The Role Of Keratinocytes Necroptosis For Inflammation Development In Contact Hypersensitivity

Background and objective:Allergic contact dermatitis is one of the most common contact hypersensitivity reactions.The typical histological features of contact hypersensitivity are epidermal necrosis,intercellular edema,dermal vasodilatation and inflammatory cells infiltration.It was previously thought that activated T lymphocytes and inflammatory cytokines and chemokines played an important role in the contact hypersensitivity reaction,while keratinocytes were the end-stage "target cells" of the inflammatory response.However,in recent years,researchers have realized that keratinocytes may play a more "active" role in the inflammatory response of the skin.Whether cell death of keratinocytes is involved in the regulation of the inflammatory response in contact hypersensitivity has not been illustrated.Therefore,this study aims to explore the specific role of keratinocytes in the inflammation of contact hypersensitivity and the mechanisms involved in regulating inflammation in contact hypersensitivity,and to further explore potential therapeutic strategies.Methods:1.CHS mice model was constructed by topically applying dinitrochlorobenzene(1chloro-2,4-dini-trochlorobenzene,DNCB)to skin of mice ears,and the model was evaluated by appearance scoring,mice ears thickness measurement and weighing,and histochemical staining of paraffin sections.2.The cell death level of epidermal keratinocytes of mice ears was evaluated by TUNEL staining.3.The phosphorylation levels of RIPK1,RIPK3 and MLKL(key regulating molecules of necroptosis)were detected by western blotting assay after protein extraction from mice ears,and the levels of intracellular MLKL phosphorylation in epidermal keratinocytes were further detected by immunofluorescence staining.4.Breeding Mlkl keratinocyte conditional knockout mice to explore the effect of deletion of MLKL-mediated necroptosis in keratinocytes on contact hypersensitivity inflammation.The level of inflammatory cell infiltration was evaluated by immunohistochemical staining for CD4,CD8,and MPO.5.Systemic administration of RIPK1 inhibitors and formulation of MLKL inhibitors into topical gels for the treatment of DNCB-induced contact hypersensitivity inflammation to evaluate the potential of targeting keratinocytes necroptosis to develop novel therapeutic agents.6.To observe whether glucocorticoids(dexamethasone cream),a classical drug used clinically for the treatment of contact dermatitis,exert therapeutic effects by inhibiting the necroptosis pathway.7.Further,we simulated the environment of keratinocytes under contact hypersensitivity inflammation by TNF-α and IFN-γ in an in vitro cultured human keratinocyte cell line HaCaT,and explored the concrete effect of dexamethasone on keratinocytes necroptosis through the in viro cell model.The death level of in vitro HaCaT cell was evaluated by Trypan blue staining and PI staining.And the phosphorylation levels of RIPK1,RIPK3 and MLKL were observed by western blotting.8.HaCaT cells with MLKL stable knockdown were constructed by lentiviral transfection,and the protein levels of High mobility group box 1(HMGB1)in nucleus,cell plasma and supernatant were detected by western blotting in the MLKL knockdown HaCaT cells in the presence or absence of TNF-α and IFN-γ,respectively.9.Recombinant HMGB1 protein was constructed and injected intracutaneously into the ear skin tissue of Mlkl keratinocyte conditional knockout mice to observe whether it could recapitulate DNCB-induced contact hypersensitivity inflammatory response.Results:1.The contact hypersensitivity(CHS)mice model was successfully constructed by applying 1%DNCB to the ear skin for initial sensitization and 0.5%DNCB to the same location for motivation 1 week later.Erythema,edema and erosion were observed on mice ear,and the thickness and the unit weight were significantly increased.H&E staining demonstrated epidermal necrosis,intercellular edema and infiltration of inflammatory cells.2.The phosphorylation levels of RIPK1,RIPK3 and MLKL,key molecules of necroptosis,were significantly increased in the mice model of contact hypersensitivity,and the necroptosis pathway was activated,and the level of cell death of keratinocytes in epidermis was confirmed by TUNEL staining.3.We did not observe similar dermatitis skin appearances in the ears of Mlkl keratinocyte conditional knockout mice.The inflammation response to DNCB-induced contact hypersensitivity,and the cell death level of epidermal keratinocytes in the ears of Mlkl keratinocyte conditional knockout mice were significantly reduced.CD4+and CD8+T lymphocyte infiltration in the dermis did not differ from controls,but the level of MPO,MMP-9 and NE were significantly reduced.4.Both intraperitoneal injection of RIPK1 inhibitor(Necrostatin-1)and topical application of MLKL inhibitor(TC13172)could both alleviate DNCB-induced contact hypersensitivity inflammation.5.Dexamethasone cream(0.075%)significantly inhibited necroptosis pathways while treating contact hypersensitivity effectively.6.In the in vitro cellular model of TNF-α and IFN-y-induced contact hypersensitivity,dexamethasone reduced the positive staining for dead cells by Typan blue and PI,and also decreased the phosphorylation levels of RIPK1,RIPK3 and MLKL.7.HMGB1 was decreased in cell plasma while increased in the supernatant of HaCaT cells after TNF-α and IFN-γ stimulation,indicating that increased secretion of HMGB1 from intracellular to extracellular.In contrast,both intracytoplasmic and released HMGB1 were significantly reduced in HaCaT cells with a stable knockdown of MLKL.8.DNCB-induced contact hypersensitivity inflammatory response reappears after intradermal injection of recombinant murine-derived HMGB1 protein into mouse ear skin tissue in Mlkl keratinocytes conditional knockout mice.Conclusions:In the inflammatory environment of contact hypersensitivity,the necroptosis pathway in keratinocytes was activated and the release of MLKL-mediated proinflammatory factor HMGB1 was increased,which amplified the inflammatory signal of contact hypersensitivity and further aggravated skin tissue damage.Inhibiting the activation of necroptosis pathway in mouse keratinocytes through various means was able to alleviate the inflammation and tissue damage in contact hypersensitivity reactions.In addition,dexamethasone,a classical topical drug for the clinical treatment of allergic contact dermatitis,can also reduce contact hypersensitivity inflammation by inhibiting keratinocytes necroptosis.