Dynamics of Polarized Macrophages and Activated CD8+ Cells in Heart Tissue of Atlantic Salmon Infected With Piscine Orthoreovirus-1

Abstract

Piscine orthoreovirus (PRV-1) infection causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is also associated with focal melanized changes in white skeletal muscle where PRV-1 infection of macrophages appears to be important. In this study, we studied the macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes during experimentally induced HSMI. The immune response in heart with HSMI lesions was characterized by CD8⁺ and MHC-I expressing cells and not by polarized macrophages. Fluorescent in situ hybridization (FISH) assays revealed localization of PRV-1 in a few M1 macrophages in both heart and skeletal muscle. M2 type macrophages were widely scattered in the heart and were more abundant in heart compared to the skeletal muscle. However, the M2 macrophages did not co-stain for PRV-1. There was a strong cellular immune response to the infection in the heart compared to that of the skeletal muscle, seen as increased MHC-I expression, partly in cells also containing PRV-1 RNA, and a high number of cytotoxic CD8⁺ granzyme producing cells that targeted PRV-1. In skeletal muscle, MHC-I expressing cells and CD8⁺ cells were dispersed between myocytes, but these cells did not stain for PRV-1. Gene expression analysis by RT-qPCR complied with the FISH results and confirmed a drop in level of PRV-1 following the cell mediated immune response. Overall, the results indicated that M1 macrophages do not contribute to the initial development of HSMI. However, large numbers of M2 macrophages reside in the heart and may contribute to the subsequent fast recovery following clearance of PRV-1 infection.

Keywords: Atlantic salmon; Piscine orthoreovirus 1; cell mediated immunity; heart and skeletal muscle inflammation; macrophage polarization.

Figure 1

Fluorescent in situ hybridization (FISH) of iNOS2, Arg2 and PRV-1 (A–F) in heart tissue at the peak of HSMI (6 wpc). (A) A limited number of iNOS2 (red) positive M1 macrophages were detected. (B) Merged image showing modest co-localization of PRV-1 and iNOS2 (inset). (C) Widely distributed Arg2 (purple) positive M2 macrophages. (D) Merged image does not show co-localization of PRV-1 and Arg2-positive M2 macrophages. (E) Phase contrast image showing aggregated blood cells in the stratum spongiosum layer. (F) PRV-1 (green) infected cells amongst clustered blood cells. Cellular nuclei stained with DAPI (blue) (Scale Bar = 100 µm). (G) Relative fold change expression (medians) of iNOS2 and Arg2 at 4 wpc and 6 wpc normalized against EF1α expression. (*) shows significant difference from the control (**p < 0.01).

Figure 2

Fluorescent in situ hybridization (FISH) of iNOS2, Arg2 and PRV-1 (A–F) in skeletal muscle at the peak of HSMI (6 wpc). (A) Few iNOS2 (red) positive M1 macrophages were detected. (B) Merged image showing co-localization (yellow in inset) of PRV-1 and iNOS2-positive M1 macrophages. (C) Sporadic presence of Arg2 (purple) positive M2 macrophages (D) Merged image does not show co-localization of PRV-1 and Arg2 (E) Phase contrast image showing aggregated blood cells in a vessel in the center. (F) A limited number of PRV-1 infected cells (green) were found (Scale Bar = 100 µm). (G) Relative fold change expression (medians) of iNOS2 and Arg2 at 4 wpc and 6 wpc normalized against EF1α. (*) show significant difference from the control (**p < 0.01).

Figure 3

Fluorescent in situ hybridization (FISH) of PRV-1, CD8α and GzmA in heart tissue at peak of HSMI associated histopathological changes (6 wpc). (A) Phase contrast image showing blood cells in the spongiosum. (B) Nuclei stained with DAPI (Blue). (C) Merged image showing co-localization of PRV-1 (green) with CD8+ (red) cells and cells expressing GzmA (purple) (arrows, yellow in inserts). GzmA stained cells were prominent in areas with dense PRV-1 staining (Scale Bar = 50 µm). (D, E) Relative fold change expression (medians) of CD8α and GzmA at 4 wpc and 6 wpc normalized against EF1α. Dots show outlier values in the respective group of fish. (*) shows significant difference from the control (*p < 0.05, **p < 0.01).

Figure 4

Fluorescent in situ hybridization (FISH) of CD8α, GzmA and PRV-1 in skeletal muscle at the peak of HSMI (6 wpc). (A) Phase contrast image showing structures of myocytes. (B) Nuclei stained with DAPI (Blue). (C) Merged image showing presence of PRV-1 (green) infected cell (arrowhead), and CD8+ cells (insets). Some cells were stained positive only for CD8α and GzmA specific transcripts (arrows) (Scale Bar = 50µm). (D, E) Relative fold expression (medians) of CD8α and GzmA at 4 wpc and 6 wpc normalized against EF1α. (*) shows significant difference from the control (*p < 0.05, **p < 0.01).

Figure 5

Fluorescent in situ hybridization (FISH) of PRV-1, MHC-I and IL-17A in heart tissue at peak of HSMI (6 wpc). (A) Phase contrast image showing an area with aggregated blood cells (star) in the spongiosum layer. (B) Merged image showing presence of PRV-1 (green) in cardiomyocytes and clotted blood cells, partially co-localizing with MHC-I (red) positive cells (insets). Some MHC-I positive cells in the aggregated blood did not co-stain for PRV-1 (arrow). IL-17A (purple) positive cells were sporadically scattered in the tissue (arrowhead). Nuclei stained with DAPI (Blue) (Scale Bar = 50 µm). (C) Relative fold change expression (medians) of MHC-I at 4 wpc and 6 wpc normalized against EF1α. Asterisk (*) shows significantly difference from the control (**p < 0.01).

Figure 6

Fluorescent in situ hybridization (FISH) of PRV-1 and MHC-I in skeletal muscle at peak of HSMI associated histopathological changes (6 wpc). (A) Phase contrast image showing myocyte structure (B) Merged image showing MHC-I (red) positive cells (arrowheads) and PRV-1 (green) infected cells (arrow). Nuclei stained with DAPI (Blue) (Scale Bar = 50 µm). (C) Relative fold change expression (medians) of MHC-I at 4 wpc and 6 wpc normalized against EF1α. Dot sign shows outlier value from the respective fish group. (*) shows significantly difference from the control (**p < 0.01).