| The p40 is a common subunit of Interleukin-12(IL-12)and IL-23,which not only can form heterodimeric IL-12 and IL-23 with p35 and p19,also can exert immune effects in the form of monomer and homodimer.At present,only one gene encoding p40 has been found in mammals,while three genes encoding p40,namely p40 a,p40b and p40 c,have been isolated and identified in some bony fish,which are caused by the whole genome replication event specific to fish.In mammals,p40 homodimer can antagonize the effect of IL-12/23 and involve in the regulation of certain autoimmune diseases.It also inhibits the generation of regulatory T cells by the production of NO.In bony fish,so far the information of p40 homodimer's function has been lacking.In this study,grass carp is used as a research model,and the recombinant p40 a,p40b and p40 c homodimer proteins are prepared to explore their immune functions.First,the recombinant homodimer proteins of grass carp p40 a,p40b and p40 c were obtained by the E.coli prokaryotic expression system and renaturation method,and their activities were verified by detecting the expression of grass carp TNF-? and IL-1? in head kidney leukocytes(HKLs).To clarify whether these p40 isoforms mimic the classical function of their mammalian homologues,three homodimer proteins of grass carp p40 s were combined with grass carp IL-23A(p19/p40a),IL-23 B and IL-23 C to treat HKLs.However,p40 dimer proteins were not found to antagonize the activity of IL-23 isoforms,and in fact,they can up-regulate the gene expression of grass carp IL-17A/F1 in an addition manner.In consistent with this,p40 a,p40b and p40 c homodimer proteins alone could up-regulate the gene expression of IL-17A/F1 in HKLs.Given that IL-1? can amplify the IL-23-induced IL-17A/F1 expression,the IL-1? immune neutralization experiment was performed,and the result showed that anti-IL-1? did not change the combined effects of p40 s and IL-23 s on IL-17A/F1 gene expression,suggesting that IL-1? was not involved in the regulation of p40 s and IL-23 s in HKLs.In fact,the phosphorylation levels of STAT3 were found to be enhanced by the combination treatment with of p40 s and IL-23 s compared with IL-23 s alone,indicating the amplification effect of the downstream signaling pathway induced by p40 proteins.In mammals,another important function of p40 homodimer is to induce the expression of iNOS.In agreement with this,three grass carp p40 homodimer proteins showed the ability to up-regulate the gene expression of grass carp iNOS in HKLs.Others in the laboratory has revealed that three grass carp p40 monomers could not induce the expression of iNOS,suggesting that the p40 monomer and its homodimer are functionally different in grass carp.Furthermore,the signaling pathway involved in the regulation of iNOS by p40 homodimers was explored.bioinformatic cis-element analysis in the promoter of grass carp iNOS showed that the transcription factors involved in the iNOS transcription were mainly STAT3 and NF-?B.Along this line,the STAT3 and NF-?B inhibitor were used to co-treat HKLs with three p40 homodimer proteins,respectively,showing that both inhibitors could inhibit the p40 homodimers-up-regulated expression of iNOS,indicating the involvement of STAT3 and NF-?B signaling pathways in the transcription of iNOS by three p40 homodimer proteins.Besides,in mammals,IRF1 is indispensable to iNOS expression,In this regard,the effects of grass carp p40 monomer and its homodimer on the IRF1 expression were examined in HKLs,founding that p40 homodimers but not monomers induced the expression of IRF1.These results suggest differential mechanism of grass carp p40 a,p40b and p40 c monomers and their homodimers on iNOS expression.In conclusion,the recombinant homodimer proteins of grass carp p40 a,p40b and p40 c were obtained for the first time in this study,and their immunological functions were preliminarily identified.These data laid the foundation for exploring the functional role of p40 a,p40b and p40 c homodimers in teleost fish. |
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