The Effects And Mechanism Of SPRY1 On The Innate Immunity System In Psoriasis

Background:Psoriasis is a polygenic disease with a psoriasis phenotype in susceptible individuals under a variety of stimuli such as trauma,infections,or drugs.The characteristic skin lesions are characterized by sharply demarcated,erythematous plaques covered by white scales,and the histopathology shows that hyperkeratosis,parakeratosis,acanthosis,dermal dilation,and perivascular infiltration of lymphocytes.Patients with psoriasis are often accompanied by co-morbidities such as hypertension,diabetes,coronary heart disease,obesity,and metabolic syndrome.Psoriasis has a long duration and recurrent attacks.It is a complex skin disease that seriously affects the physical and mental health of patients.Therefore,the study of the pathogenesis of psoriasis cannot be delayed.The dysfunction of immune system plays an important role not only in the pathogenesis of psoriasis,but it is also an important basis for the treatment of psoriasis in recent years.Among them,centered on the IL-23/T17 axis,from the innate immunity to the adaptive immune and inflammatory signal loop is the main direction of research.And its associated protein molecules have always been concerned.The SPRY1 protein belongs to the SPRY protein family and is a cysteine-rich protein with multiple structural functional domains,including a C-terminal cysteine-rich domain,a Rafl binding domain,and a highly conserved N-terminal functional domain.Previous studies have identified SPRY 1 negative feedback regulation of the RTK signaling pathway.SPRY1 protein family involved in a variety of diseases and physiological processes.Our previous study found that overexpression of SPRY1 inhibits proliferation,promotes apoptosis,and affects differentiation in keratinocytes.Furthermore,SPRY1 is differently expressed in psoriasis patients and normal individuals.However,the specific role of SPRY1 in the pathogenesis of psoriasis,whether it participates in the pathogenesis of psoriasis innate immunity and its possible effects,have not been studied in depth.Therefore,in this dissertation,we will discuss in depth the effects of SPRY1 on the pathogenesis of psoriasis,especially innate immunity.Objective:Clarify the expression differences of SPRY1 in psoriasis and normal human skin and keratinocytes;Define the correlation between SPRY1 and skin barrier function,innate immune-related genes,psoriasis-related inflammatory factors,and keratinocyte biological functions;Elucidate the role of SPRY1 in skin barriers,changes in antimicrobial peptides,and innate immunity through the K14-SPRY1 mouse model.Through the conditional overexpression of SPRY1 in K14-SPRY1 transgenic mice,to detect a new mechanism of trauma and infection induced psoriasis,and on this basis,new targets for the treatment of psoriasis will be further discovered.Methods:The localization and expression of SPRY1 in normal human skin and keratinocytes were identified by immunohistochemistry and immunofluorescence;Normal human keratinocytes were isolated and cultured,and the correlation between SPRY1 and cell differentiation was detected by WB and qRT-PCR;Immunohistochemistry and Immunofluorescence was used to detect the expression and localization of SPRY1 in lesions of psoriasis,eczema and nodular prurigo.RNAseq,qTR-PCR,and WB methods were used to identify SPRY1 in keratinocytes and normal human keratinocytes in the lesion area of psoriasis patients.LPS,Poly I:C,Hydrogen Peroxide and Inflammatory Factors(IL-6,IL-17A,IL-23,TNFa)and Psoriasis Therapies(Clobetasol,MTX,Calcipotriol,Acitretin)were co-incubated with cultured normal and lesional keratinocytes.WB and qRT-PCR methods were used to compare the effect of the above factors on the expression of SPRY1 in the corresponding keratinocytes;Detection and localization of SPRY1 with antibacterial peptide LL37 were identified in in normal and psoritatic lesions and peri-lesional keratinocytes by fluorescence detection;Expression of SPRY 1 were detected by WB and qRT-PCR after normal human keratinocytes treated by recombinant human LL37.Proteins that might interact with SPRY1 were detected by co-immunoprecipitation and mass spectrometry analysis.The epidermal barrier,differentiation,proliferation,and apoptosis related proteins and genes were detected by WB and qRT-PCR in the epidermis of WT mice and SPRY1 transgenic mice.Construction of mouse wound healing model,comparison of wound healing process between SPRY1 transgenic mice and WT mice by Masson's trichrome staining,immunohistochemistry and WB.Construction of mouse psoriatic inflammation with IMQ,then compares epidermal thickness and vascularization by HE staining of the lesion area,immunofluorescence and qRT-PCR were used to compare the expression differences of SPRY1;Using qRT-PCR and ELISA methods to compare inflammatory factors,epidermal barrier,innate immunity and other related genes and proteins.Using flow cytometry to detect changes inflammatory cells of in the dermis,skin-draining lymph nodes,and the peripheral blood after modeling.Results:1.SPRY1 is continuously expressed in the granular layer of skin and participates in the late differentiation of keratinocytes.2.The expression of SPRY1 was decreased or even lost in the lesion area of psoriasis patients,and the expression of SPRY1 in the lesion area recovered after drug treatment.3.LPS,Poly I:C,H2O2 and psoriasis-related inflammatory factors inhibit the expression of SPRY1 in normal human keratinocytes;clobetasol,MTX,calcipotriol and acitretin can rescue SPRY1 expression in psoriatic lesional keratinocyte;4.Antibacterial peptide LL37 inhibits SPRY 1 expression in normal human keratinocytes;5.Overexpression of SPRY1 can promote the apoptosis of epidermal keratinocytes,inhibit epidermal proliferation and affect differentiation in the skin of C57BL/6 mice.6.Overexpression of SPRY1 delayed the wound healing of C57BL/6 mice by7.affectting the maturation and mechanization of granulation tissue,and inhibited the crawling of hyperplastic epidermis;8.Overexpression of SPRY1 reduced the phenotype of psoriasis-like dermatitis in IMQ-induced C57BL/6 mice model and affected the expression of epidermal barrier and innate immune-related genes.In addition,it also inhibited the produce of IL-17A+y8T cells and moDCs in mice dermis.Conclusion:Lesional skin and isolated cultured keratinocytes of psoriasis patients lack expression of SPRY 1.SPRY1 inhibits keratinocytes' proliferation,promotes apoptosis,and affects late differentiation.The expression of SPYR1 reduced in normal human keratinocytes after infection,injury,or inflammation.SPRY1 expression was restored in skin and keratinocytes of psoriatic lesions after drug treatment.Overexpression of SPRY1 in epidermis affected the wound healing process of skin in mice.Overexpression of SPRY1 in epidermis releived IMQ-induced mice psoriasis-like dermatitis.