Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Abstract

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effects of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune-related genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65 × 10⁸ CFU/mL) was evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV group), recombinant plasmid pEGFP-Gal9 treatment (Gal9 group) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase-1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O. macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O. macrolepis infected with A. hydrophila in Gal9 group at 24 h was significantly lower than that in EV group (P < 0.05), and the survival rate had increased from 15 % to 35 %. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play an important regulatory role in glycolysis/gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.