Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Immunometabolism

• Topography

• Hypoxia

• Tuberculosis Granuloma

Main Findings

A hallmark of Mycobacterium tuberculosis (Mtb) infection is the formation of spatially organized granulomatous structures composed of diverse immune and stromal cell populations. While tuberculosis (TB) granulomas are central to bacterial control and host survival, their spatial and functional dynamics that govern organization and immune competence remain incompletely understood. Previous work by McCaffrey et al. (Nat Immunol, 2022) identified an immune-tolerant macrophage phenotype in human TB granulomas that was marked by PD-L1, IDO1, TGFβ, and IL-10 expression. Lymphocytes expressing IFNγ were preferentially absent from this tolerogenic niche.

In the current preprint, McCaffrey & Delmastro et al. expand upon this by dissecting the spatial immunometabolic landscape of TB granulomas in both nonhuman primates and human lung tissue. The authors have used in-situ hypoxia sensor pimonidazole to identify hypoxic region surrounding the necrotic core of granulomas in nonhuman primates. To further understand the different immune and stromal cells, immunometabolic markers and their spatial distribution, the authors have performed a multiplexed imaging method known as multiplexed ion beam imaging by time-of-flight (MIBI-TOF). For data analysis the authors have implemented spatial analysis method used to analyse maps and topographic information. In addition, the authors have also used single cell RNA-sequencing, spatial transcriptomics, immunofluorescence and epigenetic sequencing.

The inner caseous necrotic zone that contains debris from dead macrophages and neutrophils alongside Mtb is encircled by the hypoxic zone characterized by peri-necrotic macrophages with elevated glycolytic markers such as GLUT1. This region is also enriched for neutrophils, fibronectin-positive macrophages, CD11c+ and CD68+ macrophages but lacks T cells. Interestingly, macrophages in this hypoxic zone have downregulation of HLA-DR, one of the key markers involved in T cell activation. Surrounding this inner myeloid region is an outer myeloid zone enriched in strong expression of indoleamine 2,3-dioxygenase (IDO1), a rate limiting enzyme of tryptophan catabolism that usually has potent immunosuppressive effect. This IDO1+ region comprises CD14+CD11c+ macrophages and T cells, exhibiting interferon signalling, reactive oxygen species, and epigenetic signatures of immune activity. The outermost region (the cuff) is dominated by lymphocytes. Importantly, bacterial burden is highest in the hypoxic, glycolysis-dominant zone and TB granulomas with high Mtb burden show an expansion of this area. 

Hence, the authors suggest that hypoxia is the core driver for zonation in immunometabolic and epigenetic-mediated immune subversion in TB granulomas that allow bacterial growth.

Significance and Novelty

The high resolution 38-plex MIBI-TOF analysis pipeline to create topographic zonations called “buffer zones” represents a significant advancement in understanding in situ context and cellular composition of immune cells within TB granulomas. The authors have meticulously analyzed the different macrophage subtypes and their immunometabolic characteristics across the various “buffer” zones from the granuloma center to the periphery. The authors indicate that hypoxia creates a broadly immunosuppressive environment affecting both macrophages and T cells. The presence of the complement protein C1QB extending from IDO1+ zone into the lymphocytic area suggests that complement pathways are important for bacterial control. In addition, the authors propose that structural elements such as collagen deposition and vascularization could further influence the spatial dynamics of TB granulomas, with potential implications for immune activation and lesion remodelling.

Limitations and suggestions

• This study's observational nature highlights the need for more research on how hypoxia affects immune responses in TB granulomas. It is unclear if immunometabolic zonation precedes or follows necrotic core formation. Exploring whether these zones correlate with lipid-rich foamy macrophages—another hallmark of TB granulomas—could provide deeper insights.

• Exploring how host redox and metabolic mechanisms shape topographic zonation could be insightful. In situmetabolomics and metabolic analysis of organotypic macrophage cultures under different partial pressure of oxygen might provide further insights. The secretome of macrophages might provide additional insights about why T cell infiltration is halted in the hypoxic zone of TB granulomas.

• Differential spatial distribution of neutrophils among high- and low-burden granulomas may explain their role in balancing bacterial control as opposed to immune suppression in TB granulomas.

• The impact of oxygen gradients on complement-driven metabolic pathways and cytokine gradients needs deeper investigation.

• The authors could use lung tissues from responder and non-responders of antitubercular treatment and analyse from patients with HIV co-infection.

Given the dynamic host-pathogen interplay, complementing in vivo data with in vitro and ex vivo metabolomics and other functional assays may strengthen the conclusions.

Altogether, the preprint conveys a novel and multimodal framework of spatial patterning of immunometabolic zonation in TB granuloma, offering potential insights for the design of host-directed therapeutics.

Credit

Reviewed by Amitava Sinha as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.

The author declares no conflict of interests in relation to their involvement in the review.