The Possible Mechanisms That Neutrophil Extracellular Traps Modulate Psoriasiform Inflammation And Intervention Mechanisms Of Tetrahydroxy Stilbene

Objective: Psoriasis is a complex autoimmune skin disease that resident skin keratinocytes interact with immune cells with arising an inflammatory loop.During the pathogenesis of psoriasis,neutrophils are recruited and release neutrophil extracellular traps in psoriatic lesion.IL-36 cytokines,especially IL-36γ,are pivotal drivers of psoriasis.Tetrahydroxy stilbene glucoside(2,3,5,4-tetrahydroxy stilbene-2-O-β-Dglucoside,2354Glu)may recruit NETs to attenuate psoriasis whereas without further verification.Thus,we aimed to explore the putative role of NETs in ex vivo model which was characterized by excessive expression of IL-36γ and its possible regulatory mechanisms;we aimed to investigate how 2354 Glu manifested in psoriasis when partial NETs were inhibited by Cl-amidine;we intended to make a thorough inquiry in how mice in response to different application time of imiquimod.Methods: 1.Establishment of psoriasis-like ex vivo model.After anesthetizing neonatal mice less than three days old by cold,removed constant size of skin tissues were cultured in vitro.One hour before application of Poly(I:C)(25 μg/m L),ex vivo tissues were pretreated with Cl-amidine(200 μM),GSK 484(10 μM)or DNase I(5 μg/m L).24 hours later,ATP(3 m M)was administrated to ex vivo tissues.Then,supernatant and skin tissues were harvested and detected the production of NETs and inflammatory cytokines such as IL-36γ,IL-36α,IL-36β,HMGB1 and others.2.Establish model of murine response to different application time of imiquimod.C57BL/6 mice about 8～10 weeks old were divided into 5 groups randomly with 0,2,4,6,8 days of application time of 62.5 mg 5% imiquimod.Then,skin tissues,serum,livers and spleens were all collected.Keratin 1,keratin 14,keratin 17 and PCNA were observed by immunohistochemical staining while expressions of IL-36γ,HMGB1 were detected by Western blotting,RT-q PCR,immunofluorescence staining and others.Myeloperoxidase(MPO)-DNA complex in serum was tested through ELISA.3.The model of 2354 Glu intervening in psoriasis under NETs exhaustion was established by C57BL/6 mice about 8～10 weeks old.Mice were randomly divided into6 groups,which were normal group,IMQ plus 2354 Glu 100 mg/kg group,IMQ plus2354 Glu 25 mg/kg group,IMQ plus 2354 Glu 25 mg/kg plus Cl-amidine 10 mg/kg group and IMQ plus Cl-amidine 10 mg/kg group.2354Glu(100 mg/kg or 25 mg/kg)was applied intraperitoneally and 10 mg/kg Cl-amidine was administrated subcutaneously at 9:00 am for eight consecutive days.Since the fifth day,62.5 mg 5%imiquimod was applied on the murine dorsal skin for four consecutive days.Skin and serum were collected on the ninth day.ELISA was utilized to observe the expressions of IL-36γ,MPO-DNA in serum.Keratin 1,keratin 14 and PCNA in psoriatic lesions were labeled through immunohistochemical staining.The levels of inflammatory cytokines such as IL-36γ,IL-36β were detected by Western blotting,RT-q PCR,immunofluorescence staining and others.Results: 1.Excessive IL-36γ and NETs were generated in Poly(I:C)plus ATP induced ex vivo tissues.The production of NETs was inhibited by Cl-amidine,GSK 484 and DNase I,to vary degrees.Meanwhile,inflammatory factors such as HMGB1 and IL-36γ and infiltration of MPO and F4/80 in psoriatic lesions were both reduced after treated with Cl-amidine,GSK 484 and DNase I.2.The phenomenon of erythema,scales and epidermal thickening increased gradually under imiquimod application.Inflammatory cytokines such as IL-36γ,IL-36β,IL-36α and HMGB1 were accumulated in psoriatic skin in the process of imiquimod application.Neutrophils and macrophages were activated by imiquimod and migrated progressively to epidermis with the formation of Munro’s abscesses.Keratinocytes were undergoing hyper-proliferation and abnormal differentiation which were caused by imiquimod.3.2354 Glu ameliorated erythema,scales and epidermal thickening induced by imiquimod.2354 Glu had a dose dependent inhibition on the secretion of IL-36 cytokines.2354Glu improved expressions of keratin 1(the marker of parakeratosis),keratin 14(the marker of hyperkeratosis)and PCNA(the marker of proliferation)induced by imiquimod.However,exhaustion of NETs deteriorated psoriasiform symptoms,upregulated the inflammatory cytokines such as IL-36 and TNF-α.Besides,exhaustion of NETs exacerbated abnormal differentiation and hyper-proliferation of keratinocytes.Conclusion: Serving as a kind of ds RNA,Poly(I:C)combined ATP induced the secretion of IL-36γ and NETs in ex vivo model;NETs regulated the expressions of inflammatory cytokines such as IL-36γ,IL-1β and HMGB1;Inhibitory NETs decreased the infiltration of immune cells caused by Poly(I:C)plus ATP in ex vivo skin,which may be related with the regulatory effects on inflammatory cytokines of NETs;Imiquimod stimulated the abnormal differentiation and proliferation of keratinocytes;Neutrophils and macrophages were activated and accumulated in psoriatic skin;Induction of imiquimod on inflammatory cytokines such as IL-36γ and HMGB1 lasted only 7 days,which limited its application;Protective effect on psoriasis of 25 mg/kg2354 Glu relied on NETs,whereas NETs had beneficial and positive function during this process.