HMGB1 Induces The Proliferation And IL-18 Release Of Keratinocytes:A Mechanism Underlying The Pathogenesis Of Psoriasis

Background:Psoriasis is a chronic inflammatory skin disease commonly seen in department of dermatology.Clinically characterized by raised,erythematous plaques with adherent silvery scales,the skin disease leads to severely uncomfortable itching,which does great harm to the patients'physical and mental health.In psoriasis,multiple immune cells,especially T lymphocytes,are infiltrated in skin and release cytokines like TNF-?,IFN-?and IL-17 that induce inflammation and promote the proliferation of keratinocytes,which is the key event in the pathogenesis of psoriasis.However,more recent studies have proved that keratinocytes are not only responders to inflammation but also inflammation accelerators.Activated keratinocytes possess innate immune functions and can also release cytokines and chemokines,thus contributing to the formation and the maintainence of lesional inflammation.Nevertheless,the machanism underlying the activation of keratinocytes is still not elucidated.High-mobility group box 1 protein(HMGB1)is a nuclear protein that expresses in almost all types of cells.Under physiological condition,HMGB1 helps to maintain the genomic stabilization and to regulate the transcription and expression of genes.However,when the cells are under exogenous or endogenous stress,HMGB1 can be released into cytoplasm and subsequently into extracellular space,and then acts as a damage-associated molecular pattern(DAMP)molecule to promote immune response by binding to pattern recognition receptors(PRRs)on cell membrane.Once the binding takes place,HMGB1will activate inflammatory signaling pathways like NF-?B,which promtoes the production and the secretion of inflammatory factors and then leads to the formation of inflammation.In addition,extracellular HMGB1 can also promote the proliferation of cells from diverse tissues via binding to corresponding receptors.To date,researchers have proved the involvement of HMGB1 in the development of various infectious diseases(like sepsis)and autoimmune diseases(like rheumatoid arthritis),but the role of HMGB1 in cutaneous inflammatory diseases has not been well investigated.Recenly,it is reported that HMGB1 is secreted from epidermal keratinocytes in psoriatic lesion and the serum HMGB1 level is correlated with the disease severity,which implies the involvement of HMGB1 in psoriasis pathogenesis.Moreover,our research group has found that three HMGB1 receptors,including RAGE,TLR2 and TLR4,are overexpressed on the membrane of keratinocytes in psoriatic lesions.Based on these findings,we hypothesized that HMGB1 released from keratinocytes could bind to the PRRs of keratinocytes per se in an autocrine way and then activate inflammatory signaling pathways like NF-?B,which then enhances the proliferation and the innate immune functions of keratinocytes and promotes the release of inflammatory factors,thus contributing to the development of psoriasis.Objectives:The present study sought to clarify the effects of HMGB1 on the activation of keratinocytes as well as the underlying molecular mechanism,and to elucidate the role of the proliferation and the innate immune functions of keratinocytes regulated by HMGB1in the pathogenesis of psoriasis.Methods:1.Analysis on the effects of HMGB1 on the proliferation of keratinocytesNormal human keratinocytes(NHKs)were stimulated by recombinant human HMGB1(rhHMGB1).Firstly,the proliferation of NHKs was examined by CCK8 assay.Secondly,the activation of NF-?B p65 signaling pathway in NHKs under rhHMGB1stimulation was testified by immunofluorescence and western blot.Thirdly,NHKs were transfected by p65 siRNA and then stimulated by rhHMGB1,and the expression of miR-17-92 cluster was evaluated by qRT-PCR.Lastly,NHKs were transfected by miR-17-92 siRNA and then stimulated by rhHMGB1,and the proliferation of NHKs,the expression of cell cycle proteins and the distribution of cell cycle were detected by CCK8,western blot and flow cytometry,respectively.2.Analysis on the influence of HMGB1 on the innate immune functions of keratinocytesNHKs were stimulated by recombinant human HMGB1(rhHMGB1).Firstly,the expressions of inflammatory factors were screened by PCR-array,the results of which were further validated by qRT-PCR.Western blot and ELISA were used to detect the protein level and secretional level of interleukin-18 that showed the highest fold change in previous PCR-array assay.Secondly,NHKs were transfected by p65 siRNA and then stimulated by rhHMGB1,and the mRNA level,protein level and secretional level of IL-18were examined by qRT-PCR,western blot and ELISA,respectively.The binding of p65 to IL18 gene promoter region was validated by chromatin immunoprecipitation(ChIP).Thirdly,The protein levels of cleaved caspase-1,mature IL-18 in rhHMGB1-treated NHKs were examined by western blot.The assebly of inflammasomes was testified by co-immunoprecipitation(Co-IP).Lastly,NHKs were transfected by caspase-1 siRNA and then stimulated by rhHMGB1,and the protein level and the secretional level of cleaved IL-18 were measured by western blot and ELISA,respectively.3.Analysis on the role of HMGB1 in the development of dermatitis of psoriasis-like miceFirstly,imiquimod was smeared on the ears and the backs of mice to build up psoriasis mouse models,and the intervention of HMGB1/IL-18 neutralizing antibodies or corresponding isotype controls was given preventively.The inflammation level of back lesions was observed.The thickness of ears was evaluated by using vernier caliper.The histological features of lesions were investigated by H&E staining.The infilatration number of CD3~+T cells was dectected by immunofluorescence.The ratios of Th1 cells and Th17 cells were evaluated in peripheral blood samples by using flow cytometry.The numbers of IFN-?~+cells and IL-17~+cells were calculated by using immunofluorescence.The serum levels of IFN-?and IL-17 were examined by ELISA.Secondly,the epidermis samples of psoriasiform lesions were separated.The mRNA level of IL-18 in the lesional epidermis was evaluated by qRT-PCR.The protein levels of phosphorylated p65,cleaved caspase-1,IL-18 and cleaved IL-18 in the epidermis were examined by western blot.The assembly of inflammasomes in the epidermis was testified by Co-IP.The serum level of IL-18 was detected by ELISA.Lastly,completely established psoriasis-like mice were given the treatment of HMGB1/IL-18 neutralizing antibodies,and those indexes for evaluating the severity of psoriasiform dermatitis as described previously were reassessed.4.Analysis on the mechanism underlying the release of HMGB1 from keratinocytes in psoriasisFirstly,NHKs were stimulated by recombinant human IFN-?.Immunofluorescence was used to observe the location of HMGB1.Western blot was used to examine the protein levels of phosphorylated STAT1,cytosolic HMGB1 and nuclear HMGB1.ELISA was used to detect the secretional level of HMGB1.Secondly,NHKs were transfected by STAT1 siRNA and then stimulated by IFN-?.Western blot was used to testify the interference efficiency of STAT1 siRNA and evaluate the protein levels of cytosolic HMGB1 and nuclear HMGB1.Immunofluorescence was used to observe the location of HMGB1.ELISA was used to detect the secretional level of HMGB1.Results:1.HMGB1 promotes the proliferation of keratinocytes via inducing the expression of miR-17-92 clusterFirstly,CCK8 assay showed that rhHMGB induced the proliferation of NHKs.Secondly,Jaspar online bioinformatics database was used to predict the binding sites of NF-?B p65 in the promoter region of the gene that encodes miR-17-92 cluster,a microRNA cluster related to the proliferation of keratinocytes in psoriasis.Immunofluorescence and western blot showed that rhHMGB1 activated NF-?B p65signaling pathway,which subsequently induced the up-regulation of miR-17-92 cluster.Lastly,we found that the proliferation of NHKs induced by rhHMGB1 was mediated by miR-17-92 cluster,and rhHMGB1 inhibited the expression of CDKN2B and promoted cell cycle progression of NHKs via inducing miR-17-92 cluster.Taken together,our results indicate that HMGB1 facilitates the expression of miR-17-92 cluster via NF-?B p65 signaling pathway,thus promoting the proliferation and the cell cycle progression of keratinocytes.2.HMGB1 promotes the expression of IL-18 in keratinocytes via activating NF-?B p65 signaling pathwayFirstly,our PCR-array assay showed that rhHMGB1 increased the mRNA levels of multiple inflammatory factors in NHKs,in which IL-18 showed the highest fold change.Moreover,western blot and ELISA confirmed that rhHMGB1 elevated the protein level and secretional level of IL-18 in NHKs.After the transfection with p65 siRNA,the elevations of IL-18 expression in NHKs at mRNA level,protein level and secretional level induced by rhHMGB1 were abrogated.At last,our ChIP assay verified that rhHMGB1facilitated the binding of p65 to the promoter region of IL18 gene.These findings demonstrate that HMGB1 induced the expression of IL-18 via NF-?B p65 signaling pathway.3.HMGB1 facilitates the release of IL-18 from keratinocytes via activating inflammasomesFirstly,our western blot assay confirmed that the expressions of cleaved caspase-1and cleaved IL-18 were elevated in NHKs stimulated by rhHMGB1.Furthermore,Co-IP assay observed the binding of caspase-1 to ASC in NHKs under rhHMGB1 treatment.After the transfection with caspase-1 siRNA,the maturation and the secretion of IL-18 in NHKs induced by rhHMGB1 were attenuated.Collectively,our results indicate that HMGB1 promotes the maturation and the release of IL-18 in keratinocytes via inflammasome activation.4.HMGB1-IL-18 axis contributes to the formation of psoriasiform dermatitis in imiquimod-treated mice via enhancing Th17 immune responseFirstly,the preventive intervention of HMGB1/IL-18 neutralizing antibodies obviously alleviated the erythema and the scale on the back of imiquimod-treated mice and decreased the thickness of ears.Meanwhile,H&E staining showed that mice treated by HMGB1/IL-18 neutralizing antibodies had thinner lesional epidermis.Immunofluorescence assay showed that HMGB1/IL-18 neutralizing antibodies decreased the lesional infilatration of CD3~+T cells.Secondly,the flow cytometry assay found that HMGB1/IL-18 neutralizing antibodies reduced the ration of Th17 cells but not Th1 cells in the peripheral blood of mice.ELISA assay proved that HMGB1/IL-18 neutralizing antibodies lowered serum IL-17 level but not serum IFN-?level in psoriasis-like mice.Immunofluorescence showed that IL-17~+cells but not IFN-?~+cells were reduced in the lesions of mice treated by HMGB1/IL-18 neutralizing antibodies.At last,we separated the epidermis of psoriasiform lesions and found that HMGB1 neutrazling antibody suppressed the actively of NF-?B p65 signaling pathway and inflammasomes in the lesional epidermis,and decreased the mRNA level and protein level of IL-18.ELISA assay showed that serum IL-18 level was reduced in the mice treated by HMGB1 neutralizing antibody.Co-IP assay showed decreased binding effects between caspase-1 and ASC in mice with HMGB1 blockade.Taken together,our findings support that HMGB1-IL-18axis contributes to the formation of psoriasiform dermatitis in imiquimod-treated mice via facilitating Th17 immune response.5.HMGB1/IL-18 neutralizing antibodies accelerate the recovery process of mice from imiquimod-induced psoriasiform dermatitisAfter the complete formation of psoriasiform dermatitis in mice,the withdrawal of imiquimod caused less severe skin inflammation in all the mice.However,the treatment of HMGB1/IL-18 neutralizing antibodies still led to thinner mouse ears than control.Subsequent H&E staining and immunofluorescence assay showed that the thickness of lesional epidermis and the number of infiltrated CD3~+T cells in the mice were both significantly lower in the HMGB1/IL-18-blockade groups than the control groups.Our results demonstrate that HMGB1/IL-18 neutralizing antibodies could help the mice to recover from psoriasis-like dermatitis induced by imiquimod.6.IFN-?promotes the release of HMGB1 from keratinocytes via STAT1 signaling pathwayFirstly,both immunofluorescence and western blot showed the translocation of HMGB1 from nulceus to cytoplasm in keratinocytes under IFN-?stimulation.Moreover,IFN-?facilitated the secretion of HMGB1 from keratinocytes as shown by ELISA.After the transfection with STAT1 siRNA,the translocation of HMGB1 from nulceus to cytoplasm and the release of HMGB1 from keratinocytes induced by IFN-?were both significantly blocked.These results confirm that IFN-?leads to the release of HMGB1from keratinocytes by activating STAT1 signaling pathway.Conclusion:In psoriasis lesions,IFN-?facilitates the release of HMGB1 from keratinocytes,and then HMGB1 can bind to corresponding PRRs on the membrane of keratinocytes.On one hand,HMGB1 induces the expression of miR-17-92 cluster that promotes the proliferation of keartinocytes.On the other hand,HMGB1 promotes the expression and secretion of IL-18 in keratinocytes via activating NF-?B p65 signaling pathway and inflammasomes,which subsequently enhances Th17 immune response and promotes the development of psoriasis.Our study elucidates the regulation of HMGB1 on the biological behaviors of keratinocytes in psoriasis and provides new evidence for the theory about the contribution of keratinocytes to the formation of psoriatic inflammation.HMGB1 and downstream IL-18 are potential molecular targets for the therapy of psoriasis and are worth attention in the future drug discovery for psoriasis.