role in the pathogenesis of vitiligo.Melanocyte destruction is mainly caused by CD8+T cell-mediated immune damage.Previous studies have shown that keratinocytes can secrete a large number of chemokines such as CXCL10 and CXCL16 under oxidative stress,which mediate the migration of CD8+T cells to the skin through binding to the receptors,CXCR3 and CXCR6,resulting in melanocyte-specific damage.So far,there have been no studies directly addressing the relationship between NLRP1 inflammasome,CD8+T cells and CXCL10 and CXCL16[25-26].Wanget al.[17]proposed that,apart from CD8+T cell-mediated killing,the activation of NLRP1 inflammasome that leads to increased IL-1β synthesis and release may be another reason for the loss of melanocytes at the edges of vitiligo lesions.Activation of capase-1 through the inflammasome pathway also induces pyroptosis and inflammatory cell death.Studies have shown that acute lung injury in mice is caused by exogenous and endogenous stimulation of NLRP1 inflammasome,which increases caspase-1 activity towards its downstream substrates and mediates the onset of pyroptosis. One can specu-late that NLRP1 inflammasome-mediated apoptosis through caspase-1 is another way of causing melanocyte destruction,which will be an interesting research direction in future.

Conclusion

Studies have shown that NLRP1 inflammasome play an important role in vitiligo.However,because studies on NLRP1 inflammasome are limited,we still do not fully understand the mechanisms of NLRP1 inflammasome function,especially regarding their involvement in vitiligo.Further in-depth study of NLRP1 inflammasome should provide a clearer understanding of the pathogenesis and development of vitiligo and provide promising new approaches for treatment,such as interfering with the activation of NLRP1 inflammasome.Important areas that deserve further exploration include the identification of NLRP1 mutations that have a causal relationship with vitiligo susceptibility,the specific mechanism of high NLRP1 inflammasome expression in the peripheral blood of vitiligo patients and how that affects the occurrence and development of vitiligo,and the specific effects of treating vitiligo by interfering with the NLRP1 inflammasome pathway,especially the processing and secretion of IL-1β.

Acknowledgement

The work was supported by the National Natural Science Foundation of China(No.81673078).