CX3CR1 Is Involved In Skin Inflammation By Regulating Ly6C^hi Monocyte Migration And Epidermis Langerhans Cells Reconstitution In Mice

ObjectiveClassic Langerhans cell（LC）is a professional antigen presenting cell in the skin,which plays an important regulatory role in skin inflammation.However,the function and regulation mechanism of Ly6C^hi monocyte-derived LC under inflammatory conditions is not very clear.In this study,we used Cx3cr1^GFP/GFPFP/GFP transgenic mouse and nested mouse model to investigate the regulation of CX3CR1 on Ly6C^hi monocyte-derived LC and its effect on inflammation during skin inflammation.This will provide a new target for the precise treatment of skin inflammation.Methods1.Prepare ACD models of C57BL/6 and Cx3cr1^GFP/GFP mice.From day 5,dynamically detect the degree of mouse ear swelling and draw the thickness variation curve of the ear;on the 9th day,make mouse ear slices freeze sections and HE staining.2.Flow cytometry was used to detect the difference in the number of immune cells（including classical LC and gdT cells）in the epidermis of C57BL/6 and Cx3cr1^GFP/GFPFP/GFP mouse ears at steady state.3.Flow cytometric analysis of Ly6C^hi mononuclear cells in peripheral blood,spleen and bone marrow of C57BL/6 and Cx3cr1^GFP/GFP mice under steady-state and ACD conditions.4.The ACD model of C57BL/6 and Cx3cr1^GFP/GFP mice was prepared by smearing DNFB.The number of short-term LCs in the epidermis of the two mouse ears was measured by flow cytometry on the 9th day.5.C57BL/6 and Cx3cr1^GFP/GFP mouse dermatitis models were prepared by UV irradiation.The number of short-term LCs in the epidermis of the two mouse ear epidermis were measured by flow cytometry at 1,2,and 3 weeks,respectively.6.The e450-labeled C57BL/6 mouse bone marrow Ly6C^hi monocytes and CFSE-labeled Cx3cr1^GFP/GFP mouse bone marrow Ly6C^hii monocytes were intravenously injected at a ratio of 1:1 for UV challenge（wavelength:254 nm,distance 38 cm,Duration:30 minutes）of CD45.1 mice.Four days later,flow cytometry was performed to examine the origin and differences of Ly6C^hii monocytes in the epidermis of CD45.1 mouse ears.7.Flow cytometry was used to detect the expression of inflammatory factors iNOS and TNFa in the LC subgroups（long-term LC and short-term LC）of the epidermis of C57BL/6 mice.Flow cytometry was used to classify the LC subpopulations in the epidermis of C57BL/6 mouse ear slices and further detect the expression of IL-1βand other cytokines in LC subpopulations by RT-PCR.Results1.In the ACD disease model,ear swelling and inflammatory cell infiltration were significantly lighter in Cx3cr1^GFP/GFP mice compared to C57BL/6 mice.2.At steady state,there was no difference in the number of immune cells（including classical LC and gdT cells）in the epidermis of Cx3cr1^GFP/GFP mouse ear compared to C57BL/6 mice.3.In steady state,compared with C57BL/6 mice,the number of Ly6C^hi mononuclear cells in peripheral blood,spleen and bone marrow of Cx3cr1^GFP/GFP mice did not change significantly,and the number of Ly6C^lo monocytes decreased（consistent with reports in the literature）.However,in the DNFB-induced ACD model,the number of Ly6C^hii mononuclear cells in the peripheral blood and spleen of Cx3cr1^GFP/GFP mice was significantly increased,and the number of Ly6C^hi monocytes in the bone marrow did not change significantly.4.In the DNFB-induced ACD model,the number of short-term LCs in the epidermis of Cx3cr1^GFP/GFP mouse ears was significantly reduced compared to C57BL/6 mice.5.In the UV irradiation-induced dermatitis model,the number of short-term LCs in the epidermis of the Cx3cr1^GFP/GFP mouse ear was significantly reduced compared to C57BL/6 mice.6.The number of Ly6C^hi mononuclear cells derived from Cx3cr1^GFP/GFP mouse bone marrow was significantly reduced in the epidermis of CD45.1 mouse ears compared with Ly6C^hi monocytes derived from C57BL/6 mouse bone marrow.7.Flow cytometry and qRT-PCR analysis showed that short-term LC highly expressed iNOS,TNFa,IL-1βand other inflammatory factors compared with long-term LC.ConclusionWhether in the DNFB-coated or UV-irradiated mouse dermatitis model,the absence of CX3CR1 delayed the migration of Ly6C^hi mononuclear cells to the site of inflammation and differentiated into short-term LC with pro-inflammatory effects,thereby inhibiting the inflammatory response of the skin.