The Regulatory Role Of LCN2 On Keratinocytes In The Pathogenesis Of Psoriasis

Background:Psoriasis is a chronic autoimmune and inflammatory skin disease,which is divided into different subtypes including psoriasis vulgaris,pustular psoriasis,erythroderma psoriasis and arthritis psoriasis.The psoriasis vulgaris is characterized by erythematous patches or plaques,covered with silvery white scales,and often recurrent.The main pathological manifestations were significant hyperkeratosis and dyskeratosis,with epidermal hyperplasia and perivascular lymphocyte infiltration in both epidermis and dermis.Recent studies have found that aberrant activation of immunity dominated by helper Th17 cells plays a pivotal role in the pathogenesis of psoriasis.However,keratinocytes(KCs),the main target cells of psoriasis,may be the key factor that promotes the disorder of local immunity and prolongs the chronic inflammatory state of psoriasis.Lipocalin2(LCN2),also known as 24p3,neutrophil gelatinase associated lipoprotein(NGAL),is a small molecule secretory glycoprotein of lipocalin superfamily.As a lipocalin,LCN2 regulates lipid homeostasis,and participates in various metabolic diseases such as obesity,diabetes,cardiovascular disease and nonalcoholic fatty liver disease.In addition,LCN2 was highly expressed in a variety of autoimmune diseases.Several studies have highlighted a potential role for LCN2 in regulating innate immunity and inflammation.LCN2 potentiates macrophages,neutrophils and epithelial cells to secrete cytokines and chemokines,which may be one of the underlying mechanisms in the pathogenesis of metabolic and autoimmune diseases.However,the pro-inflammatory role of LCN2 and relevant metabolic function still remain obscure.Our previous studies have shown that LCN2 is an important component of neutrophil extracellular trap,but promotes the chemotaxis and activation of neutrophils in return.The above process results in the formation of neutrophil micro abscess in psoriatic lesions,contributing to the pathogenesis and development of psoriasis.Highthroughput sequencing data revealed that LCN2 levels in the serum and skin lesions of psoriasis patients were significantly increased.Several reports have already documented that psoriatic skins exhibited abnormal lipid composition and altered lipid metabolic processes during psoriasis pathogenesis.According to these studies,we hypothesize that LCN2 activates KCs through regulating intracellular lipid metabolism,thus,promoting the release of a myriad of inflammatory mediators,which contribute to the initiation and development of psoriasis.Objectives:1.To determine the expression of LCN2 receptors on psoriasis skins and their role in the pathogenesis of psoriasis;2.To explore the effects of LCN2 on keratinocytes and the underlying mechanisms;3.To explore the potential of targeting LCN2 or downstream signals for psoriasis treatment.Methods:1.Analysis of the expression pattern of LCN2 receptors on the epidermis of psoriasis patients and normal controls,and their role in IMQ-induced psoriasis-like dermatitis:The skin samples of patients with psoriasis vulgaris and healthy controls were collected,and the epidermis was isolated.The expression of LCN2 receptors in the epidermis was determined by real-time quantitative PCR(qRT-PCR),Western Blot and immunofluorescence.The psoriasis-like mouse model was established using imiquimod(IMQ),and then treated with siRNA targeting the LCN2 receptor.The ear thickness was measured every day,and the ear tissues were collected for H&E staining,qRT-PCR,flow cytometry and immunofluorescence analysis to evaluate the effect of siRNA treatment.2.Analysis of the impact of LCN2 on keratinocyte activation:Immortalized human keratinocytes(HaCaT cells)were stimulated with different concentrations of LCN2 to choose optimal concentration for stimulation.HaCaT cells were stimulated with LCN2 and collected for RNA-sequencing(RNA-seq)to assess differentially expressed genes(DEGs)between the two groups,and DEGs enriched pathways were further analyzed.In addition,the DEGs that induced by LCN2 were verified using human primary keratinocytes.3.Analysis of the underlying mechanism of LCN2 on keratinocyte activation:Total cholesterol content of KCs was detected by Amplex Red cholesterol detection kit;Atorvastatin was used for cholesterol depletion,and the effects of atorvastatin on LCN2-mediated inflammatory response in KCs was determined by qRT-PCR;The underlying mechanism of how LCN2 activates KCs was initially explored by qRT-PCR,ELISA and Western Blot,and the inhibitors and siRNA were further used for pathway confirmation.The the key gene that mediate keratinocyte activation downstream of LCN2 was indicated and overexpressed by plasmid in KCs.Then the RNA-seq was used to detect genes induced by the plasmid,and the DEGs were further analyzed for pathway analysis and compared with that induced by LCN2 stimulation.The RNA-seq results were verified by qRT-PCR.Chromatin immunoprecipitation(ChIP)and Luciferase Report assay were used to verify the transcriptional activity of the targeted gene.4.Exploration of targeting indicated gene for psoriasis treatment:IMQ-induced psoriasis-like mouse model was established.The epidermis was topically treated with inhibitor or modified siRNA of the targeted gene downstream of LCN2.Then the severity of ear skin inflammation of different groups was evaluated by ear thickness measurement,tissue H&E staining,immunofluorescence,and qRT-PCR analysis.Results:1.LCN2 receptor 24p3R was highly expressed in psoriatic lesional skins:QRTPCR,immunofluorescence and Western Blot results showed that 24p3R was highly expressed in the skin lesions of patients with psoriasis compared with that of normal control,while there was no significant difference in the expression of Megalin and Melanocortin 4 receptor(MC4R)between the two groups.2.Silencing 24p3R alleviated IMQ-induced psoriasis-like inflammation in mice:Mouse experiments showed that 24p3R depletion in epidermis reduced the ear thickness and inflammatory cell infiltration of mouse ears,as well as epidermal inflammatory mediator production induced by IMQ.3.LCN2 induced keratinocyte activation:RNA-seq results showed that LCN2 increased the expression of cytokines(Interleukin(IL)-1β,IL-23)and chemokines(CX-C motifligand(CXCL)1,CXCL10,C-C motif ligand(CCL)20)that are crucial for psoriasis development in KCs,and up-regulated genes in cholesterol synthesis pathway.In addition,we obtained consistent results in LCN2-stimulated human primary keratinocytes.4.LCN2 activates SREBP2-NLRC4 to promote keratinocyte activation in psoriasis:The results of Amplex Red cholesterol analysis showed that LCN2 slightly up-regulated the intracellular cholesterol level of KCs.However,cholesterol depletion with atorvastatin had no significant effect on LCN2-mediated keratinocyte inflammation.Among the up-regulated genes in cholesterol synthesis pathway,SREBP2 is involved in pro-inflammatory process.SREBP2 inhibition significantly inhibited keratinocyte inflammation caused by LCN2,as shown by the down-regulated expression of IL-1β,IL-23,CXCL1,CXCL10,CCL20.In addition,Western Blot,qRTPCR and ELISA results showed that LCN2-induced IL-1β expression was dependent on the activation of NLRC4 inflammasome,which was significantly restricted by SREBP2 suppression.In addition,mechanism analysis showed that LCN2-induced SREBP2 activation was via mTOR-S6K1 pathway.5.SREBP2 was the crutial transcriptional factor mediating LCN2-induced keratinocyte activation:RNA-seq and qRT-PCR results demonstrated that the NSREBP2 overexpression significantly elevated the expression of genes associated with inflammation and cholesterol synthesis,which was consistent with the results of LCN2 stimulation.Western Blot results further showed that overexpression of N-SREBP2 activated the NLRC4-IL-1β axis.SREBP2 also showed transcriptional activity determined by ChIP and Luciferase Report assay.The results showed that SREBP2 could directly bind to NLRC4 promoter region,leading to NLRC4 transcription and activation.6.Silencing SREBP2 attenuated IMQ-induced psoriasis-like inflammation in mice:Topical treatment with si-SREBP2 or betulin significantly reduced the erythema,scaling,ear thickness,infiltrated inflammatory cells as well as inflammatory mediator expression in IMQ-induced psoriasis-like mice.Conclusion:In this study,we explored the effects of LCN2 on KCs and the underlying mechanism in the pathogenesis of psoriasis.We found 24p3R,the LCN2 receptor,was highly expressed in the epidermis of psoriasis skin lesions.Both in vivo and in vitro experiments showed that LCN2 interacted with 24p3R to induce keratinocyte activation by promoting cytokine and chemokine production,contributing to immunity disorder in psoriasis.In addition,LCN2 activated genes in cholesterol synthesis pathway,among which SREBP2 was found to mediate LCN2-induced keratinocyte activation.Furthermore,we found that NLRC4 inflammasome was the downstream responser of LCN2-SREBP2 axis,whose activation was regulated by SREBP2 at transcriptional level.In conclusion,on the basis of previous researches,our study unveils the essential role of LCN2 in lipid metabolic-inflammatory crosstalk of KCs in the pathogenesis of psoriasis,and provides laboratory basis for targeted strategies for psoriasis treatment.