The Effect Of IL-19 On Keratinocyte Function In Atopic Dermatitis

Research background and purposeAtopic dermatitis(AD)is a common chronic recurrent inflammatory skin disease,with T helper 2(Th2)-based immune dysregulation and abnormal epidermal differentiation being two key factors in its pathogenesis.AD has several clinical phenotypes and endotypes.Currently,topical corticosteroids and calcineurin inhibitors are still the first-line treatment for acute flares and remission maintenance in AD,but side effects and recurrence are still two big challenges for patients and dermatologists.The advent of biological agents such as dupilumab bears the promise for future treatments of AD.However,the pathogenesis of AD is very complex,and different patients show different efficacy for the same treatment.Thus,targeted therapy based on endotypes was put forward.At the same time,a lot of related basic and clinical studies have been implemented.To further explore the heterogeneity of AD,our team performed a single-cell sequencing study in AD patients,and found that IL-19 was significantly increased in lesions of patients with chronic severe AD compared to healthy controls(unpublished data).IL-19,a member of the IL-10 cytokine family,can be produced by keratinocytes,endothelial cells,epithelial cells and monocytes,and has an immune regulatory effect on the development of various inflammatory skin diseases,neoplastic diseases and systemic inflammatory diseases.Although many studies have reported the correlation between elevated IL-19 levels in skin lesions and serum of AD patients and the severity of disease,the role of IL-19 in the pathogenesis of AD has been rarely studied.IL-19 exerts its biological effects mainly through binding to the IL-20RA/RB receptor complex.Previous studies have demonstrated that IL-20RA/RB is mainly expressed in keratinocytes among epidermal cells.Therefore,exploring the effect of IL-19 on keratinocytes is important for further understanding the pathogenesis of AD.We first performed gene ontology(GO)analysis of IL-19-related differentially expressed genes(DEGs)in AD patients.The results showed that IL-19-related DEGs were associated with keratinocytes’ differentiation and keratinization.And the data of gene set enrichment analysis(GSEA)found that IL-19-related genes were mainly enriched in the tyrosine kinase/signal transduction and transcriptional activation(JAK-STAT)pathway.Based on above,we first measured the level of differentiation markers and epithelium-derived cytokines in keratinocytes treated with IL-19 alone or IL-19 together with IL-4/IL-13.Afterwards,we further explored underlying molecular mechanism by measuring the phosphorylation of STAT3 and STAT6 in keratinocytes under the same stimulation conditions.In addition,considering external allergens are indispensable to the pathogenesis of AD,we investigated whether external stimuli,like HDM and SEB,could induce the production of IL-19 in keratinocyte.In conclusion,the purpose of this study is to explore the possible mechanism of IL-19 in the pathogenesis of AD,so as to further broaden our understanding of AD pathophysiology and provide prospects for therapy based on endotype.Research method1.AD datasets GSE32924 and GSE36842 from the GEO database were downloaded,and AD patients were divided into high and low IL-19 m RNA groups.Differentially expressed genes(DEGs)in these two groups were identified and subjected to Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)Analysis and Gene Set Enrichment Analysis(GSEA).Meanwhile,receiver operating characteristic curve(ROC)and protein-protein interaction(PPI)network were plotted in AD dataset.2.FLG,LOR,keratin-10,TSLP,IL-33,IL-25,IL-19 and the phosphorylated STAT3 and STAT6 were measured in Ha Ca T cells by RT-PCR and/or western blot before and after stimulated with IL-4/IL-13 with or without different concentrations of IL-19.The IL-19 level in Ha Ca T cells after stimulation with house dust mite(HDM)or staphylococcal enterotoxin type B(SEB)were also examined.Results1.By biological information analysis of genes in IL-19 high and low expression groups of AD patients,IL-19-related differentially expressed genes(DEGs)were identified,including positive genes(SERPINB4,LCE3 D,SERPINB3,SPRR2 B,S100A9,SPRR1 B,SPRR1A,CDSN,MMP12,SPRR2G)and negative genes(KRT35,CYP4F8,PM20D1,KRT85,RPS4Y1,ANKRD36 B,KDM5D,LOC101926960,KRT79,USP9Y).2,GO enrichment analysis revealed that of IL-19-related DEGs are closely related to keratinization and differentiation of keratinocytes.GSEA enrichment analysis showed that of IL-19-related DEGs were mainly enriched in tyrosine kinase/signal transduction and transcriptional activation(JAK-STAT),hedgehog pathway,and the TP53 pathway.3.By in vitro cellular experiments,we found that IL-19 alone reduced the production of keratin-10 and LOR,while increased the level of TSLP,IL-20RA/RB in Ha Ca T cells.When IL-19 and IL-4/IL-13 acted together,they significantly downregulated the level of epidermal differentiation proteins(FLG,LOR and keratin-10)and upregulated the production of epithelial-derived inflammatory cytokines(TSLP,IL-33 and IL-25).4.We also detected increased phosphorylated STAT3/6 in Ha Ca T cells under the stimulation of IL-19 with IL-4/IL-13 by Western Blotting.5.External allergen house dust mite(HDM)induced IL-19 production in Ha Ca T cells,while Staphylococcus aureus enterotoxin B(SEB)did not show this effect.Conclusion1.IL-19-related DEGs may be involved in keratinization and differentiation of keratinocytes through JAK-STAT,hedgehog pathway,TP53 pathway.2.IL-19 alone does not affect the proliferation and apoptosis of keratinocytes.But it has a partly effect on the differentiation and cytokine-secreting function of keratinocytes.It might get involved in the pathogenesis and progression of AD by enhancing the effect of IL-4/IL-13 on keratinocytes.