Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice.

The current study reveals that in chronic TB, the B cell-deficient µMT strain, relative to wild-type (WT) C57BL/6 mice, displays in the lungs lower levels of inflammation that are associated with decreased CD4+ T cell proliferation, diminished Th1 response, and enhanced levels of interleukin (IL)-10. The latter result raises the possibility that B cells may restrict lung expression of IL-10 in chronic TB. These observations are recapitulated in WT mice depleted for B cells using anti-CD20 antibodies. IL-10 receptor (IL-10R) blockade reverses the phenotypes of decreased inflammation and attenuated CD4+ T cell responses in B cell-depleted mice. Together, these results suggest that in chronic murine TB, B cells, by virtue of their capacity to restrict expression of the anti-inflammatory and immunosuppressive IL-10 in the lungs, promote the development of a robust protective Th1 response, thereby optimizing anti-TB immunity. This vigorous Th1 immunity and restricted IL-10 expression may, however, allow the development of inflammation to a level that can be detrimental to the host. Indeed, decreased lung inflammation observed in chronically infected B cell-deficient mice, which exhibit augmented lung IL-10 levels, is associated with a survival advantage relative to WT animals. Collectively, the results reveal that in chronic murine TB, B cells play a role in modulating the protective Th1 immunity and the anti-inflammatory IL-10 response, which results in augmentation of lung inflammation that can be host-detrimental. Intriguingly, in tuberculous human lungs, conspicuous B cell aggregates are present in close proximity to tissue-damaging lesions manifesting necrosis and cavitation, suggesting the possibility that in human TB, B cells may contribute to the development of exacerbated pathology that is known to promote transmission. Since transmission is a major hindrance to TB control, investigating into whether B cells can shape the development of severe pulmonic pathological responses in tuberculous individuals is warranted.

Hydrogen sulfide dysregulates the immune response by suppressing central carbon metabolism to promote tuberculosis.

The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognized to play a crucial role in human health. Using cystathionine γ-lyase (CSE)-deficient mice, we demonstrate an unexpected role of H2S in Mycobacterium tuberculosis (Mtb) pathogenesis. We showed that Mtb-infected CSE-/- mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitro Mtb infection of macrophages resulted in reduced colony forming units in CSE-/- cells. Chemical complementation of infected WT and CSE-/- macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulating Mtb survival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1ß, IL-6, TNF-α, and IFN-γ levels in response to Mtb infection. Notably, Mtb infected CSE-/- macrophages show increased flux through glycolysis and the pentose phosphate pathway, thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected WT mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1ß, IL-6, and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S-producing enzymes in human necrotic, nonnecrotic, and cavitary pulmonary tuberculosis (TB) lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.

Micro-Computed Tomography Analysis of the Human Tuberculous Lung Reveals Remarkable Heterogeneity in Three-dimensional Granuloma Morphology.

Rationale: Our current understanding of tuberculosis (TB) pathophysiology is limited by a reliance on animal models, the paucity of human TB lung tissue, and traditional histopathological analysis, a destructive two-dimensional approach that provides limited spatial insight. Determining the three-dimensional (3D) structure of the necrotic granuloma, a characteristic feature of TB, will more accurately inform preventive TB strategies.Objectives: To ascertain the 3D shape of the human tuberculous granuloma and its spatial relationship with airways and vasculature within large lung tissues.Methods: We characterized the 3D microanatomical environment of human tuberculous lungs by using micro computed tomography, histopathology, and immunohistochemistry. By using 3D segmentation software, we accurately reconstructed TB granulomas, vasculature, and airways in three dimensions and confirmed our findings by using histopathology and immunohistochemistry.Measurements and Main Results: We observed marked heterogeneity in the morphology, volume, and number of TB granulomas in human lung sections. Unlike depictions of granulomas as simple spherical structures, human necrotic granulomas exhibit complex, cylindrical, branched morphologies that are connected to the airways and shaped by the bronchi. The use of 3D imaging of human TB lung sections provides unanticipated insight into the spatial organization of TB granulomas in relation to the airways and vasculature.Conclusions: Our findings highlight the likelihood that a single, structurally complex lesion could be mistakenly viewed as multiple independent lesions when evaluated in two dimensions. In addition, the lack of vascularization within obstructed bronchi establishes a paradigm for antimycobacterial drug tolerance. Lastly, our results suggest that bronchogenic spread of Mycobacterium tuberculosis reseeds the lung.

A mouse model of TB-associated lung fibrosis reveals persistent inflammatory macrophage populations during treatment.

Post-TB lung disease (PTLD) causes a significant burden of global disease. Fibrosis is a central component of many clinical features of PTLD. To date, we have a limited understanding of the mechanisms of TB-associated fibrosis and how these mechanisms are similar to or dissimilar from other fibrotic lung pathologies. We have adapted a mouse model of TB infection to facilitate the mechanistic study of TB-associated lung fibrosis. We find that the morphologies of fibrosis that develop in the mouse model are similar to the morphologies of fibrosis observed in human tissue samples. Using Second Harmonic Generation (SHG) microscopy, we are able to quantify a major component of fibrosis, fibrillar collagen, over time and with treatment. Inflammatory macrophage subpopulations persist during treatment; matrix remodeling enzymes and inflammatory gene signatures remain elevated. Our mouse model suggests that there is a therapeutic window during which adjunctive therapies could change matrix remodeling or inflammatory drivers of tissue pathology to improve functional outcomes after treatment for TB infection.

B cell heterogeneity in human tuberculosis highlights compartment-specific phenotype and functional roles.

B cells are important in tuberculosis (TB) immunity, but their role in the human lung is understudied. Here, we characterize B cells from lung tissue and matched blood of patients with TB and found they are decreased in the blood and increased in the lungs, consistent with recruitment to infected tissue, where they are located in granuloma associated lymphoid tissue. Flow cytometry and transcriptomics identify multiple B cell populations in the lung, including those associated with tissue resident memory, germinal centers, antibody secretion, proinflammatory atypical B cells, and regulatory B cells, some of which are expanded in TB disease. Additionally, TB lungs contain high levels of Mtb-reactive antibodies, specifically IgM, which promotes Mtb phagocytosis. Overall, these data reveal the presence of functionally diverse B cell subsets in the lungs of patients with TB and suggest several potential localized roles that may represent a target for interventions to promote immunity or mitigate immunopathology.

Myeloid cell expression of CD200R is modulated in active TB disease and regulates Mycobacterium tuberculosis infection in a biomimetic model.

A robust immune response is required for resistance to pulmonary tuberculosis (TB), the primary disease caused by Mycobacterium tuberculosis (Mtb). However, pharmaceutical inhibition of T cell immune checkpoint molecules can result in the rapid development of active disease in latently infected individuals, indicating the importance of T cell immune regulation. In this study, we investigated the potential role of CD200R during Mtb infection, a key immune checkpoint for myeloid cells. Expression of CD200R was consistently downregulated on CD14+ monocytes in the blood of subjects with active TB compared to healthy controls, suggesting potential modulation of this important anti-inflammatory pathway. In homogenized TB-diseased lung tissue, CD200R expression was highly variable on monocytes and CD11b+HLA-DR+ macrophages but tended to be lowest in the most diseased lung tissue sections. This observation was confirmed by fluorescent microscopy, which showed the expression of CD200R on CD68+ macrophages surrounding TB lung granuloma and found expression levels tended to be lower in macrophages closest to the granuloma core and inversely correlated with lesion size. Antibody blockade of CD200R in a biomimetic 3D granuloma-like tissue culture system led to significantly increased Mtb growth. In addition, Mtb infection in this system reduced gene expression of CD200R. These findings indicate that regulation of myeloid cells via CD200R is likely to play an important part in the immune response to TB and may represent a potential target for novel therapeutic intervention.

Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue.

BACKGROUND: The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. METHODS: We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). FINDINGS: We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. INTERPRETATION: RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. FUNDING: Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Human alveolar lining fluid from the elderly promotes Mycobacterium tuberculosis intracellular growth and translocation into the cytosol of alveolar epithelial cells.

The elderly population is highly susceptible to developing respiratory diseases, including tuberculosis, a devastating disease caused by the airborne pathogen Mycobacterium tuberculosis (M.tb) that kills one person every 18 seconds. Once M.tb reaches the alveolar space, it contacts alveolar lining fluid (ALF), which dictates host-cell interactions. We previously determined that age-associated dysfunction of soluble innate components in human ALF leads to accelerated M.tb growth within human alveolar macrophages. Here we determined the impact of human ALF on M.tb infection of alveolar epithelial type cells (ATs), another critical lung cellular determinant of infection. We observed that elderly ALF (E-ALF)-exposed M.tb had significantly increased intracellular growth with rapid replication in ATs compared to adult ALF (A-ALF)-exposed bacteria, as well as a dampened inflammatory response. A potential mechanism underlying this accelerated growth in ATs was our observation of increased bacterial translocation into the cytosol, a compartment that favors bacterial replication. These findings in the context of our previous studies highlight how the oxidative and dysfunctional status of the elderly lung mucosa determines susceptibility to M.tb infection, including dampening immune responses and favoring bacterial replication within alveolar resident cell populations, including ATs, the most abundant resident cell type within the alveoli.

NAD(H) homeostasis underlies host protection mediated by glycolytic myeloid cells in tuberculosis.

Mycobacterium tuberculosis (Mtb) disrupts glycolytic flux in infected myeloid cells through an unclear mechanism. Flux through the glycolytic pathway in myeloid cells is inextricably linked to the availability of NAD+, which is maintained by NAD+ salvage and lactate metabolism. Using lung tissue from tuberculosis (TB) patients and myeloid deficient LDHA (LdhaLysM-/-) mice, we demonstrate that glycolysis in myeloid cells is essential for protective immunity in TB. Glycolytic myeloid cells are essential for the early recruitment of multiple classes of immune cells and IFNγ-mediated protection. We identify NAD+ depletion as central to the glycolytic inhibition caused by Mtb. Lastly, we show that the NAD+ precursor nicotinamide exerts a host-dependent, antimycobacterial effect, and that nicotinamide prophylaxis and treatment reduce Mtb lung burden in mice. These findings provide insight into how Mtb alters host metabolism through perturbation of NAD(H) homeostasis and reprogramming of glycolysis, highlighting this pathway as a potential therapeutic target.

Detection of Mycobacterium tuberculosis in human tissue via RNA in situ hybridization.

Rationale: Accurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify Mycobacterium tuberculosis ( Mtb ) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of Mtb mRNA. Objectives: To employ RNAscope to determine the spatial distribution of Mtb mRNA within tuberculous human tissue, to appraise ZN-negative tissue from confirmed TB patients, and to provide proof-of-concept of RNAscope as a platform to inform TB diagnosis and Mtb biology. Methods: We examined ante- and postmortem human TB tissue using RNAscope to detect Mtb mRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). Measurements and main results: We adapted RNAscope for Mtb and identified intact and disintegrated Mtb bacilli and intra- and extracellular Mtb mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. Conclusions: RNAscope has diagnostic potential and can guide therapeutic intervention as it detects Mtb mRNA and morphology in ZN-negative tissues from TB patients, and Mtb mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of Mtb bacilli exist in vivo .

A high-resolution 3D atlas of the spectrum of tuberculous and COVID-19 lung lesions.

Our current understanding of the spectrum of TB and COVID-19 lesions in the human lung is limited by a reliance on low-resolution imaging platforms that cannot provide accurate 3D representations of lesion types within the context of the whole lung. To characterize TB and COVID-19 lesions in 3D, we applied micro/nanocomputed tomography to surgically resected, postmortem, and paraffin-embedded human lung tissue. We define a spectrum of TB pathologies, including cavitary lesions, calcium deposits outside and inside necrotic granulomas and mycetomas, and vascular rearrangement. We identified an unusual spatial arrangement of vasculature within an entire COVID-19 lobe, and 3D segmentation of blood vessels revealed microangiopathy associated with hemorrhage. Notably, segmentation of pathological anomalies reveals hidden pathological structures that might otherwise be disregarded, demonstrating a powerful method to visualize pathologies in 3D in TB lung tissue and whole COVID-19 lobes. These findings provide unexpected new insight into the spatial organization of the spectrum of TB and COVID-19 lesions within the framework of the entire lung.

Aggregated Mycobacterium tuberculosis Enhances the Inflammatory Response.

Mycobacterium tuberculosis (Mtb) bacilli readily aggregate. We previously reported that Mtb aggregates lead to phagocyte death and subsequent efficient replication in the dead infected cells. Here, we examined the transcriptional response of human monocyte derived macrophages to phagocytosis of aggregated Mtb relative to phagocytosis of non-aggregated single or multiple bacilli. Infection with aggregated Mtb led to an early upregulation of pro-inflammatory associated genes and enhanced TNFα signaling via the NFκB pathway. These pathways were significantly more upregulated relative to infection with single or multiple non-aggregated bacilli per cell. Phagocytosis of aggregates led to a decreased phagosome acidification on a per bacillus basis and increased phagocyte cell death, which was not observed when Mtb aggregates were heat killed prior to phagocytosis. Mtb aggregates, observed in a granuloma from a patient, were found surrounding a lesion cavity. These observations suggest that TB aggregation may be a mechanism for pathogenesis. They raise the possibility that aggregated Mtb, if spread from individual to individual, could facilitate increased inflammation, Mtb growth, and macrophage cell death, potentially leading to active disease, cell necrosis, and additional cycles of transmission.

Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology.

Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis Infection.

Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth-/- mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin-ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.