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.
Highlights
Macrophages are some of the first cells that respond to infection and disease
To provide further confirmation that the head kidney leukocytes (HKLs) become more macrophage-like during culture time, we examined changes in the mRNA expression of two macrophage markers, macrophage receptor with collagenous structure and major histocompatibility complex II (MHC II) [10,33,34,35,36,37,38] in the samples used for miRNA qPCR validation
We found a decrease in miR-150 expression in Day 5 macrophage-like HKLs compared to Day 1 monocyte-like cells, suggesting that if miR-150 has the same role in Atlantic salmon as it does in other vertebrates, both the expression changes and the morphology analysis indicate that the HKLs become more macrophage-like and less monocyte-like during culture time. miR-126, along with a subset of miRNAs, is enriched in human and murine hematopoietic stem cells (HSCs), compared to other cells in the bone marrow, and its expression is decreased in differentiated cells of the lymphoid and myeloid lineages, as determined by a bidirectional miR-126 reporter vector [63]
Summary
Macrophages are some of the first cells that respond to infection and disease. They are critical in mounting and resolving an immune response during tissue injury and/or pathogen infection [1]. M1 macrophages are induced by cytokines primarily secreted by Th1 cells, including IFN-γ and TNF-α, and are involved in the inflammatory response, producing reactive oxygen species (ROS) and inflammatory cytokines. M2 macrophages are induced by cytokines including IL-4, IL-13 and TGF-β and are involved in wound healing, repair and immune suppression [4,5,6]. While our knowledge of fish macrophage biology is expanding, macrophage differentiation and activation across all teleost species, including the Atlantic salmon, an economically important farmed fish, remain to be adequately described
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