RNA sequencing provides exquisite insight into the manipulation of the alveolar macrophage by tubercle bacilli

Abstract

Mycobacterium bovis, the agent of bovine tuberculosis, causes an estimated $3 billion annual losses to global agriculture due, in part, to the limitations of current diagnostics. Development of next-generation diagnostics requires a greater understanding of the interaction between the pathogen and the bovine host. Therefore, to explore the early response of the alveolar macrophage to infection, we report the first application of RNA-sequencing to define, in exquisite detail, the transcriptomes of M. bovis-infected and non-infected alveolar macrophages from ten calves at 2, 6, 24 and 48 hours post-infection. Differentially expressed sense genes were detected at these time points that revealed enrichment of innate immune signalling functions, and transcriptional suppression of host defence mechanisms (e.g., lysosome maturation). We also detected differentially expressed natural antisense transcripts, which may play a role in subverting innate immune mechanisms following infection. Furthermore, we report differential expression of novel bovine genes, some of which have immune-related functions based on orthology with human proteins. This is the first in-depth transcriptomics investigation of the alveolar macrophage response to the early stages of M. bovis infection and reveals complex patterns of gene expression and regulation that underlie the immunomodulatory mechanisms used by M. bovis to evade host defence mechanisms.

Alveolar macrophage chromatin is modified to orchestrate host response toMycobacterium bovisinfection

“…Previous studies have shown that bAM undergo extensive gene expression reprogramming following infection of M. bovis (Nalpas et al, 2015), with approximately one third of the genome significantly differentially expressed within bovine macrophages 24 hours after infection (Malone et al, 2018). Changes of this magnitude are comparable to those observed in previous experiments that have examined the chromatin remodelling that accompanies mycobacterial infection of macrophages, where trimethylation of lysine 4 of Histone H3 (H3K4me3) was shown to correlate with active transcription (Arts et al, 2018; Bouttier et al, 2016).…”

“…( C ) Correlation and Venn diagram for both RNA-seq studies. The x-axis of the scatter plot represents the log 2 fold change for each of the 232 genes from this study and the y-axis represents the log 2 fold change for each of the 232 genes from the previous study (Nalpas et al, 2015). The Venn diagram shows the global overlap of differentially expressed genes from both studies with an FDR cut off of <0.1.…”

“…Existing published RNA-seq data generated by our group using M. bovis -infected ( n = 10) and control non-infected bAM ( n = 10) at 24 hpi (Nalpas et al, 2015), was also examined in light of the results from the present study. For the 232 genes identified here, a Pearson correlation coefficient of 0.85 was observed for two data sets (Figure 3D), thus demonstrating that gene expression differences between M. bovis -infected and control non-infected bAM are consistent across experiments, even where samples sizes are markedly different.…”

“…Considering the dramatic perturbation of the vertebrate macrophage by intracellular mycobacteria, we and others have demonstrated that bovine and human alveolar macrophage transcriptomes are extensively reprogrammed in response to infection with M. bovis and M. tuberculosis (Jensen et al, 2018; Lavalett et al, 2017; Malone et al, 2018; Nalpas et al, 2015; Papp et al, 2018; Vegh et al, 2015). These studies have also revealed that differentially expressed gene sets and dysregulated cellular networks and pathways are functionally associated with many of the macrophage processes described above that can control or eliminate intracellular microbes.…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…Mtb devoid of a capsule induced a strong pro-inflammatory response, and downregulated for example the expression of the IFNγ receptor, which could promote survival and persistence of Mtb [356,357]. Another study looking into the transcriptional response of bovine alveolar macrophages after M. bovis infection obtained conclusions different from other studies with similar settings, partly attributed to the use of detergent-free cultivated bacilli, but unfortunately a direct comparison of the transcriptomics upon infection of Mtb cultivated with or without detergent was not performed here [358]. Looking into the immunological response after BCG-vaccination and Mtb-challenge of mice, encapsulated BCG triggered a more potent immune response as compared to non-encapsulated (detergent-cultivated) BCG, featuring polyfunctional T cells, higher IFNγ production and lower CFU burden [359].…”