Functional Study Of B Cells And Immunoglobulins In The Pharyngeal Mucosa And Ocular Conjunctiva Of Rainbow Trout

Fish live in a microbial-rich and-complex aquatic environment.Mucosal surfaces of teleost fish are subjected to different kinds of insults from external pathogens due to direct contact with aquatic environment.In the long-term evolutionary process,teleost fish have evolved an effective mucosal immune system to counter these environmental challenges,which act as the first line of defense against external threats.Mucosa-associated lymphoid tissue(MALT)can initiate sophisticated innate and adaptive immune responses to resist the invasion of pathogenic microorganisms.As important physiological organs of bony fish,pharynx cavity and eyes are directly exposed to water environment and threatened by a vast number of pathogens.From the aspects of structural biology,cell biology and molecular biology,we found a diffuse mucosal associated lymphoid tissue(D-MALT)in pharyngeal mucosa(PM)and ocular conjunctiva(OC),and abundant B cells and immunoglobulin(Ig)are present in their mucosa and mucosal secretions,respectively.but the role of fish B cells and Igs to protect against infection by pathogens and the mechanism of immune response in pharyngeal and ocular mucosal surfaces are thus far unknown.To interrogate whether MALT appears in the teleost pharyngeal cavity,and local B cell and Igs immune responses,here,we developed an infection model in which the rainbow trout(Oncorhynchus mykiss)was exposed to Ichthyophthirius multifiliis(Ich)parasite.Our results indicated that the teleost PM is different from that of mammals.Instead of lymph nodes,fish have a D-MALT in PM,which can locally produce a strong innate and adaptive immune response to the infection with parasite Ich.Moreover,we provided the first demonstration that s Ig T act as the main players involved in immune responses in the PM of rainbow trout as opposed to Ig M and Ig D.Critically,we showed that Ig T~+B cells are locally proliferated and generate parasite-specific Ig T in the PM of rainbow trout,thus providing the first evidence of locally induced mucosal Igs responses in the PM of early vertebrates.Finally,we discovered that teleost Ig T plays a crucial role in immune exclusion by coating the symbiotic bacteria in the PM.In mammals,the ocular surface is regarded as a mucosal tissue supported by a dedicated mucosal immune system,which is an important entry point for various pathogens,such as protozoa,viruses,bacteria,and fungi.B cells and Igs play vital roles in protecting the OC from invading pathogens.Since fish lack eyelids,eyes are more vulnerable to the threat of aquatic pathogens.In order to reveal the adaptive immune mechanism of trout's OC and the immune function of Ig in maintaining eye health,we performed a systematic study on the organizational structure of trout's OC,and found that fish lack lacrimal glands,but they still have conjunctiva,which harbors goblet cells,is considered as the workshop of tear production.Moreover,we used two models of rainbow trout(Oncorhynchus mykiss)that infected with the Ich or F.columnare to evaluate the local B cells and Igs responses in the OC.To our knowledge,our findings are the first to reveal a well-defined diffuse MALT in the trout's OC,which mounts robust local innate and adaptive immune responses upon infection with parasite Ich or F.columnare.Importantly,fish ocular mucus contains abundant Igs,in which the Ig T is the main player in the fish immune response to parasitic and bacterial antigens in the OC.Here,we also demonstrated Ig T~+B cells in the OC is the result of local proliferation,and then secrete large amunts of parasitic and bacteria-specific s Ig T,which is transported by p Ig R.Thus,different types of immune molecules(s Ig A versus s Ig T)and lymphoid structures(organized versus diffuse)evolved in OC of mammals and fish,but they shared analogous strategies to maintain the immune system homeostasis.