The Comparative Study On The Inducible Expression Of Interleukin 12 In Grass Carp Macrophages And Dendritic-like Cells

In mammals,interleukin 12(IL-12)is a heterodimer composed of IL-12p35 and IL-12p40 subunits,which is produced by innate immune cells,especially macrophages and dendritic cells.It plays an important role in the development of type I helper T cells from Na?ve CD4~+T cells,inducing abundant production of IFN-?to resist against pathogens in immune system.Then,IL-12 was proposed to be a bridge between innate and adaptive immune.Notably,in many teleosts including grass carp,there are several isoforms of IL-12 arising from whole genome dulplication.IL-12 of fish is more complicated than mammal's.But the information of protein expression and the source of cells about IL-12 isoforms is lacking.Therefore,this dissertation used Western Blotting to investigate the inducible expression patterns of IL-12 isoforms at protein levels and the related signal pathways in grass carp macrophages and dendritic-like cells in response to heat-inactivated Aeromonas hydrophila(HI A.hydrophila)and recombinant grass carp IL-1?(rgcIL-1?(8)treatment(13)In detail,we firstly isolated the grass carp macrophages by adherent method,which were identified by the morphologic observation.Meanwhile,grass carp dendritic-like cells were obtained and identified by the morphologic features and cell surface marker genes(such as cd80/86,cd83,and mhc2dab)expression.In order to detect the protein expression levels of IL-12p35 subunits,grass carp IL-12p35a and IL-12p35b polyclonal antibodies produced by custom synthesis were verified to specially recognize IL-12p35a and IL-12p35b,respectively.Secondly,we examined the inductive expression of IL-12isoforms in grass carp macrophages and dendritic-like cells,founding that HI A.hydrophila could apparently induce the protein synthesis and secretion of IL-12isoforms consisting of IL-12p35a in a short time as well as the protein synthesis of IL-12p40a/b/c subunits in macrophages,which indicated IL-12 might participate in immune regulation during the early stage of infection with Aeromonas hydrophila.To further study the related mechanism that HI A.hydrophila rapidly induced the protein expression of IL-12 in grass carp macrphages,we found it was unaffected by inhibiting transcription using actinomycin D(Act D).In dendritic-like cells,HI A.hydrophila significantly induced IL-12p40b and IL-12p40c protein synthesis as seen in macrophages,but the inductive protein expression of IL-12p35a and IL-12p40a was not observed in dendritic-like cells,which was different from the findings in macrophages.It suggested that HI A.hydrophila exerted the different stimulant function on the two innate cells and illustrated that the isoforms of IL-12 composed of IL-12p35a might be borne mainly by macrophages with the infection of Aeromonas hydrophila.We could not determine the IL-12p35b subunit protein due to the low recognition ability of IL-12p35b polyclonal antibody to target proteins under the treatment of HI A.hydrophila.Finally,IL-1?was perceived as a classic pro-inflammmatory cytokine in the early phase of inflammation with the Aeromonas hydrophila infection,so it is well beneficial for further understanding the characteristic of IL-12 production to reveal whether IL-1?regulates the protein expression of IL-12 isoforms in grass carp.Thereby,we detected the protein expression of IL-12 subunits in grass carp macrophage treated by rgcIL-1?and found that it increased the protein synthesis and secretion of IL-12isoforms consisting of IL-12p35a rather than that of IL-12p35b.The further mechanismic study found that the protein IL-12 synthesis and secretion of IL-12p35a induced by rgcIL-1?was inhibited the NF-?B inhibitor,indicating the involvement of NF-?B signal pathway in mediating protein expression of IL-12p35a in macrophage.Additionally,the protein synthesis of IL-12p40a/b/c subunits in macrophage induced by rgcIL-1?were obviously variant,which suggested possible different mechanisms of the protein synthesis of IL-12p40 subunits in grass carp.In summary,this thesis systematically compared the inducible protein expression of IL-12 isoforms in grass carp macrophages and dendritic-like cells for the first time in teleost.The protein expression profiles of grass carp IL-12p35a were different in these two innate immune cells,suggesting that the IL-12 isoforms production composed of IL-12p35a might be borne mainly by macrophages under Aeromonas hydrophila infection.In addition,rgcIL-1?might regulate grass carp IL-12 production via NF-?B signal pathway in macrophages.These contributed to understanding the position of IL-12 isoforms in fish innate immunity.