AUTHOR=Qiu Xingyang , Sun Hao , Wang Dan , Ren Jingqi , Wang Xinyan , Zhang Anying , Yang Kun , Zhou Hong 

TITLE=Stimulus-Specific Expression, Selective Generation and Novel Function of Grass Carp (Ctenopharyngodon idella) IL-12 Isoforms: New Insights Into the Heterodimeric Cytokines in Teleosts

JOURNAL=Frontiers in Immunology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.734535

DOI=10.3389/fimmu.2021.734535

ISSN=1664-3224

ABSTRACT=<p>Interleukin-12 (IL-12) is a heterodimeric cytokine composed of a p35 subunit specific to IL-12 and a p40 subunit shared with IL-23. In this study, we unveiled the existence of two <italic>p35</italic> paralogues in grass carp (named <italic>gcp35a</italic> and <italic>gcp35b</italic>). Notably, <italic>gcp35a</italic> and <italic>gcp35b</italic> displayed distinct inducible expression patterns, as poly I:C merely induced the gene expression of <italic>gcp35a</italic> but not <italic>gcp35b</italic>, while recombinant grass carp interferon-gamma (rgcIfn-γ) only enhanced the transcription of <italic>gcp35b</italic> but not <italic>gcp35a</italic>. Moreover, the signaling mechanisms responsible for the inducible expression of <italic>gcp35a</italic> and <italic>gcp35b</italic> mRNA were elucidated. Because of the existence of three grass carp <italic>p40</italic> genes (<italic>gcp40a, gcp40b</italic> and <italic>gcp40c</italic>) and two <italic>p35</italic> paralogues, six gcIl-12 isoforms were predicted by 3D modeling. Results showed that gcp40a and gcp40b but not gcp40c had the potential for forming heterodimers with both gcp35 paralogues <italic>via</italic> the disulfide bonds. Non-reducing electrophoresis experiments further disclosed that only gcp40b but not gcp40a or gcp40c could form heterodimers with gcp35 to produce secretory heterodimeric gcp35a/gcp40b (gcIl-12AB) and gcp35b/gcp40b (gcIl-12BB), which prompted us to prepare their recombinant proteins. These two recombinant proteins exhibited their extensive regulation on Ifn-γ production in various immune cells. Intriguingly, both gcIl-12 isoforms significantly enhanced the transcription of <italic>il-17a/f1</italic> and <italic>il-22</italic> in lymphocytes, and their regulation on <italic>il-17a/f1</italic> expression was mediated by Stat3/Rorγt signaling, supporting the potential of gcIl-12 isoforms for inducing Th17-like responses. Additionally, stimulatory effects of gcIl-12 isoforms on <italic>il-17a/f1</italic> and <italic>ifn-γ</italic> expression were attenuated by gcTgf-β1 <italic>via</italic> suppressing the activation of Stat3 signaling, implying that their signaling could be manipulated. In brief, our works provide new insights into the inducible expression pattern, heterodimeric generation and functional novelty of Il-12 isoforms in teleosts.</p>