Immunoglobulin M-based local production in skin-associated lymphoid tissue of flounder (Paralichthys olivaceus) initiated by immersion with inactivated Edwardsiella tarda

Abstract

Fish skin, the mucosal site most exposed to external antigens, requires protection by an efficient local mucosal immune system. The mucosal reserve of IgM is recognized as an immune strategy that blocks pathogen invasion to maintain homeostasis, whereas the mechanism of skin-associated local IgM production induced by mucosal antigens is not well know. In this study, we found that the skin of flounder (Paralichthys olivaceus) was equipped with the immune cellular and molecular basis for processing mucosal antigens and triggering local specific responses, i.e., CD4⁺ Zap-70⁺ T cells, CD4⁻ Zap-70⁺ T/NK cells, IgM⁺ MHCII⁺ B cells, PNA⁺ MHCII⁺ antigen-presenting cells, UEA-1⁺ WGA⁺ and UEA-1⁺ WGA⁻ antigen-sampling cells, as well as secreted IgM and pIgR, as demonstrated by indirect immunofluorescence assay using different antibodies and lectins. After immersion immunization with inactivated Edwardsiella tarda, qPCR assay displayed up-regulation of immune-related genes in flounder skin. Flow cytometry analysis and EdU labeling demonstrated that the mucosal inactivated vaccine induced local proliferation and increased amounts of cutaneous IgM⁺ B cells. Skin explant culture proved the local production of specific IgM in the skin, which could bind to the surface of E. tarda. ELISA, laser scanning confocal microscopy, and Western blot revealed that, in addition to the elevated IgM levels, pIgR protein level was significantly up-regulated in skin tissue and surface mucus containing the pIgR (secretory component, SC)-tetrameric IgM complex, indicating that mucosal vaccine stimulated up-regulation of IgM and pIgR, which were secreted as a complex into skin mucus to exert the protective effects as secretory IgM. These findings deepen the understanding of IgM-based local responses in the mucosal immunity of teleosts, which will be critical for subsequent investigation into the protective mechanism of mucosal vaccines for fish health.