Establishment Of Animal Model Of Linear IgA Bullous Dermatosis

LABD is a rare autoimmune subepidermal bullous disease that is characterized by IgA deposition in the basement membrane zone.It occurs in children and adults.Currently the pathogenesis of LABD is not clear.The work was completed in University of North Carolina at Chapel Hill Dermatology Laboratory,which had successfully established a variety of animal models of autoimmune subepidermal bullous disease and had extremely experienced in animal experiments.In this study,the pathogenic mechanism of IgA autoantibodies was initially explored by gradually establishing animal model of LABD.Part 1 Purify and characterize NC16A-specific IgAObjective:To purify and characterize NC16A-specific IgA from LABD patients.Methods:NC16A-specific IgA was purified by two affinity columns,first by commercial human IgA-affinity column to isolate total IgA from LABD patients and then by home-made NC16A-afficity column.The purity of NC16A-specific IgA was determined by commercial IgA-specific ELISA,and then further characterized for antigen binding by indirect IF and western blotting.Results:The concentration of purified antibody was 100ug/ul.Indirect immunofluorescence using NC16A mouse skin as substrate showed linear deposition of IgA in the basement membrane.Recombinant protein GST-NC16A-WB results showed that a protein band of 37KDa.Conclusion:In this experiment,NC16A-specific IgA was successfully purified by affinity chromatography,laying a foundation for the further experiments.Part 2 Necessity of human neutrophils in the formation of subepidermal blisters in LABDObjective:To verify the role of human neutrophils in the formation of subepidermal blisters in LABD.Methods:Isolated human and mouse neutrophils,divided NC16A neonatal mice into 3 groups:the first group injected NC16A IgA into mouse upper back,the second group injected pathogenic IgA and mouse neutrophils at the same time,the third group injected pathogenic IgA and human-derived neutrophils.Direct immunofluorescence and HE staining were taken from the local skin to detect the presence of IgA deposition and epidermis-dermis separation in the basement membrane zone.What's more,determined the appropriate antibody amount to cause LABD on mouse.Results:100-250ug NC16A-specific IgA were appropriate for mouse to induce subepidermal blisters.Only the mouse which injected NC16A IgA antibody and human-derived neutrophils showed subepidermal blisters after 24 hours,the MPO value gradually increased with neutrophil activation.Conclusion:In the presence of human neutrophils,NC1 6A IgA can cause subepidermal blister formation in NC16A mice.Part 3 Develop humanized Fc?RI/NC16A mouse modelObjective:To test whether LABD IgA autoantibodies induce subepidermal blisters in double humanized NC16A and IgA receptor Fc?R(hFcaR)mice(termed NC16A/hFcaR).Methods:Transgenic technology was used to transfer Fc?R1 and NC16A into mice,establish double-humanized FcaR1/NC16A mice,and then injected NC16A IgA antibody to transgenic mice,and observed mouse within 0-48 hours.The skin biopsies were taken and analyzed by routine histology(to identify inflammation and lesional site),direct IF(to identify IgA deposition at the basement membrane zone),Indirect IF(to identify and localize infiltrating immune cells),and MPO assay(to quantify infiltrating neutrophils).Results:Twenty-four hours after the injection of anti-NC16A IgA antibody,the transgenic mice showed the formation of subepidermal blisters and neutrophil infiltration in ears,and IgA deposition in the basement membrane zone.The MPO value gradually increased and were significantly higher than the control group.Conclusion:LABD IgA caused neutrophil activation by cross-linking with Fc?RI on the surface of neutrophils,which can cause subepidermal blisters in Fc?RI/NC16A mice,successfully built a transgenic LABD mouse modelPart 4 Establishment of humanized FcaRI/NC16A/CXCR2 KO mouse model Objective:To analyze local immune response of IgA and neutrophils by CXCR2 knock out mouse model.Methods:Transgenic technology was used to knock out CXCR2 and built FcaRI/NC16A/CXCR2 KO mouse.Results:Successfully obtained FcaRI/NC16A/CXCR2 KO mice.Conclusion:There were three possible outcomes after the injection of anti-NC16A IgA antibody to CXCR2 KO mice.In addition to CXCR2,there may be other pathways in LABD IgA-induced neutrophils recruitment and activation.