In vitro innate immune stimulation using beta-glucan in head kidney adherent macrophage-like cells derived from rohu, Labeo rohita

• β−1,3-glucan upregulated cytokines ( il6, tnfα, ifnγ ) in Labeo rohita head kidney adherent macrophages-like cells. • mtor, hif1α, and hdac12 mRNA expression increased post β−1,3-glucan exposure. • Metabolite changes include elevated lactate and LDH, and decreased glucose, indicating innate immune memory in these adherent macrophage-like cells. Innate immune memory is acquired through metabolic and epigenetic alteration of innate immune cells, including macrophages, leading to non-specific cross-protection against various pathogens, subsequently improving host survival. The present study evaluated the induction of trained immunity in rohu through β-1,3-glucan treatment on its head kidney originated adherent macrophage-like cells. For this study, isolated head kidney adherent macrophage-like cells through Histopaques-1077, cultured in l-15 media, have been treated using β-1,3-glucan and followed by evaluation of metabolites viz. , glucose, LDH and lactate and expression pattern analysis of cytokines ( tnfα, il6 , and ifnγ ) and related markers ( mtor , mammalian target of rapamycin and hif1α , hypoxia-inducible factor) of trained immunity, including epigenetic marker ( hdac12 , histone deacetylase) at different time points (1,3, 6, 12, 24, 48 and 72 h) . Increased glycolysis in the cells post-β-glucan restimulation was confirmed by the high LDH, lactate and decreased glucose level in the cell culture supernatant. The expression patterns of mtor, hif1α, and hdac12 were found upregulated post β-1,3-glucan restimulation in vitro . Further, a significantly upregulated expression profile of proinflammatory cytokines ( tnfα, il6, and ifnγ ) was observed. Overall, the study highlighted the expression patterns of trained immunity markers and the enhanced metabolic activity in cells stimulated with β-1,3-glucan, further confirming the crucial role of trained immunity in fish when exposed to a potential ligand. These findings validate existing reports on trained immunity in teleost fish through metabolic and epigenetics approaches. However, further research is needed to understand the responses and functions of trained immune cells during infection.