Project description:Using the zebrafish-M. marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-seq analysis of zebrafish granulomas as well as analysis of M. tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation.
Project description:The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. 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. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for 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 hallmark structure of mycobacterial infection.
Project description:Induced lymphoid structures associated with pulmonary granulomas have been observed during TB in both humans and mice and could orchestrate host defense. In order to determine whether granulomas perform lymphoid functions, mice lacking secondary lymphoid organs (SLOs) were infected with Mycobacterium tuberculosis (MTB). As in wild type mice (WT), granulomas developed, MTB was controlled, and antigen-specific T cell responses including T central memory (TCM) cells were generated in the complete absence of SLOs. Moreover, T cell activation correlated with granuloma formation in these mice and TCM cells accumulated in lungs and participated in protection against secondary MTB infection. Lung granulomas may therefore represent tertiary lymphoid organs (TLOs) capable of priming robust T cell responses and mediating host defense during chronic MTB at the site of infection.
Project description:Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. We conducted spatial and single-cell RNA sequencing of clinical granuloma biopsy specimens from tuberculosis patients. We found that granulomas consist of concentric transcriptional laminae surrounding areas of central necrosis. Gene expression programs associated with regional architecture were conserved among samples. We identified distinct populations of granuloma-associated stromal cells, fibroblasts, lymphocytes, mast cells, dendritic cells, neutrophils and macrophages. Populations further differentiate into multiple granuloma-specific states that correlate with location. We used inferential analysis to predict dominant granuloma cell-cell interactions, the activity of major signaling pathways, and differential transcription factor activities. Using spatial deconvolution, we mapped a conserved pattern of cellular organization dominated by macrophages rich in osteopontin/SPP1 expression. Trajectory analysis of macrophage subtypes mapped their differentiation and supported the importance of SPP1 to granuloma macrophage polarization. Cumulatively, we have identified a dominant macrophage granuloma population as well as its central regulatory gene in human samples and confirmed its importance to granuloma biology in vivo.
Project description:Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. We conducted spatial and single-cell RNA sequencing of clinical granuloma biopsy specimens from tuberculosis patients. We found that granulomas consist of concentric transcriptional laminae surrounding areas of central necrosis. Gene expression programs associated with regional architecture were conserved among samples. We identified distinct populations of granuloma-associated stromal cells, fibroblasts, lymphocytes, mast cells, dendritic cells, neutrophils and macrophages. Populations further differentiate into multiple granuloma-specific states that correlate with location. We used inferential analysis to predict dominant granuloma cell-cell interactions, the activity of major signaling pathways, and differential transcription factor activities. Using spatial deconvolution, we mapped a conserved pattern of cellular organization dominated by macrophages rich in osteopontin/SPP1 expression. Trajectory analysis of macrophage subtypes mapped their differentiation and supported the importance of SPP1 to granuloma macrophage polarization. Cumulatively, we have identified a dominant macrophage granuloma population as well as its central regulatory gene in human samples and confirmed its importance to granuloma biology in vivo.
Project description:Tuberculous meningitis is one of the fatal forms of extra pulmonary disease associated with high mortality and severe neurological defects in affected individuals. We have carried out transcriptome level analysis using whole human genome microarrays to identify differential expression of genes between tuberculous meningitis and normals. In our gene expression analysis, we found 2,434 genes that were differentially erexpressed with 2 or more than 2 fold changes between tuberculous meningitis compared to normal cases. Most of the genes encoded many of the proteins, which involves metabolism, energy pathways, cell growth and/or maintenance, transport and cell communication and signal transduction. We have performed immunohistochemistry for the validation of some of the novel candidates identified in our microarray studies.!Series_overall_design = Present study carried out mRNA expression profiling of five samples from patients diagnosed with tuberculous meningitis and four head injury cases were used as controls. We have used 4X44K arrays from agilent plaform. To validate our microarray results, we have done Immunohistochemistry on 15 TBM cases with control groups. Present study carried out mRNA expression profiling of five samples from patients diagnosed with tuberculous meningitis and four head injury cases were used as controls. We have used 4X44K arrays from agilent plaform. To validate our microarray results, we have done Immunohistochemistry on 15 TBM cases with control groups.!Series_type = Expression profiling by array
Project description:Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. Using the Mycobacterium marinum-zebrafish model we found that mycobacterial infection induces spp1 expression in macrophages and that spp1 ablation results in granuloma formation defects and reduced survival in adult animals. To determine the impact of mycobacterial infection on the transcriptional response of nascent granuloma macrophages we conducted bulk RNA-seq on flow-sorted infected and uninfected macrophages isolated from infected larvae or uninfected controls.
Project description:Mycobacterium tuberculosis infection in humans triggers formation of granulomas, tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. We find that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. Using the zebrafish-Mycobacterium marinum model, we identify fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function results in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. In humans, we find that granuloma macrophages are similarly transformed. Long considered largely through the prism of immune signaling pathways, granuloma macrophages are reprogrammed by epithelial modules that alter the trajectory of mycobacterial infection.