| Fish evolved a series of mucosal immune tissues to adapt to the complex aquatic environments,which not only acts as a barrier against pathogens directly,but also prevented various pathogens re-invasion by activating acquired mucosal immune responses.An appropriate pathogenic infection model is essential for studying the mucosal immune system of fish.Cryptocaryon irritans is a serious ectoparasitic pathogen which mainly parasitic on fish skin,fin and gill,can also induce acquired immune responses in fish.Due to its unique parasitic characteristics,C.irritans can be a good pathogen model to study mucosal immunity in fish.Therefore,the research on the mucosal immune response of fish against C.irritans can not only reveal the immune mechanism of fish against C.irritans infection,but also provide a theoretical basis for the immune prevention of this parasitic disease.In this study,a mouse anti-grouper IgM polyclonal antibody was prepared,which can specifically recognize IgM in grouper serum and mucus,and can also label cells.The cells sorted by this antibody have lymphocyte morphology and express characteristic markers of fish IgM~+B cells(IgM,Ig D and MHC II).IgM in grouper serum is mainly tetramer,but there are also a small amount of dimer,while IgM in mucus is mainly tetramer.Glycosylation analysis showed that grouper IgM heavy chain could be glycosylated.Grouper IgM is abundantly expressed in various tissues,especially in head kidney,spleen,pyloric caecum and intestine.IgM positive cells were also detected in these tissues by immunohistochemistry.In addition,it was also found that 12.78±0.33%IgM~+cells in HKL had the activity of phagocytosis of large particles.To study the function of IgM in anti-C.irritans immune response,we established an C.irritans immune model.After three immunizations,groupers can resist about 90%of C.irritans re-infection.Significant increase of specific or total IgM level were found in the serum and mucus of immune grouper,which can be synthesized and secreted in the head kidney and skin.IgM antibody-secreting cells are significantly increased in the gill of first infection groupers and the immune groupers.In addition,passive immunity confirmed that specific IgM in the blood can be transported to skin mucus,suggesting that specific IgM antibodies produced by systemic immune tissues can be transported to mucosal immune tissues.Experiments in vitro and in vivo confirmed that IgM could specifically bind to C.irritans.The infection rate is significantly increased in IgM-RNA interference immune grouper,indicating that IgM is involved in anti-C.irritans immune response.The recombinant GDCI3 protein was also used to immunize grouper by intraperitoneal injection,and the production of specific IgM was also found in serum and mucus,suggesting that the injection of C.irritans recombinant protein could also elicit anti-C.irritans immune response.To further investigate how IgM initiates an immune response against C.irritans,we conducted a preliminary study of the BCR signaling pathway.The calcium flux of HKL was increased followed by the stimulation with anti-IgM antibody,suggesting that the grouper has an intact BCR signaling pathway.Six upstream genes of grouper BCR signal pathways were identified,the tissue distribution,subcellular localization and activation of NF-?B were also analyzed.Significant regulation of the BCR signaling pathway was found in C.irritans infected groupers,suggesting that the BCR signal pathway may be activated during the infection.Futhermore,we successfully prepared a polyclonal antibody against BTK,one of the key genes in the BCR signal pathway,and analyzed the effects of sodium vanadate/hydrogen peroxide and Ibrutinib on BTK phosphorylation.Finally,it was also found that the infection of C.irritans caused phosphorylation of BTK in gill and skin,indicating that BTK was activated during C.irritans infection. |
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