Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells

Abstract

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

Keywords: Atlantic salmon; cell differentiation; head kidney culture; macrophages; miRNA; sequencing.

Figure 1

Influence of culture time on the morphology and function of Atlantic salmon adherent head kidney leukocytes (HKLs). Representative images of Giemsa-stained HKLs cultured for (A) 1 day and (B) 5 days. Arrows indicate spread cells (S; cells with pseudopodia present) and non-spread, round cells (R; cells with no pseudopodia present). Scale bar on the bottom left of panel (A) and panel (B) is equal to 20 μm. (C) Mean percentage of round vs. spread cells in Day 1 and Day 5 cultures, where *** indicates a significant difference of p < 0.0001 by chi-square test. (D) Percentage of phagocytic HKLs on Day 1 and Day 5 of culture. (E) Percentage of HKLs producing reactive oxygen species (ROS), as determined by flow cytometry. (F) Maximum rate of ROS production (pmol/(s*million cells)), as determined by Oroboros respirometry. Day 1 control value: 0.089 pmol/(s*million cells); Day 5 control value: 0.105 pmol/(s*million cells). Data shown as the mean +/− SE; different lowercase letters indicate a significant difference of p < 0.05 as determined by a repeated measures one-way ANOVA for phagocytosis data and a paired Student’s T-test for ROS data, n = 4. PMA: phorbol myristate acetate.

Figure 2

miRNA expression and diversity (average normalized read counts of 370 miRNAs) in Day 1 and Day 5 Atlantic salmon adherent HKLs. The top 20 most abundant miRNAs in Day 1 and Day 5 are indicated. * indicates miRNAs present in the top 20 of one day but not the other day.

Figure 3

Results from hierarchical clustering of differentially expressed (DE) miRNAs from Day 1 and Day 5 HKLs shown as a heatmap. miRNAs counts per million were normalized and clustered using Pearson correlation and complete linkage hierarchical clustering. F indicates fish number; D indicates Day 1 or Day 5 (for example, F1D1 is Fish 1 Day 1).

Figure 4

qPCR analysis of DE miRNAs identified by small RNA sequencing. Data shown as the mean log₂ relative quantity (RQ) ± SE., n = 5. Significance determined by a Student’s T-test, * p < 0.05 and ** p < 0.01. (A) miR-146a, (B) miR-146b, (C) miR-155, (D) miR-221, (E) miR-126, (F) miR-150, (G) miR-139, (H) miR-2188 and (I) miR-200ae.