Two Distinct Interferon-γ in the Orange-Spotted Grouper ( Epinephelus coioides ): Molecular Cloning, Functional Characterization, and Regulation in Toll-Like Receptor Pathway by Induction of miR-146a.

Interferon gamma (IFN gamma) is a Th1 cytokine that is critical for innate and adaptive immunity. Toll-like receptors (TLRs) signaling pathways are critical in early host defense against invading pathogens. miR-146a has been reported to participate in the regulation of host immunity. The known mechanisms of integrations between the IFN. and TLR signaling pathways are incompletely understood, especially in teleosts. In this study, orange-spotted grouper (Epinephelus coioides) IFN gamma 1 and IFN gamma 2, their biological activities, especially their involvements in TLR pathway, were explored. We identified and cloned two IFN. genes of E. coioides, namely EcIFN gamma 1 and EcIFN gamma 2. The produced recombinant E. coioides IFN gamma 1 (rEcIFN gamma 1) and IFN gamma 2 (rEcIFN gamma 2) proteins showed functions, which are similar to those of other bony fishes, such as enhancing nitric oxide responses and respiratory burst response. rEcIFN gamma 2 could regulate TLR pathway by enhancing the promoter activity of miR-146a upstream sequence and thus increasing the expression level of miR-146a, which possibly targets TNF receptor-associated factor 6 (TRAF6), a key adapter molecule in TLR signaling pathway. Taken together, these findings unravel a novel regulatory mechanism of anti-inflammatory response by IFN gamma 2, which could mediate TLR pathway through IFN gamma 2-miR-146a-TRAF6 negative regulation loop. It is suggested that IFN gamma 2 may provide a promising therapeutic, which may help to fine tune the immune response.