Comparative multi-omics of the macrophage response to infection with Mycobacterium tuberculosis complex bacteria reveals pathogen-driven epigenomic reprogramming

Reviewer #1 (Public review):

This manuscript by Hall et al. uses a multi-omic approach to investigate how distinct members of the Mycobacterium tuberculosis complex (MTBC: M. bovis, M. tuberculosis, the attenuated M. bovis BCG vaccine strain, and gamma-irradiated M. bovis) affect bovine alveolar macrophage epigenetic and transcriptional responses after 24 hours. The investigators used RNA-Seq, ATAC-Seq, and ChIP-Seq to assess differential gene expression in each complex type and integrated gene transcription with chromatin accessibility and epigenetic modifications, highlighting key immune response genes/pathways upregulated in response to infection and pathogen-specific host adaptation mechanisms. The analysis also revealed that the most pronounced transcriptional and epigenetic responses were in the M. bovis-infected cells compared with the other complex types. Comparing top genes associated with M. bovis infection of macrophages to a GWAS data set revealed 4 key genes associated with increased susceptibility to infection.

Overall, this is a technically sound manuscript that contains highly interesting and useful data on bovine innate immune responses to different types of Mycobacterium tuberculosis, which are important to the immunology and infectious disease community as well as the livestock industry. However, in its current format, the manuscript presents the data/figures in a way that is not particularly informative (despite the rich data set) and is too descriptive. We also have some general concerns and suggestions listed below.

Reviewer #2 (Public review):

Summary:

In this manuscript, Hall et al. present a rigorous and comprehensive multi-omic comparative analysis investigating how host-adapted and non-host-adapted mycobacteria reconfigure the bovine host immune response. By utilizing RNA-seq, ATAC-seq, and ChIP-seq across four histone marks (H3K4me3, H3K4me1, H3K27ac, and H3K27me3) plus CTCF binding, the authors track the regulatory dynamics of primary bovine alveolar macrophages (bAM) challenged with Mycobacterium bovis (MBO), Mycobacterium tuberculosis (MTU), M. bovis BCG, and gamma-irradiated (killed) M. bovis (IRR).

The study highlights a profound, pathogen-driven epigenomic reprogramming that is largely unique to the host-adapted virulent pathogen (M. bovis). Crucially, the authors integrate these regulatory networks with existing Holstein-Friesian GWAS datasets to prioritize novel candidate genes (ERBB4, LRCH1, MRTFA, and RNPC3) associated with M. bovis infection susceptibility. This work represents a significant advancement in our understanding of host-pathogen interactions and animal resilience to bovine tuberculosis (bTB).

Strengths:

(1) The manuscript addresses a major socioeconomic problem in global livestock agriculture and human zoonotic health. By profiling host-adapted vs. non-host-adapted and live vs. dead bacilli, it provides fundamental insights into mycobacterial virulence mechanisms and evolutionary adaptation.

(2) The multi-omic approach is robustly executed, and the sample size (n=6 for RNA-seq, n=3 for ChIP/ATAC-seq subgroups) is highly appropriate for primary livestock cell cultures.

(3) The inclusion of live virulent, live attenuated, non-host-adapted, and killed strains allows the authors to dissect whether host responses are driven by passive PAMP recognition or active, pathogen-directed virulence factors.

(4) The parallel mapping of four distinct histone marks alongside chromatin accessibility mapping (ATAC-seq) yields a highly refined picture of enhancer and promoter dynamics.

Weaknesses:

(1) The profound transcriptional response of the gamma-irradiated (IRR) M. bovis group (3,320 DEGs vs. 2,312 for MTU) is intriguing but lacks a deep biochemical and functional explanation.

(2) While the paper provides a clear atlas of epigenetic alterations, the underlying mycobacterial effectors driving these specific chromatin alterations remain largely correlative.

Reviewer #3 (Public review):

Summary:

Hall et al. use a multi-omics approach to investigate the responses of bovine alveolar macrophages to Mycobacterium tuberculosis infections and the underlying mechanisms that are shared between bacterial family members. The study is of particular importance for multiple reasons, including the impact of bovine infections on food supply (and resulting economic impacts) as well as the use of bovines as a large animal model to investigate the effects of M. tuberculosis infection. The authors isolated bovine alveolar macrophages and exposed them to infection with M. bovis, M. bovis BCG, irradiated M. bovis, or M. tuberculosis. 24 hours post-infection, samples were analyzed by RNAseq, ChIP-seq, and ATAC-seq to look at alterations in the transcriptome and epigenome. Through fluorescence imaging-based analysis, the authors show equivalent infection (bacterial uptake) in all groups, except for the irradiated M. bovis. Principal component analysis of the transcriptomic data demonstrated strong segregation for the M. bovine-infected cells compared to the other groups, which had some intermixing in the PCA. Among the differentially expressed genes, the cytokine IL36G was significantly upregulated across all four groups. This is significant as this cytokine enhanced autophagy in macrophages and subsequent M. tuberculosis killing activity. To further investigate the transcriptional changes, ChIP-seq and ATACseq were utilized to investigate chromatin changes in the form of differential affinity binding sites (DABS) and differential open chromatin regions (DOCR). TPMRSS2, a protease that plays an important role in multiple types of infections (tuberculosis, COVID, etc.), was found to be a significant DOCR in both the M. bovis and M. tuberculosis challenge groups, conferring enhanced TPMRSS2 expression in both of these groups. Using the integrative approach between all the omics data, the authors found that CD274, which encodes the PD-L1 protein, was upregulated in all four groups. PD-L1 is known to play an immunosuppressive role, and PD-L1+ macrophages have been shown to create "cold" microenvironments that would likely favor the mycobacterium. Lastly, SNP analysis found variants in four genes (ERBB4, LRCH1, MRTFA, RNPC3) that could serve as susceptibility genes.

Strengths:

Overall, this study demonstrates that challenge with M. bovis elicits a more extensive remodeling of chromatin and subsequent gene expression changes in macrophages compared to the other closely related strains. The work demonstrates the power of functional genomics and its utility in investigating the underlying changes that can affect responses to infections and subsequent outcomes. While the study lacks functional validation, the cohesive dataset is quite compelling, and from it, the authors draw conservative conclusions and are frank about their study limitations.

Weaknesses:

Lack of functional validation of some of the targets found.