Leukocytes play a crucial role in the defense against bacterial pathogens. When encountering a microorganism, leukocyte cells adjust their bioenergetic processes to activate defense mechanisms. Mammalian macrophages shift their metabolism to glycolysis upon polarization, but little is known about fish leukocytes’ bioenergetics. Beta-glucan, a component of yeast cell walls, is widely used in aquaculture as an immunostimulant and is a promising product for the channel catfish industry to reduce disease impacts. This study evaluated the metabolic pathways profile of adherent and nonadherent channel catfish leukocytes fed with different dietary beta-glucan concentrations.
Channel catfish (~35 g) were stocked in 16 tanks (30 L) at a stocking density of 10 fish per tank.
Fish were fed with 0, 50, 100, and 200 µg of beta-glucan per g of body weight per day for 21 days. The head kidney of six fish per group was collected ten days after the beta-glucan exposure. The leukocytes were isolated from the head kidneys and incubated in 6-well plates with modified AIM V culture medium at 28 ºC with 5% CO2 overnight to allow adherent cells (mostly macrophages) to attach to the bottom of the well. The next day, non-adherent cells (granulocytes, lymphocytes, and NK-cells) were separated. Both cell types were plated in a Seahorse XF 96 Cell Culture microplate coated with Cell-Tak at a concentration of 8 × 104 cells per well. The Seahorse Mito Stress Test was performed at 28ºC. Reagents and their concentrations were injected into each well in the following order: oligomycin (1.5 μM), FCCP (0.5 μM), and Rotenone/Antimycin (1 μM). The Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured using the Seahorse XF Pro before and after the reagents injection.
Adherent cells from fish fed 100 µg of beta-glucan exhibited the highest OCR, followed by those fed 50 µg, the control group, and 200 µg. In contrast, the ECAR was highest in fish fed 50 µg, followed by 100 µg, the control, and 200 µg. Nonadherent cells from fish fed 200 µg of beta-glucan showed the highest baseline OCR, followed by 50 µg, 100 µg, and the control. ECAR was variable across treatments in non-adherent cells from fish fed 50, 100, and 200 µg of beta-glucan compared to the control group. Overall, ECAR was higher in the treatment 200 µg compared to the control group.
Studying leukocyte metabolism during activation helps understand how these cells manage their metabolic pathways to eliminate pathogens. In conclusion, beta-glucan has modulated adherent cells and nonadherent leukocytes metabolism differentially. These findings provide insight into beta-glucan-induced immune mechanisms underlying immunostimulant efficacy in channel catfish.