Rabies virus uniquely reprograms the transcriptome of human monocyte-derived macrophages

Abstract

Macrophages are amongst the first immune cells that encounter rabies virus (RABV) at virus entry sites. Activation of macrophages is essential for the onset of a potent immune response, but insights into the effects of RABV on macrophage activation are scarce. In this study we performed high-throughput sequencing on RNA extracted from macrophages that were exposed to RABV for 48 hours, and compared their transcriptional profiles to that of non-polarized macrophages (M0), and macrophages polarized towards the canonical M1, M2a and M2c phenotypes. Our analysis revealed that RABV-stimulated macrophages show high expression of several M1, M2a and M2c signature genes. Apart from their partial resemblance to these phenotypes, unbiased clustering analysis revealed that RABV induces a unique and distinct polarization program. Closer examination revealed that RABV induced multiple pathways related to the interferon- and antiviral response, which were not induced under other classical polarization strategies. Surprisingly, our data show that RABV induces an activated rather than a fully suppressed macrophage phenotype, triggering virus-induced activation and polarization. This includes multiple genes with known antiviral (e.g. APOBEC3A, IFIT/OAS/TRIM genes), which may play a role in anti-RABV immunity.

Keywords: Lyssavirus; RNA sequencing; innate immunity; macrophage polarization; rabies virus (RABV); transcriptomics.

Figure 1

Experimental layout, initial analysis on sample variation and confirmation of the polarization model. A schematic overview of the presented transcriptome study is shown in (A), indicating the experimental timeline, the experimental groups and the number of reads and genes retrieved from the Illumina sequencing. Clustering of the individual samples was based on calculated Pearson’s correlation coefficients (B). The number of substantially expressed genes (mean RPKM > 0.5 of the three individual samples per phenotype), is presented in the Venn diagram in (C). The expression of signature M1, M2a or M2c genes in all individual samples is shown, in log₂ RPKM, in (D). The dotted line indicates the threshold for substantial expression, at 0.5 RPKM or -0.3 log₂. For clarity, samples with an expression level of <0.5 RPKM are all placed below this line.

Figure 2

Initial analysis of the similarities and differences between the M0, M1, M2a, M2c and RABV macrophage gene expression profiles. All genes that are substantially expressed in at least one of the phenotypes (mean RPKM > 0.5) were submitted in a principal component analysis (A), showing the two components responsible for the majority of variation (PC1 and PC2) and the top 10 active variables that contribute the most to this variation (highest cos₂ values, grey arrows). The expression levels of these 10 genes for every individual samples in shown in the right graph, in log₂ RPKM. The number of differentially expressed genes (DEGs, p_adj < 0.05, log₂ fold change < -1 or > 1) between the five different macrophage phenotypes is shown in (B), as well as the number of upregulated or downregulated DEGs when compared to M0 (C).

Figure 3

Clustering of all DEGs of the five studied macrophage phenotypes, and the major differences in enriched pathways between M1 and RABV macrophages. All genes that were differentially upregulated in at least one phenotype were clustered (A). A cluster of highly upregulated genes of RABV macrophages (highlighted in red) was submitted to pathway enrichment analysis. Pathway enrichment analyses were also performed on genes that were differently upregulated in RABV macrophages but not in M1 macrophages, and vice versa (B).

Figure 4

Visualization of the most significantly regulated genes of RABV-macrophages, and the induction of multiple virus response-related pathways. A volcano plot highlights the genes with the lowest adjusted p-value (p_adj , genes in italics) and the highest upregulation (log₂ fold change, genes in bold) of RABV macrophages compared to M0 macrophages (A). Genes in red have a log₂ fold change between -1 and 1 and are not differentially expressed. Log₂ fold change in M1, M2a and M2c macrophages of the highlighted genes in (A) is shown in (B). Asterisks (*) in B indicate that expression levels in RABV macrophages are significantly higher than in M1 macrophages. Clustering analysis (C) and pathway enrichment analysis (D) of the differentially and substantially expressed (RPKM > 0.5) genes of RABV macrophages. The top 20 induced pathways of RABV are compared to that of the other phenotypes, and p-values are shown if the specified pathway was present in the top 20 of the specified macrophage phenotype.