The Mechanism By Which DDX5 Regulates The Splicing Of Interleukin-36 Receptor In Keratinocytes Aggravates Atopic Dermatitis

Keratinocytes,as a major cell type in skin epidermis,are key to maintain skin barrier intact and control the balance of inflammatory responses in skin.Defects of keratinocytes lead to numerous diseases,such as psoriasis and atopic dermatitis.Alternative splicing（AS）contributes to transcriptomic and proteomic diversity.It is known that the abrrent alternative splicing often leads to different diseases.However,whether the abbrent alternative splicingin keratinocytes would be associated with the pathogenesis of atopic dermatitis（AD）remains unknown.We have found that the RNA helicase DDX5 is a shared splicing factor between AD and psoriasis,and DDX5 defect aggravets psoriasis.Therefore,we hypothesised that DDX5 might be involed in the pathogenesis of AD.To determine the role of DDX5 in AD,we first detected the expression of DDX5in lesional skin of patients with AD and found that the m RNA and protein levels of DDX5 were reduced in the lesional skin of AD patients compared to that of normal patients.To further verify the expression of DDX5 in AD,we analyzed the expression of DDX5 in the ears and skin of AD mice induced by MC903 and found that DDX5 is also reduced in lesional skin of AD mice.Immunofluorescent staining showed that DDX5 was predominantly decreased in epidermal keratinocytes of AD mice.Moreover,DDX5 was reduced in lesional skin of mice with tape stripping,suggesting that the damage of skin barrier causes the reduction of DDX5.To confirm that decreased DDX5 in keratinocytes is related to the pathogenesis of AD,we generated mice with specific ablation of DDX5 in keratinocytes(hereafter referred to Ddx5^?/kc)and then developed AD models in these mice by MC903.Ddx5^?/kc mice were hihghly susceptible to MC903 and showed increased ear thickness and much severe inflammation.To screen the molecule that can inhibit the expression of DDX5 in AD,we used a panel of cytokines that are highly expressed in lesional skin of AD to stimulate primary human keratinocytes.Among these cytokines,only IL-17D inhibited the m RNA and protein expression of DDX5 in keratnocytes.We then further analyazed the expression of of DDX5 in lesinoal skin of WT and Il17d^–/–AD mice,and found that the deficiency of IL-17D restored DDX5 expression in lesional skin of AD was increased compared to WT AD mice.Next,we explore whether DDX5 can aggravate atopic dermatitis by affecting the alternative splicing of key genes.We tested the splicing of 6 receptors that regulate inflammatory responses in AD,and found that DDX5 was involved in the splicing of IL-36R and IL-7R pre-m RNA.The deficiency of DDX5 leads to increased IL-36R but decreased soluble IL-36R（s IL-36R）in keratinocytes.In contrast,DDX5 deficiency results in decreased IL-7R but increased soluble IL-7R（s IL-7R）.s IL-36R inhibited the expression of IL-36-induced cytokines and chemokines in keratinocytes.The intradermal administration of s IL-36R alleviated the pathologies of Ddx5^?/kc AD mice.Moreover,the coculture of DDX5^–/–keratinocytes and T cells showed that increased s IL-7R might promote the proliferation of T cells and then maintain their survival.All these data suggest that DDX5 defect leads to decreased s IL-36R but increased s IL-7R,both of which aggrevate the inflammation in AD.In summary,our resultsdemonstrate that IL-17D inhibits the expression of DDX5in AD,and decreased DDX5 leads to abnormal splicing of IL-36R and IL-7R pre-m RNA,which results in decreased s IL-36R but increased s IL-7R in keratinocytes.The decrease in s IL-36R amplifies the inflammatory response to IL-36 in keratinocytes and increases the expression of cytokines and chemokines,which recruit leukocytes into lesional skin of AD.Increased s IL-7R might therefore promote the proliferation of T cells and maintain their survival.Our data reveal that DDX5 regulates the splicing of IL-36R and IL-7R pre-m RNA to aggrevate AD,and provide new insights into the treatment of AD.