RIPK1 And The Novel Inhibitor NHWD-1062 Regulate The Outcome Of Psoriasis Through KCs

Objectives: Psoriasis is an immune-mediated,recurrent,chronic inflammatory disease,while Receptor-interacting serine /threonine-protein kinase 1(RIPK1),an important upstream kinase,is a major regulator of tumor necrosis factor receptor 1(TNFR1)signaling and regulates multiple signaling pathways involved in inflammation and cell death processes.At present,the clinical efficacy of the RIPK1 inhibitor is limited and the regulatory mechanism is unclear in the treatment of psoriasis.The aim of this study is to investigate the specific mechanism by which RIPK1 and its kinase inhibitor NHWD-1062 regulate inflammation in psoriasis lesions,providing a scientific basis for the clinical translation of NHWD-1062 for the treatment of psoriasis.Methods: Immunofluorescence,immunohistochemistry,GEO database analysis,gene knockdown or overexpression,transcriptome sequencing,animal experiments,q PCR,Western blot,dual luciferase reporter gene assay and other methods were used to conduct this research.Results:(1)RIPK1 was highly expressed in skin lesions of patients with psoriasis compared with healthy skin of patients without psoriasis;RIPK1was highly expressed in skin lesions compared with non-lesioned areas of patients with psoriasis.(2)RNA-seq results showed that there were 463 up-regulated and664 down-regulated genes in Ha Ca T cells after knockdown of RIPK1.Differential gene heat map results showed that psoriasis-related genes(S100 family protein,IL-1 family protein,CCL2,CCL22,and IVL)were down-regulated after knockdown of RIPK1.In vitro validation of RNA-seq results suggested that knockdown of RIPK1 could down-regulate the m RNA levels of S100A7,S100A8,S100A9,IL-1β and IVL in Ha Ca T cells,which was consistent with the RNA-seq results.(3)TNF-α and IL-17 A successfully induced psoriasis-like inflammation in KCs in vitro.Knockdown of RIPK1 down-regulates m RNA levels of psoriasis-associated pro-inflammatory mediators TNF-α,IL-1β,IL-6,S100A7,S100A8,S100A9,CCL2,CXCL2 and IL-8 induced by TNF-α and IL-17 A and is able to inhibit the proliferation of KCs.(4)NHWD-1062 did not affect the activity of KCs in vitro and effectively inhibited the phosphorylation of RIPK1 in KCs.NHWD-1062 inhibited the m RNA levels of psoriasis-associated pro-inflammatory cytokines and chemokines TNF-α,IL-1β,IL-6,CCL2,IL-8,IL-17 a and S100a8 in KCs in vitro and the proliferation of KCs induced by TNF-αand IL-17 A,and also found that the anti-inflammatory ability of NHWD-1062 in KCs was better than that of Nec-1s(a RIPK1 kinase inhibitor used in cell model studies).(5)The inhibition potency of NHWD-1062 on RIPK1 kinase in vivo was verified by a TNF-α-induced mouse SIRS model,and found that NHWD-1062 could effectively prevent TNF-α-induced loss of body temperature in mice,and its potency was better than that of GSK’772.In an IMQ-induced psoriasis-like mouse model,gavage of NHWD-1062 effectively ameliorated the inflammatory phenotype of psoriasis-like skin,downregulated the expression of RIPK1 and its phosphorylation in mouse skin lesions,and inhibited the overproliferation of mouse epidermis as well as the m RNA levels of psoriasis-related pro-inflammatory mediators(IL-1β、IL-17a、IL-22、S100a8、S100a9 and Cxcl2)in skin lesions.(6)RNA-seq analysis of RIPK1-regulated downstream pathways and KEGG pathway enrichment analysis revealed that these down-regulated genes were involved in NF-κB,TLRs and TNF signaling pathways.Among them,TLR1 in TLRs signaling pathway was most significantly down-regulated.(7)In vitro validation of RNA-seq results revealed that knockdown of RIPK1 in KCs or inhibition of RIPK1 using NHWD-1062 can inhibit the phosphorylation of P65 and the expression of TLR1 protein levels.Dual luciferase reporter gene assay showed that P65 can target and regulate the promoter region of TLR1 and enhance the expression of TLR1.(8)In vivo validation of RNA-seq results revealed that NHWD-1062 inhibited the phosphorylation of P65 and the protein level of TLR1 in IMQ-induced psoriatic lesions in mice.Analysis of the GEO database revealed that the m RNA levels of P65 and TLR1 were higher in psoriasis lesions than in non-lesion areas,and the m RNA levels of RIPK1,P65,and TLR1 in psoriasis lesions were positively correlated.(9)In vitro exploration of TLR1 regulation in KCs revealed that activation of TLR1 enhanced transcription of psoriasis-associated pro-inflammatory mediators in KCs and promoted proliferation of KCs at basal levels and at inflammatory levels induced by TNF-α and IL-17 A.Conclusion:(1)NHWD-1062,a small molecule RIPK1 kinase inhibitor developed by our team,can effectively inhibit the kinase activity of RPIK1 in KCs,and also has good kinase inhibitory potency in vivo of mice.(2)Inhibition of RIPK1 kinase activity by NHWD-1062 or knockdown of RIPK1 down-regulates the m RNA levels of psoriatic-associated pro-inflammatory cytokines and chemokines in KCs and inhibits the proliferation of KCs.(3)NHWD-1062 alleviates IMQ-induced psoriasis-like skin inflammation by inhibiting the m RNA levels of psoriasis-associated pro-inflammatory factors in mouse skin lesions.(4)RIPK1 and its kinase inhibitor NHWD-1062 can regulate psoriasis-like inflammation in KCs via the RIPK1/NF-κB/TLR1 axis.