The Functional Study Of Immunologlobulins In Buccal Mucosa Of Rainbow Trout In Anti-flavobacterium Columnare Infection

The buccal mucosa in vertebrates is a critical mucosal barrier that protects buccal healthy.Since the buccal mucosa is continuously exposed to a plethora of triggers including diverse commensal microbial communities and dietary and air-or waterborne antigens that may cause infection,vertebrates have evolved an effective innate and adaptive immune system to protect the buccal surface.Our previous studies have shown that diffuse MALT appears in teleost buccal mucosa and IgT plays a crucial role alike to mammalian IgA in response upon parasite infection.In addition,IgT coats a large number of buccal microorganisms and plays an important role in maintaining homeostasis.However,very little is known about the evolutionary roles of buccal B cells and mucosal immunoglobulins(Igs)in immune defense against bacterial pathogen.Here we developed a bath infection model that rainbow trout experimentally exposed to Flavobacterium columnare(F.columnare),which is well known as a mucosal pathogen.We found that F.columnare successfully invaded the trout buccal mucosa.On this basis,we studied the pathological changes of the buccal mucosa after infection,the expression of immune-related gene,the proliferation of B cells,and the secretion of pathogen-specific Igs.The results are as follows:(1)The classical phenotype of columnaris disease appeared in the trout after infection,characterized by gill necrosis and fin rot.We observed that F.columnare gathered especially on the mucus cells of the buccal epithelium on the first day after challenge,and the localization of this bacterial pathogen gradually moved down to the middle layer of the buccal epithelium over time.(2)The number of mucus cells decreased significantly at different time points post-infection.And we found strong inflammatory response in buccal mucosa after infection,the expression levels of immune-related genes including antibacterial peptides,cytokines,chemokines and complement factors,were significantly upregulated in trout buccal mucosa following F.columnare infection.(3)We charactered that a higher percentage of IgT~+B-cells than IgM~+B-cells in an epithelial layer of trout buccal mucosa post infection.By in vivo proliferation assays,we found significant proliferative IgT~+B-cell responses in trout BM,suggesting that the accumulation of IgT~+B-cells in the buccal mucosa after bacterial infection is due to local proliferation.(4)We detected significant bacterial-specific IgT mainly in buccal mucus,while bacterial-specific IgM in serum in trout exposed to F.columnare.In addition,the production of high titers of bacteria-specific IgT in buccal explant cultures confirmed the local production of bacteria-specific IgT.(5)The significantly increased numbers of pIgR~+cells and transcript levels of pIgR in the BM from bacterial-infected trout suggested an important role of pIgR in the transport of Igs into the buccal mucus.Our study showed that F.columnare could successfully colonize the epithelium of trout buccal mucosa though immersion infection,and cause strong innate and adaptive immune responses in buccal mucosa,in which IgT and IgT~+B cells play an important role in the fight against bacterial infection.These results have further broadened the understanding of buccal mucosal immunity of teleost fish.