Effects Of Exogenous Electronic Field On Migration And Immunomodulatory Functions Of Skin Dendritic Cells In Mice

Objective: As the body’s first line of defense against external pathogenic microorganisms or physical and chemical stimuli,the skin plays an important role in exerting immune function and maintaining immune homeostasis.As the strongest antigen-presenting cells,dendritic cells(DC)include Langerhans cells of the epidermis and dendritic cells of the dermis,and their functions mainly include antigen uptake,activation and maturation,migration to local draining lymph nodes,antigen-presenting and initiation of T cell immunity,among which the process of targeting DC cell migration can affect the occurrence and response of immune response.The exogenous electric field is different from the weak and ununified directionality of the human physiological electric field,and its galvanotaxis on cells is more directional,and whether the exogenous electric field has the same electrotaxaxis effect on the migration of skin DC needs to be explored.At the same time,as an immune cell,under the treatment of an exogenous electric field,whether its immune-related physiological functions will also undergo certain change,and whether it can regulate the immune response is the purpose of our research on this topic.Methods:Part Ⅰ: 1.Extrtreatment and induction of differentiation of BMDC cells.BMDC was extracted by mouse bone marrow cells,and GM-CSF was added to the medium to induce differentiation.It could be used for experiments after induction of 7-10 days.2.Configuration and application of exogenous electric field treatment experimental device.3.Cell directional migration trajectory drawing and parameter analysis under the treatment of electric field.The cell device was placed on the inverted microscope and the cell migration under the treatment of the electric field was observed in real time.The voltage difference of the electric field was 1V/cm for 2h.The obtained continuous photographed pictures were imported into Fiji software for trajectory map depiction,which could draw a dot and line map of cell migration and at the same time obtained the coordinate data of cells.We then imported data into Chemotaxis And Migration Tool software to draw and statistically analyze the parameters of cell motion,including the directedness of cell movement,track speed and displacement speed.4.WB detected the changes and activation of related proteins under the treatment of electric field,including protein changes with different voltage differences(0.5h-2h)under a certain voltage difference(1V/cm)or different voltage differences(1V/cm,2V/cm,5V/cm)under a certain duration(0.5h).5.Changes of cytoskeletal proteins under the treatment of electric field.We co-stained BMDC with Phalloidin and p-Cofilin antibody after treatment of electric field and obtained immunoconfocal detection of corresponding fluorescence and colocalization as well as layer-by-layer scanning to detect the difference in stress fiber formation in different parts of cells.6.After the treatment of BMDC by PI3K-AKT pathway inhibitor Wortmannin,cell trajectory mapping and statistical analysis were performed.7.P110γflox/flox;CD11c-Cre mouse was constructed,which were specifically knocked out the P110γ gene of dendritic cells for further BMDC cell culture,mouse ear epidermal piece isolation and subsequent animal experiments.8.Cell migration trajectory plotting and statistical analysis of P110γ knockout BMDC cells and wild-type BMDC were done after electric field treatment(1V2H)at the same time.9.Comparative analysis of morphology and cytoskeletal proteins of P110γ-/-and WT BMDC cells.The morphological differences were compared by the staining of the cell morphology of the two and the Langerhans cells in the mouse ear epidermis sheet.Meanwhile,we performed immunofluorescence double-staining with Phalloidin and p-AKT to detect the changes of cytoskeletal proteins.10.The effect of electric field treatment on BMDC cytochemical chemotaxis.We used the Transwell chamber,the same number of BMDCs(including WT and P110γ-/-BMDC)that have undergone or not undergone electric field treatment were cultured in the upper chamber,and the same amount of normal medium or medium with CCL21 were added to the lower chamber.We then detected the cell numbers in the lower chamber of the migrated cells after 12 h.11.Function of electric field treatment on competitive migration of BMDC in vivo.BMDC after electric field treatment and NC group were respectively labeled with different dyes,mixed at the ratio of 1:1 and injected into mouse paw pads.After 18 hours,mice were sacrificed and single-cell suspension was prepared by ipsilateral popliteal lymph nodes(draining lymph nodes)and inguinal lymph nodes(non-draining lymph nodes).The fluorescence ratio of the two labeled dyes was detected by flow cytometry,and the Homing index value was statistically analyzed.Part Ⅱ: 1.Detection of BMDC antigen phagocytosis by electric field treatment(1V2H or5V30min).FITC-Dextran was dissolved in medium and added to BMDC cells after electric field treatment or NC group cells for 2h.Cells were washed clean and then flow cytometry was utilized to detect the mean fluorescence intensity of FITC.2.The effect of electric field treatment on the activation and maturation ability of BMDC cells.BMDC(1V2H)and NC group cells after electric field treatment were made into single-cell suspension and the expression of CD80,CD86 and MHCII molecules were detected by flow cytometry after multicolor staining.3.Effect of electric field treatment on T cell proliferation.Mouse splenic CD4+ and CD8+ T cells were extracted respectively,BMDC and T cells after electric field treatment were co-cultured according to the ratio of 1:5 respectively,and the proliferation of T cells was detected by flow cytometry after 3 days.4.BMDC proteomic analysis under the treatment of electric field.BMDC cells were divided into WT-NC,WT-1V2 H,P110γ-NC and P110γ-1V2 H groups.TMT-labeled quantitative proteomic analysis was carried out,and differential proteins were obtained after enrichment analysis,including subcellular localization analysis,GO analysis,KEGG analysis,etc.Part Ⅲ: 1.Effects of electric field treatment on contact hypersensitivity(CHS)in mice.Wild-type mice were divided into NC group(simple sensitization group),D-3 group(electrical stimulation after 3 days of sensitization),D0 group(electrical stimulation on the day of sensitization)and D+3 group(electrical stimulation 3 days before sensitization),a total of four groups with positive electrode on abdomen,negative electrode on mouse back.Mice were treated with 0.5% DNFB on abdominal sensitization and 6 days later,0.25% DNFB was used on the left ear of the mouse,no treatment in the right ear.After 24 hours,mice were acrificed and both ears were collected for HE staining.The thickness of the epidermis swelling of the mouse ear was measured,and the differences between left and right ear were counted and analyzed.2.Effects of P110γ-/-or switching electric field direction on CHS in mice.The experiments were divided into NC group(simple sensitization group),WT-D+3 group,P110γ-D+3 group and R-D+3group(negative electrode on abdomen and positive electrode on back),and the differences in epidermis thickness between left and right ears were statistically analyzed after electrical stimulation.3.Antigen phagocytosis of BMDC detection 3 days after electric field treatment.Three days after electric field treatment(5V30min),BMDC was treated with FITC-Dextran to measure antigen phagocytosis and the mean fluorescence intensity of FITC was detected by flow cytometry.4.Effect of P110γ-/-on G protein-coupled receptor signaling pathway.WT BMDC or P110γ-/-BMDC was respectively treated with CCL21 for 1 min or 5 min to detect the changes of downstream ERK,P38 and cytoskeletal protein.WB was used to detect the phosphorylation level of ERK and P38 and the mean fluorescence intensity of the cytoskeletal protein F-actin was detected by flow cytometry.Therefore,the pathway activation hindered or delayed by P110γ gene knockout could be judged.Results: 1.Exogenous electric field promoted the directional migration of BMDC cells,and their directionality,trajectory velocity and displacement velocity were enhanced.2.WB results showed that after the electric field treatment,ERK,P38 and PI3K-AKT signaling pathways were activated and phosphorylation levels were enhanced.Meanwhile,cell migration related indexes changed,and the expression amount of CCR7 protein was related to the electric field strength and electric field action time.3.The electric field treatment promoted the expression of BMDC cytoskeletal protein F-actin,induced the polymerization of cell stress fibers,which was mainly formed at the bottom of the cell.4.After treatment with Wortmannin,a PI3K-AKT signaling pathway inhibitor,its galvanotaxis was weakened,and its directionality,trajectory velocity and displacement velocity were lower than those in the WT electric stimulation group.5.After P110γ gene knockout,cell galvanotaxis was weakened and cell migration ability was reduced.6.After P110γ gene knockout,the cell morphology of BMDC cells and LC morphology in mouse ear epidermis differentiated.The cell soma became round,the cell dendrites were elongated.Meanwhile,the cytoskeletal protein changed,and the level of p-AKT decreased after the treatment of electric field.7.Electric field treatment could help enhance the chemical chemotaxis ability of BMDC cells,and this effect disappeared after P110γ gene knockout.8.Competitive migration experiments in vivo by mouse paw pad injection showed that the electric field treatment could promote the migration of cells in vivo,and P110γ gene knockout would affect the in vivo migration of cells.9.The exogenous electric field did not affect the antigen phagocytosis and cell maturity of BMDC cells after instant treatment,and only the expression of MHCII molecules of BMDC cells increased.10.Co-culture of BMDC-T cells after the treatment of electric field showed that electric field treatment of BMDC inhibited the proliferation of CD4+ T cells and promoted the proliferation of CD8+ T cells.11.BMDC proteomics analysis after electric field treatment showed that the differential protein enrichment analysis between groups mainly focused on Lysosome,Phagosome,Antigen processing and presentation,Fc gamma R-mediated phagocytosis,Regulation of actin cytoskeleton,Gap junction,Th17 cell differentiation,Th1 and Th2 cell differentiation,Tight junction,etc.12.Electrical stimulation 3 days before sensitization(D+3 group)in mice could enhance the intensity of CHS response.13.After P110γ gene knock out or switching the direction of the electric field,the intensity of CHS was weakened compared with the D+3 group.14.After 3 days of electric field treatment,the antigen phagocytosis ability of BMDC cells was enhanced.15.P110γ gene knockout affected the activation of P38 and cytoskeletal protein signaling pathways downstream of G protein-coupled receptors.Conclusion: 1.Exogenous electric field promoted the directional migration of BMDC cells.2.The immediate effect of the exogenous electric field did not affect the antigen phagocytosis ability of BMDC cells as well as cell activation and maturation.However,it can enhance the antigen phagocytosis ability 3 days after electrical stimulation.3.Electrical stimulation 3 days before sensitization enhanced the intensity of contact hypersensitivity reactions in mice,and P110γ gene knockout or switching electrode direction can weaken the reaction intensity.