Interrogation of Mycobacterial Granulomas with Single-Cell Resolution

Abstract

Tuberculous granulomas – the central immune structures of tuberculosis – are the primary site of host-pathogen interactions. These are complex, multi-cellular host immune structures that dictate disease progression and physiology. Due to the heterogenous nature of the cells that contribute the granuloma, signaling cues from diverse populations of cells can be overlooked by bulk analyses. While macrophages and monocytes make up the majority of cells in a given granuloma, other myeloid cells, lymphocytes, and stromal are known to play important roles in infection trajectory and control. A hallmark of tuberculous granulomas are epithelioid macrophages. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition or whether small populations of cells can determine cell signaling at the level of a granuloma.Using the zebrafish-Mycobacterium marinum model of infection together with single-cell RNA-seq analysis of experimentally accessible and genetically tractable zebrafish granulomas we identified 1) the basis of granuloma macrophage transformation and 2) a novel granuloma-associated fibroblast cell. Even in the presence of robust type 1 immune responses that are typical for tuberculous granulomas, countervailing type 2 signals associate with macrophage epithelialization. We find that stat6-mediated type 2 immune signaling is required for macrophage epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell-autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the granuloma structure of mycobacterial infection. In addition, we analyzed wildtype adult mycobacterial granulomas and granulomas from the genetically susceptible lta4h mutant with single-cell resolution in zebrafish. lta4h is implicated in vertebrate control of mycobacterial infection as well as human TB severity. We find that eicosanoid perturbations disrupt the formation of a fibroblast cell layer at the granuloma periphery, which is associated with containment of infection and conserved in human TB granulomas. This unique fibroblast population is marked by a transcriptional profile with mesenchymal and stem-like properties of dermal fibroblasts, including expression of the aldehyde dehydrogenase aldh1a3 and apolipoprotein D. The expression of apod in these cells is associated with ROS regulation in the granuloma. Diverse tuberculous granulomas across patients and tissues in humans also generate these unusual fibroblast populations. These findings establish that crosstalk between stromal and immune cells, shaped by host lipid mediators, contributes to divergent pathological outcomes during mycobacterial infection.