Frequency of CD4+ regulatory T cells and modulation of CD4+T lymphocyte activation in pleural tuberculoma.

BACKGROUND: The expression of regulatory T cells (Tregs) is elevated in patients with active tuberculosis (TB) or multidrug-resistant TB. However, it remains uncertain whether Tregs mediate immune suppression in pleural tuberculoma (PTM). METHODS: Peripheral venous blood and clinical data were collected from 56 PTM patients and 50 healthy volunteers. The expression of CD38 and HLA-DR expression in T cell subsets and Tregs was determined by flow cytometry. RESULTS: PTM patients had significantly more Tregs than the matched healthy controls. The expression of CD4+T cells normalized after treatment. Although the median proportions of CD3+T, CD4+T, and CD8+T lymphocytes did not differ significantly between PTM patients and healthy controls, the CD4/CD8 ratio was higher in PTM patients. Moreover, the proportion of CD4+T lymphocytes expressing activation markers, including HLA-DR and CD38, was higher in PTM patients than healthy controls. Treg expression was positively associated with the level of CD4+T lymphocyte activation. CONCLUSIONS: The increased expression of Tregs seen in PTM patients, and subsequent decrease after treatment, indicate that Tregs play an important role in the immune reactivity of PTM.

Neutrophil Dynamics Affect Mycobacterium tuberculosis Granuloma Outcomes and Dissemination.

Neutrophil infiltration into tuberculous granulomas is often associated with higher bacteria loads and severe disease but the basis for this relationship is not well understood. To better elucidate the connection between neutrophils and pathology in primate systems, we paired data from experimental studies with our next generation computational model GranSim to identify neutrophil-related factors, including neutrophil recruitment, lifespan, and intracellular bacteria numbers, that drive granuloma-level outcomes. We predict mechanisms underlying spatial organization of neutrophils within granulomas and identify how neutrophils contribute to granuloma dissemination. We also performed virtual deletion and depletion of neutrophils within granulomas and found that neutrophils play a nuanced role in determining granuloma outcome, promoting uncontrolled bacterial growth in some and working to contain bacterial growth in others. Here, we present three key results: We show that neutrophils can facilitate local dissemination of granulomas and thereby enable the spread of infection. We suggest that neutrophils influence CFU burden during both innate and adaptive immune responses, implying that they may be targets for therapeutic interventions during later stages of infection. Further, through the use of uncertainty and sensitivity analyses, we predict which neutrophil processes drive granuloma severity and structure.

Spatial and temporal localization of immune transcripts defines hallmarks and diversity in the tuberculosis granuloma.

Granulomas are the pathological hallmark of tuberculosis (TB) and the niche where bacilli can grow and disseminate or the immunological microenvironment in which host cells interact to prevent bacterial dissemination. Here we show 34 immune transcripts align to the morphology of lung sections from Mycobacterium tuberculosis-infected mice at cellular resolution. Colocalizing transcript networks at <10 µm in C57BL/6 mouse granulomas increase complexity with time after infection. B-cell clusters develop late after infection. Transcripts from activated macrophages are enriched at subcellular distances from M. tuberculosis. Encapsulated C3HeB/FeJ granulomas show necrotic centers with transcripts associated with immunosuppression (Foxp3, Il10), whereas those in the granuloma rims associate with activated T cells and macrophages. We see highly diverse networks with common interactors in similar lesions. Different immune landscapes of M. tuberculosis granulomas depending on the time after infection, the histopathological features of the lesion, and the proximity to bacteria are here defined.

Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling.

Mycobacterial infection leads to the formation of characteristic immune aggregates called granulomas, a process accompanied by dramatic remodeling of the host vasculature. As granuloma angiogenesis favors the infecting mycobacteria, it may be actively promoted by bacterial determinants during infection. Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. cis-Cyclopropanation of mycobacterial mycolic acids by pcaA drives the activation of host Vegf signaling within granuloma macrophages. Cyclopropanation of the mycobacterial cell wall glycolipid trehalose dimycolate is both required and sufficient to induce robust host angiogenesis. Inducible genetic inhibition of angiogenesis and Vegf signaling during granuloma formation results in bacterial growth deficits. Together, these data reveal a mechanism by which PcaA-mediated cis-cyclopropanation of mycolic acids promotes bacterial growth and dissemination in vivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.

In vivo inhibition of tryptophan catabolism reorganizes the tuberculoma and augments immune-mediated control of Mycobacterium tuberculosis.

Mycobacterium tuberculosis continues to cause devastating levels of mortality due to tuberculosis (TB). The failure to control TB stems from an incomplete understanding of the highly specialized strategies that M. tuberculosis utilizes to modulate host immunity and thereby persist in host lungs. Here, we show that M. tuberculosis induced the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan catabolism, in macrophages and in the lungs of animals (mice and macaque) with active disease. In a macaque model of inhalation TB, suppression of IDO activity reduced bacterial burden, pathology, and clinical signs of TB disease, leading to increased host survival. This increased protection was accompanied by increased lung T cell proliferation, induction of inducible bronchus-associated lymphoid tissue and correlates of bacterial killing, reduced checkpoint signaling, and the relocation of effector T cells to the center of the granulomata. The enhanced killing of M. tuberculosis in macrophages in vivo by CD4+ T cells was also replicated in vitro, in cocultures of macaque macrophages and CD4+ T cells. Collectively, these results suggest that there exists a potential for using IDO inhibition as an effective and clinically relevant host-directed therapy for TB.

Mycobacterium tuberculosis: Manipulator of Protective Immunity.

Mycobacterium tuberculosis (MTB) is one of the most successful pathogens in human history and remains a global health challenge. MTB has evolved a plethora of strategies to evade the immune response sufficiently to survive within the macrophage in a bacterial-immunological equilibrium, yet causes sufficient immunopathology to facilitate its transmission. This review highlights MTB as the driver of disease pathogenesis and presents evidence of the mechanisms by which MTB manipulates the protective immune response into a pathological productive infection.

Inflammation and the coagulation system in tuberculosis: Tissue Factor leads the dance.

Mycobacterium tuberculosis, the causative agent of tuberculosis, drives the formation of granulomas, structures in which both immune cells and the bacterial pathogen cohabit. The most abundant cells in granulomas are macrophages, which contribute as both cells with bactericidal activity and as targets for M. tuberculosis infection and proliferation during the entire course of infection. The mechanisms and factors involved in the regulation and control of macrophage microenvironment-specific polarization and plasticity are not well understood, as some granulomas are able to control bacteria growth and others fail to do so, permitting bacterial spread. In this issue of the European Journal of Immunology, Venkatasubramanian et al. [Eur. J. Immunol. 2016. 46: 464-479] show that mice lacking the tissue factor gene in myeloid cells have augmented M. tuberculosis growth and increased inflammation in the lungs. This suggests that tissue factor, an initiator of coagulation, is important for the generation of fibrin, which supports granuloma formation. This article demonstrates for the first time the involvement of tissue factor in inducing effective immunity against M. tuberculosis, and sheds new lights on the complex interplay between host inflammatory response, the coagulation system, and the control of M. tuberculosis infection.

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection.

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF(Δ) ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL-10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2-like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)-2 and MMP-9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.

Looking within the zebrafish to understand the tuberculous granuloma.

Tuberculosis is characterized by the formation of complex immune cell aggregates called granulomas, which for nearly a century have been viewed as critical host-beneficial structures to restrict bacterial growth and spread. A different view has now emerged from real-time visualization of granuloma formation and its consequences in the optically transparent and genetically tractable zebrafish larva. Pathogenic mycobacteria have developed mechanisms to use host granulomas for their expansion and dissemination, at least during the innate phases of infection. Host processes that are intended to be beneficial-death of infected macrophages and their subsequent phagocytosis by macrophages that are newly recruited to the growing granuloma-are harnessed by mycobacteria for their own benefit. Mycobacteria can also render the granuloma a safe-haven in the more advanced stages of infection. An understanding of the host and bacterial pathways involved in tuberculous granuloma formation may suggest new ways to combat mycobacterial infection.

The role of B cells and humoral immunity in Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a serious threat to public health, causing 2 million deaths annually world-wide. The control of TB has been hindered by the requirement of long duration of treatment involving multiple chemotherapeutic agents, the increased susceptibility to Mycobacterium tuberculosis infection in the HIV-infected population, and the development of multi-drug resistant and extensively resistant strains of tubercle bacilli. An efficacious and cost-efficient way to control TB is the development of effective anti-TB vaccines. This measure requires thorough understanding of the immune response to M. tuberculosis. While the role of cell-mediated immunity in the development of protective immune response to the tubercle bacillus has been well established, the role of B cells in this process is not clearly understood. Emerging evidence suggests that B cells and humoral immunity can modulate the immune response to various intracellular pathogens, including M. tuberculosis. These lymphocytes form conspicuous aggregates in the lungs of tuberculous humans, non-human primates, and mice, which display features of germinal center B cells. In murine TB, it has been shown that B cells can regulate the level of granulomatous reaction, cytokine production, and the T cell response. This chapter discusses the potential mechanisms by which specific functions of B cells and humoral immunity can shape the immune response to intracellular pathogens in general, and to M. tuberculosis in particular. Knowledge of the B cell-mediated immune response to M. tuberculosis may lead to the design of novel strategies, including the development of effective vaccines, to better control TB.

On the horizon: flexible immune recognition outside lymphocytes.

Since decades there is consensus among immunologists that in jawless and jawed vertebrates flexible immune recognition is strictly confined to the lymphoid lineage. In jawed vertebrates the adaptive immune system is represented by two lineages of lymphocytes, B cells and T cells that express recombinatorial antigen receptors of enormous diversity known as immunoglobulins and the T cell receptor (TCR). The recent identification of recombined immune receptors that are structurally based on the TCR in subpopulations of neutrophils and eosinophils (referred to here as TCR-like immunoreceptors, "TCRL") provides unexpected evidence for the existence of flexible host defense mechanisms beyond the realm of lymphocytes. Consistent with this, subpopulations of monocytes and macrophages from humans and mice now have also been shown to constitutively express recombined TCR-like immunoreceptors. Available in vitro evidence suggests that the TCRL in macrophages may exert functions as facilitators of phagocytosis and self-recruitment. More importantly, our recent findings that the macrophage-TCRL is implicated in granuloma formation in tuberculosis and the neutrophil-TCRL is associated with autoimmune hemolytic anemia establish for the first time a link between myeloid recombinatorial immune receptors and clinical disease. The discovery of recombined TCR-like immune receptors in granulocytes and macrophages extends the principle of combinatorial immune recognition to phagocytic cells. Conceptually, this unifies the two hitherto disparate cardinal features of innate and adaptive immunity, phagocytic capacity and recombinatorial immune recognition on a common cellular platform. Moreover, it strongly suggests that flexible host defense in vertebrates may operate on a broader cellular basis than currently thought.

Differential risk of tuberculosis reactivation among anti-TNF therapies is due to drug binding kinetics and permeability.

Increased rates of tuberculosis (TB) reactivation have been reported in humans treated with TNF-α (TNF)-neutralizing drugs, and higher rates are observed with anti-TNF Abs (e.g., infliximab) as compared with TNF receptor fusion protein (etanercept). Mechanisms driving differential reactivation rates and differences in drug action are not known. We use a computational model of a TB granuloma formation that includes TNF/TNF receptor dynamics to elucidate these mechanisms. Our analyses yield three important insights. First, drug binding to membrane-bound TNF critically impairs granuloma function. Second, a higher risk of reactivation induced from Ab-type treatments is primarily due to differences in TNF/drug binding kinetics and permeability. Apoptotic and cytolytic activities of Abs and pharmacokinetic fluctuations in blood concentration of drug are not essential to inducing TB reactivation. Third, we predict specific host factors that, if augmented, would improve granuloma function during anti-TNF therapy. Our findings have implications for the development of safer anti-TNF drugs to treat inflammatory diseases.

Right atrial tuberculoma: a diagnosis too late.

Solitary intra-cardiac cavity tuberculoma is extremely rare and often only diagnosed during a post-mortem. We report a case of right atrial tuberculoma causing right atrial outflow tract obstruction in an immune-compromised man. The diagnosis of cardiac tuberculoma was made through the detection of mycobacterium tuberculosis DNA by tuberculosis-polymerase chain reaction in the pericardial fluid. The patient succumbed five days after admission but an autopsy was refused by his family.

Cystic pulmonary tuberculoma.

Pulmonary tuberculosis (TB) is a medical and social problem, particularly in developing countries. Early diagnosis and treatment is important. Chest radiography is usually the first diagnostic tool when there is a suspicion of pulmonary TB. A computed tomography (CT) scan provides more accurate information on the extent and distribution of pulmonary TB. We present here a young, immunocompetent male patient with unusual imaging findings for pulmonary TB. We discuss the clinical presentation and management.

Transcriptional reprogramming in nonhuman primate (rhesus macaque) tuberculosis granulomas.

BACKGROUND: In response to Mtb infection, the host remodels the infection foci into a dense mass of cells known as the granuloma. The key objective of the granuloma is to contain the spread of Mtb into uninfected regions of the lung. However, it appears that Mtb has evolved mechanisms to resist killing in the granuloma. Profiling granuloma transcriptome will identify key immune signaling pathways active during TB infection. Such studies are not possible in human granulomas, due to various confounding factors. Nonhuman Primates (NHPs) infected with Mtb accurately reflect human TB in clinical and pathological contexts. METHODOLOGY/PRINCIPAL FINDINGS: We studied transcriptomics of granuloma lesions in the lungs of NHPs exhibiting active TB, during early and late stages of infection. Early TB lesions were characterized by a highly pro-inflammatory environment, expressing high levels of immune signaling pathways involving IFNgamma, TNFalpha, JAK, STAT and C-C/C-X-C chemokines. Late TB lesions, while morphologically similar to the early ones, exhibited an overwhelming silencing of the inflammatory response. Reprogramming of the granuloma transcriptome was highly significant. The expression of approximately two-thirds of all genes induced in early lesions was later repressed. CONCLUSIONS/SIGNIFICANCE: The transcriptional characteristics of TB granulomas undergo drastic changes during the course of infection. The overwhelming reprogramming of the initial pro-inflammatory surge in late lesions may be a host strategy to limit immunopathology. We propose that these host profiles can predict changes in bacterial replication and physiology, perhaps serving as markers for latency and reactivation.

Host-detrimental role of Esx-1-mediated inflammasome activation in mycobacterial infection.

The Esx-1 (type VII) secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium marinum. However, the molecular events and host-pathogen interactions underlying Esx-1-mediated virulence in vivo remain unclear. Here we address this problem in a non-lethal mouse model of M. marinum infection that allows detailed quantitative analysis of disease progression. M. marinum established local infection in mouse tails, with Esx-1-dependent formation of caseating granulomas similar to those formed in human tuberculosis, and bone deterioration reminiscent of skeletal tuberculosis. Analysis of tails infected with wild type or Esx-1-deficient bacteria showed that Esx-1 enhanced generation of proinflammatory cytokines, including the secreted form of IL-1beta, suggesting that Esx-1 promotes inflammasome activation in vivo. In vitro experiments indicated that Esx-1-dependent inflammasome activation required the host NLRP3 and ASC proteins. Infection of wild type and ASC-deficient mice demonstrated that Esx-1-dependent inflammasome activation exacerbated disease without restricting bacterial growth, indicating a host-detrimental role of this inflammatory pathway in mycobacterial infection. These findings define an immunoregulatory role for Esx-1 in a specific host-pathogen interaction in vivo, and indicate that the Esx-1 secretion system promotes disease and inflammation through its ability to activate the inflammasome.