Tobacco smoke exposure recruits inflammatory airspace monocytes that establish permissive lung niches for Mycobacterium tuberculosis.

Tobacco smoking doubles the risk of active tuberculosis (TB) and accounts for up to 20% of all active TB cases globally. How smoking promotes lung microenvironments permissive to Mycobacterium tuberculosis (Mtb) growth remains incompletely understood. We investigated primary bronchoalveolar lavage cells from current and never smokers by performing single-cell RNA sequencing (scRNA-seq), flow cytometry, and functional assays. We observed the enrichment of immature inflammatory monocytes in the lungs of smokers compared with nonsmokers. These monocytes exhibited phenotypes consistent with recent recruitment from blood, ongoing differentiation, increased activation, and states similar to those with chronic obstructive pulmonary disease. Using integrative scRNA-seq and flow cytometry, we identified CD93 as a marker for a subset of these newly recruited smoking-associated lung monocytes and further provided evidence that the recruitment of monocytes into the lung was mediated by CCR2-binding chemokines, including CCL11. We also show that these cells exhibit elevated inflammatory responses upon exposure to Mtb and accelerated intracellular growth of Mtb compared with mature macrophages. This elevated Mtb growth could be inhibited by anti-inflammatory small molecules, providing a connection between smoking-induced pro-inflammatory states and permissiveness to Mtb growth. Our findings suggest a model in which smoking leads to the recruitment of immature inflammatory monocytes from the periphery to the lung, which results in the accumulation of these Mtb-permissive cells in the airway. This work defines how smoking may lead to increased susceptibility to Mtb and identifies host-directed therapies to reduce the burden of TB among those who smoke.

Phosphatidylinositolmannoside vaccination induces lipid-specific Th1-responses and partially protects guinea pigs from Mycobacterium tuberculosis challenge.

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals' lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Compartmentalized T cell profile in the lungs of patients with HIV-1-associated pulmonary Kaposi sarcoma.

ABSTRACT: Pulmonary Kaposi sarcoma (pKS) caused by Human herpesvirus 8 (HHV-8) is a devastating form of KS in patients with advanced acquired immunodeficiency syndrome (AIDS) and is associated with increased morbidity and mortality. Blood T cells play a central role in the response of HIV-1 and HHV-8. However, little information is available on T cells in the alveolar space of HIV-1-associated pKS patients.Therefore, we examined CD8+ and CD4+ T cells in the alveolar space in comparison with the blood of patients with pKS. We recruited 26 HIV-1 positive patients with KS, including 15 patients with pKS. Bronchoalveolar lavage (BAL) cells and blood mononuclear cells were analyzed for T cell memory phenotypes, surface markers associated with exhaustion, and intracellular cytokine staining (ICS) using flow cytometry. HIV-1 and HHV-8 viral loads were measured in plasma by quantitative PCR.BAL T cells showed reduced inflammatory capacities and significantly diminished polyfunctionality compared to blood T cells from patients with pKS. This was not accompanied by increased expression of exhaustion markers, such as TIM-3 and PD-1.More importantly, we found a negative correlation between the production of MIP1-ß and TNF-α in T cells in BAL and blood, indicating compartmentalised immune responses to pKS and accentuated chronic HIV-1/HHV-8 pathogenesis via T cells in the lungs of people with pKS.

Smoking and Human Immunodeficiency Virus 1 Infection Promote Retention of CD8+ T Cells in the Airway Mucosa.

Smoking and human immunodeficiency virus 1 (HIV-1) infection are risk factors for chronic obstructive pulmonary disease (COPD), which is among the most common comorbid conditions in people living with HIV-1. HIV-1 infection leads to persistent expansion of CD8+ T cells, and CD8+ T cell-mediated inflammation has been implicated in COPD pathogenesis. In this study, we investigated the effects of HIV-1 infection and smoking on T-cell dynamics in patients at risk of COPD. BAL fluid, endobronchial brushings, and blood from HIV-1 infected and uninfected nonsmokers and smokers were analyzed by flow cytometry, and lungs were imaged by computed tomography. Chemokines were measured in BAL fluid, and CD8+ T-cell chemotaxis in the presence of cigarette smoke extract was assessed in vitro. HIV-1 infection increased CD8+ T cells in the BAL fluid, but this increase was abrogated by smoking. Smokers had reduced BAL fluid concentrations of the T cell-recruiting chemokines CXCL10 and CCL5, and cigarette smoke extract inhibited CXCL10 and CCL5 production by macrophages and CD8+ T-cell transmigration in vitro. In contrast to the T cells in BAL fluid, CD8+ T cells in endobronchial brushings were increased in HIV-1-infected smokers, which was driven by an accumulation of effector memory T cells in the airway mucosa and an increase in tissue-resident memory T cells. Mucosal CD8+ T-cell numbers inversely correlated with lung aeration, suggesting an association with inflammation and remodeling. HIV-1 infection and smoking lead to retention of CD8+ T cells within the airway mucosa.

PET Imaging Reveals Early Pulmonary Perfusion Abnormalities in HIV Infection Similar to Smoking.

Chronic obstructive pulmonary disease (COPD) is the most common noninfectious pulmonary disease among people living with HIV, independent of smoking. However, the cause for this enhanced susceptibility remains unclear, and the effects of HIV on pulmonary perfusion and ventilation are unknown. Methods: We used PET/CT in 46 smokers and nonsmokers, 23 of whom had documented HIV infection. Emphysema was assessed by CT and perfusion by 13N (13NN) PET scans. After removal of image noise, vertical and axial gradients in perfusion were calculated. We tested for differences in the total spatial heterogeneity of perfusion (CV2Qtotal) and its components (CV2Qtotal = CV2Qvgrad [vertical gradient] + CV2Qzgrad [axial gradient] + CV2Qr [residual heterogeneity]) among groups. Results: There were no significant differences in demographic parameters among groups, and all subjects had minimal radiographic evidence of emphysema. Compared with controls, nonsmokers living with HIV had a significantly greater CV2Qr/CV2Qtotal (0.48 vs. 0.36, P = 0.05) and reduced CV2Qvgrad/CV2Qtotal (0.46 vs. 0.65, P = 0.038). Smokers also had a reduced CV2Qvgrad/CV2Qtotal, however, there was no significant difference in CV2Qvgrad/CV2Qtotal between smokers living with and without HIV (0.39 vs. 0.34, P = 0.58), despite a decreased vertical perfusion gradient (Qvgrad) in smokers living with HIV. Conclusion: In nonsmokers living with well-controlled HIV and minimal radiographic emphysema, HIV infection contributes to pulmonary perfusion abnormalities similar to smokers. These data indicate the onset of subclinical pulmonary perfusion abnormalities that could herald the development of significant lung disease in these susceptible individuals.

Heterogeneous GM-CSF signaling in macrophages is associated with control of Mycobacterium tuberculosis.

Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.

Age-associated B cells expanded in autoimmune mice are memory cells sharing H-CDR3-selected repertoires.

Age-associated B cells (ABCs) represent a distinct cell population expressing low levels of CD21 (CD21-/low ). The Ig repertoire expressed by ABCs in aged mice is diverse and exhibits signs of somatic hypermutation (SHM). A CD21-/low B-cell population is expanded in autoimmune diseases, e.g. systemic lupus erythematosus, as well as in lupus-prone NZB/W mice and in mice lacking a pre-B cell receptor (SLC-/- ). However, the nature of the CD21-/low B cells (hereafter ABCs) in autoimmunity is not well understood. Here we show that in young SLC-/- mice, the vast majority of the ABCs express memory B-cell (MBC) markers in contrast to wild-type controls. A similar population is present in lupus-prone MRL mice before and at disease onset. In SLC-/- mice, a majority of the ABCs are IgM+ , their VH genes have undergone SHM, show clonal diversification and clonal restriction at the H-CDR3 level. ABC hybridomas, established from SLC-/- mice, secrete typical lupus autoantibodies, e.g. anti-Smith antigen, and some of those that bind to DNA comprise a H-CDR3 that is identical to previously described IgM anti-DNA antibodies from lupus-prone mice. Together, these results reveal that ABCs in autoimmune mice are comprised of autoreactive MBCs expressing highly restricted H-CDR3 repertoires.

M. tuberculosis-Induced Necrosis of Infected Neutrophils Promotes Bacterial Growth Following Phagocytosis by Macrophages.

Neutrophils represent the main infected cell population in the lungs of active tuberculosis patients. Efficient removal of infected and dying neutrophils is required to protect the surrounding tissue from bioactive neutrophil molecules and subsequent pathological sequelae. While the removal of apoptotic M. tuberculosis (Mtb)-infected cells, or efferocytosis, is considered beneficial for host defense, little is known about Mtb-infected necrotic neutrophils. We found that Mtb induces necrosis of human neutrophils in an ESX-1-dependent manner, and neutrophil-produced reactive oxygen species (ROS) drive this necrosis. Neutrophil necrosis was required for Mtb growth after uptake of infected neutrophils by human macrophages. Pharmacological inhibition of ROS production could prevent necrosis and restore the capability of macrophages to control Mtb growth, thereby identifying a potential host-directed therapy target. Taken together, necrosis represents the starting point for a vicious cycle including the uptake of infected necrotic cells by other phagocytes, Mtb growth therein, and sustained infection.

Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils.

Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.

Censoring of autoreactive B cell development by the pre-B cell receptor.

Antibody diversity occurs randomly as B cells recombine their immunoglobulin (Ig) heavy- and light-chain genes during development. This process inevitably generates reactivity against self structures, and several mechanisms prevent the development of autoreactive B cells. We report here a role for the pre-B cell receptor, composed of Ig heavy and surrogate light chains, in the negative selection of cells expressing Ig heavy chains with the potential to generate autoantibodies. Surrogate light-chain-deficient (SLC-/-) mice harbored elevated levels of antinuclear antibodies (ANAs) in their serum and showed evidence of escape of pre-B cells expressing prototypic autoantibody heavy chains from negative selection, leading to mature autoantibody secreting CD21-CD23- B cells in the periphery. Thus, the pre-B cell receptor appears to censor the development of certain autoantibody-secreting cells and may represent an important factor in multifactorial autoimmune diseases.