Identification of grass carp IL-10 receptor subunits: Functional evidence for IL-10 signaling in teleost immunity

Abstract

Although the functions of teleost IL-10 have been preliminarily determined, functional evidence for its receptor signaling is lacking. Particularly, the identity of fish IL-10 receptor 2 (IL-10R2) is ambiguous. Cytokine receptor family member b4 (CRFB4) and CRFB5 are likely the ortholog of mammalian IL-10R2. In this study, grass carp CRFB4 (gcCRFB4) and gcCRFB5 cDNAs were isolated and characterized. The relatively high expression levels of grass carp IL10 receptor 1 (gcIL-10R1), gcCRFB4 and gcCRFB5 in immune tissues and cells implied their importance in fish immunity. Accordingly, gcIL-10R1, gcCRFB4 and gcCRFB5 were overexpressed in a grass carp kidney cell line to identify the IL-10 receptor subunits upon grass carp IL-10 (gcIL-10) treatment. Results showed that gcIL-10R1 was essential for gcIL-10 stimulation on STAT3 activation and grass carp suppressor of cytokine signaling 3 (gcSOCS3) promoter activity, and also indicated that gcCRFB4 but not gcCRFB5 might be the ortholog of mammalian IL-10R2. Furthermore, mutation of a putative STAT3-binding element in gcSOCS3 promoter attenuated the stimulation of gcIL-10 on gcSOCS3 promoter activity, indicating that gcIL-10 may modulate gcSOCS3 transcription at least partly via STAT3 activation. This notion was further supported by our observation that gcIL-10 was able to induce STAT3 phosphorylation and STAT3 inhibitor could abolish the upregulation of gcSOCS3 mRNA expression by gcIL-10 in grass carp head kidney leukocytes. Taken together, this study for the first time functionally characterized the teleost IL-10 receptor subunits and clarified the conservation of fish IL-10 signaling during evolution, thus laying the ground for further understanding the critical immune events led by IL-10 in teleost.