Spatial Resolution of Mycobacterium tuberculosis Bacteria and Their Surrounding Immune Environments Based on Selected Key Transcripts in Mouse Lungs

Abstract

Mycobacterium tuberculosis (Mtb) bacilli are the causative agent of tuberculosis (TB), a major killer of mankind. Although it is widely accepted that local interactions between Mtb and the immune system in the tuberculous granuloma determine whether the outcome of infection is controlled or disseminated, these have been poorly studied due to methodological constraints. We have recently used a spatial transcriptomic technique, in situ sequencing (ISS), to define the spatial distribution of immune transcripts in TB mouse lungs. To further contribute to the understanding of the immune microenvironments of Mtb and their local diversity, we here present two complementary automated bacteria-guided analysis pipelines. These position 33 ISS-identified immune transcripts in relation to single bacteria and bacteria clusters. The analysis was applied on new ISS data from lung sections of Mtb-infected C57BL/6 and C3HeB/FeJ mice. In lungs from C57BL/6 mice early and late post infection, transcripts that define inflammatory macrophages were enriched at subcellular distances to bacteria, indicating the activation of infected macrophages. In contrast, expression patterns associated to antigen presentation were enriched in non-infected cells at 12 weeks post infection. T-cell transcripts were evenly distributed in the tissue. In Mtb-infected C3HeB/FeJ mice, transcripts characterizing activated macrophages localized in apposition to small bacteria clusters, but not in organized granulomas. Despite differences in the susceptibility to Mtb, the transcript patterns found around small bacteria clusters of C3HeB/FeJ and C57BL/6 mice were similar. Altogether, the presented tools allow us to characterize in depth the immune cell populations and their activation that interact with Mtb in the infected lung.

The use of multiplex imaging techniques to characterize tuberculous granuloma heterogeneity

“…Techniques used References The highly heterogeneous granulomas were found and grouped into three categories as big organized, intermediate organized and small clusters based on the size, shape and composition of the granulomas. The organized granulomas present in round structure with a necrotic center and lymphocytic cuff, while the small clusters are in uneven structure consisting of epithelioid cells ( Magoulopoulou et al, 2022). Both approaches of multiplex imaging reveal a diverse distribution of immune cells, suggesting that some of the TB granulomas recruit more immune cells than others.…”

“…Integration of these two complementary techniques allows the tissue to be mapped and the spatial associations between the components in the tissue studied. Magoulopoulou et al, 2022 used this strategy to study leprosy granulomas, and developed a quantitative analysis strategy to characterize the immune landscape of human leprosy granulomas in further detail. A total of 12 cell subtypes were identified in leprosy legions using scRNA‐seq, including macrophages, T cells, keratinocytes, and fibroblasts, and the expression of a group of genes encoding proteins mediating immunoregulatory responses was observed across different types of leprosy granuloma (Magoulopoulou et al, 2022).…”

“…Magoulopoulou et al, 2022 used this strategy to study leprosy granulomas, and developed a quantitative analysis strategy to characterize the immune landscape of human leprosy granulomas in further detail. A total of 12 cell subtypes were identified in leprosy legions using scRNA‐seq, including macrophages, T cells, keratinocytes, and fibroblasts, and the expression of a group of genes encoding proteins mediating immunoregulatory responses was observed across different types of leprosy granuloma (Magoulopoulou et al, 2022). The main advantage of this strategy is the potential for further utilization in organ mapping and discovering novel therapeutic targets for immune therapy (Di Bella et al, 2021; Gao et al, 2022; Qi et al, 2022; Su et al, 2022).…”

“…To further characterize the interactions between pathogen Mtb and granulomas during TB progression, wild type mice and Mtb‐susceptible mice were compared before and 12 weeks‐post infection (Magoulopoulou et al, 2022). Spatial sequencing was used to localize the pathogens and immune transcript and, while T cell transcripts were spread equally across the tissue, active macrophages were co‐localized with small bacteria aggregates rather than developed granulomas (Magoulopoulou et al, 2022).…”

“…Associations between the cell subtypes present in the granuloma and the immune cellular response have also been confirmed (McCaffrey et al, 2022). Recently, a combination of spatial transcriptomics and single‐cell data revealed the spatial architecture and cell population present in TB granulomas (Esaulova et al, 2021; Magoulopoulou et al, 2022). In addition, multiplex imaging approaches can be used to study latent tuberculosis infection (LTBI), and can be used to distinguish LTBI from active pulmonary TB (ATB) (Esaulova et al, 2021).…”