Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

Abstract

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including γδ T cells, and candidate populations of monocytes/macrophages and dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver immune and non-immune cells in host defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

Keywords: bacterial infection; immune-metabolism cross talk; liver; salmonid fish; single cell transcriptomics.

Figure 1

Major cell types in the Atlantic salmon liver defined by 47,982 nuclear transcriptomes. (A) UMAP highlighting five main liver cell-type clusters according to a priori defined marker genes (see Supplementary Table 2 ). (B) Bubble plots showing the expression of a priori marker genes for the five main liver cell types, including mean expression (bubble intensity) and percentage of nuclei expressing gene (bubble size). (C) Heatmap of the top 20 differentially expressed genes per each liver cell type defined against the background of all other cell types. (D) Violin plots demonstrating the expression of the most specific marker genes per each of the main liver cell types (colours are matched to the colours of the 5 cell lineages defined in part A).

Figure 2

Higher resolution atlas of Atlantic salmon liver cells defined by snRNA-Seq. (A) Unbiased graph-based clustering reveals varying heterogeneity levels across the major liver cell types, presented on a UMAP. Each cell population retains the gene signature of the parent lineage ( Supplementary Figure 2 ), while also displaying its own distinct transcriptomic profile, presented here as a heatmap (B), inclusive of the top 10 marker genes based on differential gene expression against all other cell clusters. The colour bars above columns on the heatmap illustrate the cell types to which the genes shown were identified as markers (matched to part A).

Figure 3

Striking remodeling of the hepatocyte transcriptome in response to bacterial infection. (A) UMAP visualisation of 41,792 hepatocyte nuclei, with sub-clustering performed using the most variable genes restricted to this cell lineage. (B) Shows the same UMAP with nuclei coloured by infection state (top) and the proportion of nuclei originating from each sample after normalising for different nuclei numbers across samples (bottom). (C) Pearson correlation of the expression values for the top 2,000 most variable genes across the nine hepatocytes populations H1-H9. (D) Violin plots of mean expression for the 20 most down-regulated (left) and up-regulated (right) genes based on log-fold change in H4 vs. H1. (E) Heatmap of the top 20 most upregulated and top 20 most downregulated genes in H4 relative to H1, illustrating a gradient of expression from H1 → H2 → H3 → H4 (F) Example enriched GO terms in H4 based on all up-regulated and all down-regulated genes in H4 vs. H1 (full data provided in Supplementary Tables 10 and 11 ).

Figure 4

Heterogeneity in Atlantic salmon immune cells. (A) UMAP visualization of 1,620 immune nuclei. (B) Heatmap of top 5 markers genes for each immune sub-cluster, sorted by log2 fold change. (C) Number of cell-specific genes up-regulated by infection in immune sub-clusters (D) Examples of genes showing cell-specific up-regulation in response to Aeromonas infection across the breath of immune cell heterogeneity identified.