Identification And Function Of IgM~+ B Cell Subsets In Ctenopharyngodon Idella

Teleost fish,as the more ancient vertebrates that live in water for whole life,have gradually evolved a serious of complete and effective immune systems over hundreds of millions of years,which was used to defend the invasion of different aquatic pathogens.As the main effector cells in teleost adaptive immunity,B cells can secrete antibodies to target pathogens and then promote the clearance of pathogenic bacteria.However,recent studies mostly focus on fish B cells serving as the antibody-secreting cell（ASC）,few studies have deeply clarified the phenotypes and functions on different B cell subsets in teleost fish,especially about the regulatory B cell（Breg）subset.In this study,chitosan oligosaccharide（COS）,a small-molecule immune inducer,was used to stimulate the immunoglobulin（Ig）M-bearing（Ig M⁺）B cells in grass carp（Ctenopharyngodon idella）,and then a subpopulation of proliferative plasmablasts that specifically responded to COS was successfully identified.COS can induce the proliferation and differentiation of splenic Ig M⁺B cells into Ig M^lo and Ig M^hi B cell subsets in grass carp.The Ig M^lo B cells were further identified as short-lived plasmablasts that secreted natural Ig M with binding-abilities to lipopolysaccharide（LPS）and peptidoglycan（PGN）.Moreover,the mannose receptor（MR）and integrins were serving as the binding-receptors of COS on Ig M^lo plasmablasts.The MR synergized with integrins to trigger intracellular signal transduction to boost plasmablasts generation and proliferation.Notably,Ig M^lo plasmablasts originally generated in spleen but they could migrate into blood to secrete natural Ig M,which augmented the serum bactericidal activity.Taken together,this study revealed the cellular and molecular mechanisms of COS-enhanced humoral immunity in fish.In addition to being ASC,teleost B cells have been reported to have a phagocytic ability and the ability of being antigen-presenting cell（APC）,but the ability with serving as Breg has never described in fish.Whether would fish B cells develop a type of regulatory B cell subset like that in mammals?In present study,we used monoclonal antibodies of mouse anti-grass carp Ig M and mouse anti-grass carp interleukin 15receptorα（IL-15Rα;CD25-like）to successfully separate and identify a subset of Ig M⁺CD25-like⁺Breg from grass carp.This Breg subset can secrete anti-inflammatory cytokines like interleukin-10（IL-10）to negatively regulate inflammatory response.It was found that CD25-like⁺Breg were mostly distributed in peripheral blood,accounting for 20%of the total blood Ig M⁺B cells of grass carp.In addition,recombinant protein IL-35 of grass carp can induce this Breg generation in vitro.Further studies have shown that CD25-like⁺Breg can effectively impede the chemotaxis and activation of neutrophils and even the release of reactive oxygen species（ROS）.We have also established a Trinitro-benzene-sulfonic acid（TNBS）-induced inflammation model in experiments in vivo,and we found that CD25-like⁺Breg has gradually increased with the inflammation occurrence,development,and repair in peripheral blood and intestine.CD25-like⁺Breg can impede the excessive activation of neutrophils and T helper cell 17（Th17）via producing IL-10 to alleviate inflammatory response and promote intestinal repair in the local inflammatory position.In conclusion,we have identified a subpopulation of CD25-like⁺Breg in teleost fish for the first time and clarified their immunosuppressive functions which will be filling the gap in this field of fish regulatory B cells.This study is the first to systematically investigate the phenotypes and functions of the teleost plasmablast subset and regulatory B cell subset,as well as the cellular and molecular mechanisms of their induction and differentiation.Secondly,the specific functions of two subsets of fish B cells were deeply clarified and evaluated when they underwent specifically infected conditions.These collective results have been laying a solid theoretical foundation for further research on the immune function of teleost B cell subsets.