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.

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.

Integrated plasma proteomics identifies tuberculosis-specific diagnostic biomarkers.

BACKGROUNDNovel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODSWe applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTSTB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSIONThis biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDINGMedical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Cancer immunotherapy with immune checkpoint inhibitors and infections: A particular focus on mycobacterial infections.

Cancer treatment is undergoing a major transformation with the advent of immunotherapy with immune checkpoint inhibitors. These drugs, which have a different mechanism of action from conventional cytotoxic chemotherapy, are transforming treatment paradigms for many patients suffering from advanced cancer. On the other hand, they are often complicated by specific adverse events, known as immune-related adverse events (irAEs). Infections occurring during immunotherapy with immune checkpoint inhibitors have recently received increasing attention and sometimes are seen as part of irAEs. Amongst these, mycobacterial infections have attracted particular attention. Recent reports have shown that infections occurring during immunotherapy can not only be caused by immunosuppression, but in addition new type of infections are observed that are not caused by immunosuppression. Specifically, tuberculosis (TB) has recently been shown to develop as a result of an imbalance in immunoregulation and an excessive immune response. This review highlights reports of infections during immunotherapy with immune checkpoint inhibitors, followed by a focus on the association with TB and nontuberculous mycobacteria. It concludes with a discussion of the possible mechanisms of pathogenesis and the implications for clinical practice.

Mycobacterium tuberculosis senses host Interferon-γ via the membrane protein MmpL10.

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens. Several cytokines are known to increase virulence of bacterial pathogens, leading us to investigate whether Interferon-γ (IFN-γ), a central regulator of the immune defense against Mtb, has a direct effect on the bacteria. We found that recombinant and T-cell derived IFN-γ rapidly induced a dose-dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was not observed in attenuated Bacillus Calmette-Guérin (BCG), and did not occur for other cytokines tested, including TNF-α. IFN-γ binds to the cell surface of intact Mtb, but not BCG. Mass spectrometry identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner of IFN-γ, supported by molecular modelling studies. IFN-γ binding and the OCR response was absent in Mtb Δmmpl10 strain and restored by complementation with wildtype mmpl10. RNA-sequencing and RT-PCR of Mtb exposed to IFN-γ revealed a distinct transcriptional profile, including genes involved in virulence. In a 3D granuloma model, IFN-γ promoted Mtb growth, which was lost in the Mtb Δmmpl10 strain and restored by complementation, supporting the involvement of MmpL10 in the response to IFN-γ. Finally, IFN-γ addition resulted in sterilization of Mtb cultures treated with isoniazid, indicating clearance of phenotypically resistant bacteria that persist in the presence of drug alone. Together our data are the first description of a mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies.

Anti-Tuberculosis Activity of Three Carbapenems, Clofazimine and Nitazoxanide Using a Novel Ex Vivo Phenotypic Drug Susceptibility Model of Human Tuberculosis.

We evaluated a novel physiological 3-D bioelectrospray model of the tuberculosis (TB) granuloma to test the activity of a known anti-TB drug, clofazimine; three carbapenems with potential activity, including one currently used in therapy; and nitazoxanide, an anti-parasitic compound with possible TB activity (all chosen as conventional drug susceptibility was problematical). PBMCs collected from healthy donors were isolated and infected with M. tuberculosis H37Rv lux (i.e., luciferase). Microspheres were generated with the infected cells; the anti-microbial compounds were added and bacterial luminescence was monitored for at least 21 days. Clavulanate was added to each carbapenem to inhibit beta-lactamases. M. tuberculosis (MTB) killing efficacy was dose dependent. Clofazimine was the most effective drug inhibiting MTB growth at 2 mg/L with good killing activity at both concentrations tested. It was the only drug that killed bacteria at the lowest concentration tested. Carbapenems showed modest initial activity that was lost at around day 10 of incubation and clavulanate did not increase killing activity. Of the carbapenems tested, tebipenem was the most efficient in killing MTB, albeit at a high concentration. Nitazoxanide was effective only at concentrations not achievable with current dosing (although this might partly have been an artefact related to extensive protein binding).

The paradox of immune checkpoint inhibition re-activating tuberculosis.

By attenuating T-cell activation, immune checkpoints (ICs) limit optimal anti-tumour responses and IC inhibition (ICI) has emerged as a new therapy for a broad range of cancers. T-cell responses are indispensable to tuberculosis (TB) immunity in humans. However, boosting T-cell immunity in cancer patients by blocking the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) axis can trigger re-activation of latent TB. This phenomenon appears to contradict the prevailing thought that enhancing T-cell immunity to Mycobacterium tuberculosis will improve immune control of this pathogen. In support of this anecdotal human data, several murine studies have shown that PD-1 deficiency leads to severe TB disease and rapid death. These observations warrant a serious reconsideration of what constitutes effective TB immunity and how ICs contribute to it. Through restraining T-cell responses, ICs are critical to preventing excessive tissue damage and maintaining a range of effector functions. Bolstering this notion, inhibitory receptors limit pathology in respiratory infections such as influenza, where loss of negative immune regulation resulted in progressive immunopathology. In this review, we analyse the mechanisms of ICs in general and their role in TB in particular. We conclude with a reflection on the emerging paradigm and avenues for future research.

Serial interferon-gamma release assay in lung cancer patients receiving immune checkpoint inhibitors: a prospective cohort study.

Recent advancements in cancer immunotherapy using immune checkpoint inhibitors (ICIs) have received considerable attention. Although advantageous, ICI therapies cause unique immune-related adverse events (irAEs) in some patients. Moreover, infectious diseases, such as tuberculosis, have been recognized as emerging concerns during immunotherapy. We aimed to evaluate the interferon-gamma release assay (IGRA) conversion rate and active tuberculosis incidence during immunotherapy to elucidate the incidence of tuberculosis reactivation after ICI therapy induction.We prospectively assessed IGRA results in lung cancer patients who received ICI monotherapy before ICI treatment and at 6 and 12 months after ICI treatment. We also assessed computed tomography findings to determine the presence of active tuberculosis when positive IGRA results were obtained. The ICIs used were nivolumab, pembrolizumab, atezolizumab, and durvalumab.In all, 178 patients were prospectively recruited between March 2017 and March 2020. Of these, 123 completed serial IGRAs, of whom 18, 101, and 4, respectively, had positive, negative, and indeterminate IGRAs at baseline. Three and four patients, respectively, showed IGRA reversion and conversion during immunotherapy. One patient with a sustained, stable positive IGRA and one with IGRA conversion developed active pulmonary tuberculosis during immunotherapy.We found that 3.3% and 1.6% of the patients developed IGRA conversion and active tuberculosis, respectively. Of the four patients who developed IGRA conversion, one developed active pulmonary tuberculosis during immunotherapy. Another patient with sustained, stable positive IGRA developed active tuberculosis. Physicians should be alert to tuberculosis development during ICI therapy, and IGRA testing is a useful tool to assess the risk of developing active tuberculosis.

Semi-quantitative detection of inflammatory biomarkers using a laser-patterned multiplexed lateral flow device.

Inflammatory markers including C-reactive protein (CRP) and procalcitonin (PCT) have been shown to be useful biomarkers to improve triage speed and prevent the inappropriate use of antibiotics for infections such as pneumonia. Here, we present a novel and exciting solution to guide the administration of antibiotic treatment via rapid, semi-quantitative and multiplexed detection of CRP and PCT using an advanced lateral flow device (LFD) designed to have multiple parallel flow-paths, produced via the precise laser-based partitioning of the single flow-path of a standard LFD. Each flow-path within this multiplexed LFD has a unique detection capability which permits tailored detection of CRP within a predefined cut-off range (20 µg/mL - 100 µg/mL) and PCT above a pre-defined threshold (0.5 ng/mL). We demonstrate the use of this LFD in the successful detection of CRP and PCT semi-quantitatively within spiked human serum samples. This multiplexed near-patient assay has potential for development into a rapid triage and treatment of patients with suspected pneumonia.

Infections due to dysregulated immunity: an emerging complication of cancer immunotherapy.

Immune checkpoint inhibitors (ICIs) have revolutionised cancer treatment. However, immune-related adverse events (irAEs) are a common side effect which can mimic infection. Additionally, treatment of irAEs with corticosteroids and other immunosuppressant agents can lead to opportunistic infection, which we have classed as immunotherapy infections due to immunosuppression. However, emerging reports demonstrate that some infections can be precipitated by ICIs in the absence of immunosuppressive treatment, in contrast to the majority of reported cases. These infections are characterised by a dysregulated inflammatory immune response, and so we propose they are described as immunotherapy infections due to dysregulated immunity. This review summarises the rapidly emerging evidence of these phenomena and proposes a new framework for considering infection in the context of cancer immunotherapy.

Understanding the tuberculosis granuloma: the matrix revolutions.

Mycobacterium tuberculosis (Mtb) causes the human disease tuberculosis (TB) and remains the top global infectious pandemic after coronavirus disease 2019 (COVID-19). Furthermore, TB has killed many more humans than any other pathogen, after prolonged coevolution to optimise its pathogenic strategies. Full understanding of fundamental disease processes in humans is necessary to successfully combat this highly successful pathogen. While the importance of immunodeficiency has been long recognised, biologic therapies and unbiased approaches are providing unprecedented insights into the intricacy of the host-pathogen interaction. The nature of a protective response is more complex than previously hypothesised. Here, we integrate recent evidence from human studies and unbiased approaches to consider how Mtb causes human TB and highlight the recurring theme of extracellular matrix (ECM) turnover.

Integrated transcriptomic analysis of human tuberculosis granulomas and a biomimetic model identifies therapeutic targets.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.

Tissue-resident-like CD4+ T cells secreting IL-17 control Mycobacterium tuberculosis in the human lung.

T cell immunity is essential for the control of tuberculosis (TB), an important disease of the lung, and is generally studied in humans using peripheral blood cells. Mounting evidence, however, indicates that tissue-resident memory T cells (Trms) are superior at controlling many pathogens, including Mycobacterium tuberculosis (M. tuberculosis), and can be quite different from those in circulation. Using freshly resected lung tissue, from individuals with active or previous TB, we identified distinct CD4+ and CD8+ Trm-like clusters within TB-diseased lung tissue that were functional and enriched for IL-17-producing cells. M. tuberculosis-specific CD4+ T cells producing TNF-α, IL-2, and IL-17 were highly expanded in the lung compared with matched blood samples, in which IL-17+ cells were largely absent. Strikingly, the frequency of M. tuberculosis-specific lung T cells making IL-17, but not other cytokines, inversely correlated with the plasma IL-1ß levels, suggesting a potential link with disease severity. Using a human granuloma model, we showed the addition of either exogenous IL-17 or IL-2 enhanced immune control of M. tuberculosis and was associated with increased NO production. Taken together, these data support an important role for M. tuberculosis-specific Trm-like, IL-17-producing cells in the immune control of M. tuberculosis in the human lung.

Animal Models of Ocular Tuberculosis: Implications for Diagnosis and Treatment.

The pathogenesis of ocular tuberculosis (TB) has remained unclear due to the challenges of performing mechanistic studies on clinical samples. Animal models have the potential to bridge these gaps by providing information about ocular dissemination and localization of mycobacteria, innate and adaptive immune response, and granuloma formation in the eye. Here, we critically review various animal models of ocular TB from the early 20th century to date and derive novel insights into pathogenesis of ocular TB that have direct implications on the diagnosis and treatment of this disease. Future directions on experimental approaches to understanding pathogenesis of ocular TB are also discussed.

Tuberculosis associated with Triplet therapy for lung cancer.

We report the first case of TB associated with triplet therapy (chemotherapy and immunotherapy concurrently) for lung cancer, developing just 44 days after treatment initiation. We feel that several important learning points arise from the discussion that are likely to be very relevant to the broad readership of Thorax, and have important clinical and scientific implications. In the three discussion paragraphs, we highlight that: 1) Triplet therapy is now standard first-line treatment for inoperable lung cancer. 2) TB reactivation is increasingly recognised as an adverse effect of immune checkpoint inhibition, but sending diagnostic samples is critical to avoid a missed diagnosis. 3) These insights from novel cancer immunotherapies are challenging the traditional views of the host-pathogen interaction in TB, with wide implications for future control strategies. We propose that the cases reported in the literature are likely to be the tip of the iceberg as most people with lung cancer managed with antiprogrammed death-1 agents who develop new lung lesions will be treated with standard antibiotics and then palliated when they do not respond.

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α.

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-α is responsible for accelerated Mtb growth, and TNF-α neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-α immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-α concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-α secretion.

In Vitro Granuloma Models of Tuberculosis: Potential and Challenges.

Despite intensive research efforts, several fundamental disease processes for tuberculosis (TB) remain poorly understood. A central enigma is that host immunity is necessary to control disease yet promotes transmission by causing lung immunopathology. Our inability to distinguish these processes makes it challenging to design rational novel interventions. Elucidating basic immune mechanisms likely requires both in vivo and in vitro analyses, since Mycobacterium tuberculosis is a highly specialized human pathogen. The classic immune response is the TB granuloma organized in three dimensions within extracellular matrix. Several groups are developing cell culture granuloma models. In January 2018, NIAID convened a workshop, entitled "3-D Human in vitro TB Granuloma Model" to advance the field. Here, we summarize the arguments for developing advanced TB cell culture models and critically review those currently available. We discuss how integrating complementary approaches, specifically organoids and mathematical modeling, can maximize progress, and conclude by discussing future challenges and opportunities.

Interleukin-17 regulates matrix metalloproteinase activity in human pulmonary tuberculosis.

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin-17 (IL-17) is key in host defence in TB but its role in TB-driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T-helper 17 (TH -17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH -17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT-qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho-western analysis and siRNA. IL-17 is expressed in TB patient granulomas and MMP-3 is expressed in adjacent pulmonary epithelial cells. IL-17 had a divergent, concentration-dependent effect on MMP secretion, increasing epithelial secretion of MMP-3 (p < 0.001) over 72 h, whilst decreasing that of MMP-9 (p < 0.0001); mRNA levels were similarly affected. Both IL-17 and IL-22 increased fibroblast Mtb-dependent MMP-3 secretion but IL-22 did not modulate epithelial MMP-3 expression. Both IL-17 and IL-22, but not IL-23, were significantly up-regulated in BALF from TB patients. IL-17-driven MMP-3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL-17 drives airway stromal cell-derived MMP-3, a mediator of tissue destruction in TB, alone and with monocyte-dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.