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. 2020 Oct 15:8:580848.
doi: 10.3389/fcell.2020.580848. eCollection 2020.

Adherent Intestinal Cells From Atlantic Salmon Show Phagocytic Ability and Express Macrophage-Specific Genes

Youngjin Park  1 , Qirui Zhang  2 , Geert F Wiegertjes  3 , Jorge M O Fernandes  1 , Viswanath Kiron  1
Affiliations

Adherent Intestinal Cells From Atlantic Salmon Show Phagocytic Ability and Express Macrophage-Specific Genes

Youngjin Park et al. Front Cell Dev Biol. .

Abstract

Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of Escherichia coli BioParticlesTM. AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells.

Keywords: Atlantic salmon; ImageStream®X; RNA-Seq; adherent; intestinal cells; macrophages; phagocytosis.

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Figures

FIGURE 1
FIGURE 1
Cell populations from Atlantic salmon distal intestine. (A) Live cells are shown in a brightfield (BF) area (cell size) vs. side scatter (SSC) intensity plot (cell granularity). Representative images (40 × objective, scale bar = 10 μm) from each gate are shown separately. Intestinal cells were identified based on nucleus morphologies. Using propidium iodide, live or no-stained cells (B) and dead or nucleus-stained cells (C) are shown. (D) A representative image captured by fluorescent imager (20 × objective, scale bar = 25 μm) shows salmon intestinal cell population. LYM, lymphocytes; MON, monocytes/macrophages; R, region: R1, macrophage-like cells; R2, unknown cells; R2-1, R2-2, presumptive granulocytes; R2-3, presumptive vacuolated cells.
FIGURE 2
FIGURE 2
Examination of adherent cell population from Atlantic salmon head kidney and distal intestine by imaging flow cytometry. We compared cell populations from three cell suspensions; (A and E) whole leukocytes (HKL and DIL, respectively), (B and F) supernatants (HS and DS, respectively) and (C and G) adherent cells (AKC and AIC, respectively) in a brightfield (BF) area (cell size) vs. side scatter (SSC) intensity plot (cell granularity) using IFC. The bar plots (D and H) indicate percent of the cells in the red gates in supernatants (B and F) vs. those in adherent cells (C and G, respectively). Statistically significant differences (p < 0.001) are indicated using asterisks. Bar plots show the mean ± SD (n = 6).
FIGURE 3
FIGURE 3
Phagocytic activity of whole leukocytes and adherent cells from Atlantic salmon head kidney. Percent of phagocytic cells or phagocytic ability (A) and mean number of bio-particles (BP) ingested per phagocytic cell or phagocytic capacity (B) are shown. (C) Representative cell images show cells with no BP, and 1BP, 2BP, and >3BP. Statistically significant differences (p < 0.05) are indicated using asterisks. Bar plots show the mean ± SD (n = 6). All cell images were captured with 40 × objective. Scale bar = 10 μm. BF, brightfield; 1 BP, 2 BP, and > 3 BP, 1–3 internalized bio-particles.
FIGURE 4
FIGURE 4
Phagocytic activity of whole leukocytes and adherent cells from Atlantic salmon distal intestine. Percent of phagocytic cells or phagocytic ability (A) and mean number of bio-particles (BP) ingested per phagocytic cell or phagocytic capacity (B) are shown. (C) Representative cell images show cells with no BP, and 1BP, 2BP, and >3BP. Statistically significant differences (p < 0.05) are indicated using asterisks. In addition, oval shaped phagocytes (D) and doublets containing a phagocyte that interacts with a lymphocyte-like cell (E) were found among the adherent intestinal cells. Bar plots show the mean ± SD (n = 6). All cell images were captured with 40 × objective. Scale bar = 10 μm. BF, brightfield; 1 BP, 2 BP, and >3 BP, 1–3 internalized bio-particles.
FIGURE 5
FIGURE 5
The expression of genes linked to macrophages, dendritic cells and endothelial cells in the adherent cells from Atlantic salmon distal intestine and head kidney. Employing the normalized read counts from DESeq2 analyses, the expression of genes related to macrophages (A), dendritic cells (B) and endothelial cells (C) were determined, and the expression levels between the adherent cells from salmon distal intestine (AIC) and head kidney (AKC) were compared. Statistically significant differences (p < 0.05) are indicated using asterisks. Bar plots show the mean ± SD. Sample size = 6.
FIGURE 6
FIGURE 6
The expression of genes related to T and B cells in the adherent cells from Atlantic salmon distal intestine and head kidney. Employing the normalized read counts from DESeq2 analyses, the expression of T (A) and B cells (B) associated genes in the adherent cells from salmon distal intestine (AIC) and head kidney (AKC) were compared. Statistically significant differences (p < 0.05) are indicated using asterisks. Bar plots show the mean ± SD (n = 6).
FIGURE 7
FIGURE 7
The expression of cytokine-related genes in the adherent cells from Atlantic salmon distal intestine and head kidney. Employing the normalized read counts from DESeq2 analyses, the expression of cytokine-related genes in the adherent cells from salmon distal intestine (AIC) and head kidney (AKC) were compared. Statistically significant differences (p < 0.05) are indicated using asterisks. Bar plots show the mean ± SD (n = 6).
FIGURE 8
FIGURE 8
The expression of genes related to chemokines, mucins and toll-like receptors in the adherent cells from Atlantic salmon distal intestine and head kidney. Employing the normalized read counts from DESeq2 analyses, the expression of genes related to chemokines (A), mucins (B) and toll-like receptors (C) in the adherent cells from salmon distal intestine (AIC) and head kidney (AKC) were compared. Statistically significant differences (p < 0.05) are indicated using asterisks. Bar plots show the mean ± SD (n = 6).
FIGURE 9
FIGURE 9
Heatmap of the expression of selected genes in the adherent cells from Atlantic salmon distal intestine and head kidney. Box plots indicate the expression of genes related to macrophages, dendritic cells, endothelial cells, T and B cells. The color gradient from light yellow to dark red in the heatmap indicates increasing transcript levels.
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