Adherent Intestinal Cells from Atlantic Salmon Show Phagocytic Ability and Express Macrophage Markers

Atlantic salmon (average 550g) were used in this study. The fish were starved for 24 h and were sacrificed with an overdose of MS-222. Then head kidney (HK) and distal intestine (DI) samples were collected. Immune cells from HK and DI were isolated and cultured at 12°C in Leibovitz’s L-15 Medium (L-15). Both HK and DI leukocytes were allowed to adhere on a petri dish with 2 mL L-15+ for 2 days at 12°C. After removing the supernatant containing non-adherent cells, the petri dish with the adherent cells was placed on ice for 10 min, and the cells were detached by washing three times with 1.5 mL ice-cold PBS supplemented with 5 mM EDTA. The cells obtained were centrifuged (500 × g, 5 min, 4°C) and re-suspended in 1 mL L-15+. Then, the cells were counted using a portable cell counter. Total RNA was extracted from adherent cells from HK or DI (< 500,000 cells). To compare the expression of marker genes between adherent cells from HK and DI, 12 libraries were prepared (6 replicates for each). Transcriptomic analysis revealed that adherent intestinal cells (AICs) expressed genes associated with macrophages, T cells, and endothelial cells. In addition, AIC had higher expression of TNF and TGFβ-related genes that can be linked to macrophages and T cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related markers. Twelve samples were prepared including 6 from distal intestine and 6 from head kidney.