In silico agent-based modeling approach to characterize multiple in vitro tuberculosis infection models.

In vitro models of Mycobacterium tuberculosis (Mtb) infection are a valuable tool for examining host-pathogen interactions and screening drugs. With the development of more complex in vitro models, there is a need for tools to help analyze and integrate data from these models. To this end, we introduce an agent-based model (ABM) representation of the interactions between immune cells and bacteria in an in vitro setting. This in silico model was used to simulate both traditional and spheroid cell culture models by changing the movement rules and initial spatial layout of the cells in accordance with the respective in vitro models. The traditional and spheroid simulations were calibrated to published experimental data in a paired manner, by using the same parameters in both simulations. Within the calibrated simulations, heterogeneous outputs are seen for bacterial count and T cell infiltration into the macrophage core of the spheroid. The simulations also predict that equivalent numbers of activated macrophages do not necessarily result in similar bacterial reductions; that host immune responses can control bacterial growth in both spheroid structure dependent and independent manners; that STAT1 activation is the limiting step in macrophage activation in spheroids; and that drug screening and macrophage activation studies could have different outcomes depending on the in vitro culture used. Future model iterations will be guided by the limitations of the current model, specifically which parts of the output space were harder to reach. This ABM can be used to represent more in vitro Mtb infection models due to its flexible structure, thereby accelerating in vitro discoveries.

Mycobacterium tuberculosis resisters despite HIV exhibit activated T cells and macrophages in their pulmonary alveoli.

To understand natural resistance to Mycobacterium tuberculosis ( Mtb ) infection, we studied people living with HIV (PLWH) in an area of high Mtb transmission. Given that alveolar leukocytes may contribute to this resistance, we performed single cell RNA-sequencing of bronchoalveolar lavage cells, unstimulated or ex vivo stimulated with Mtb . We obtained high quality cells for 7 participants who were TST & IGRA positive (called LTBI) and 6 who were persistently TST & IGRA negative (called resisters). Alveolar macrophages (AM) from resisters displayed more of an M1 phenotype relative to LTBI AM at baseline. Alveolar lymphocytosis (10%-60%) was exhibited by 5/6 resisters, resulting in higher numbers of CD4 + and CD8 + IFNG -expressing cells at baseline and upon Mtb challenge than LTBI samples. Mycobactericidal granulysin was expressed almost exclusively by a cluster of CD8 + T cells that co-expressed granzyme B, perforin and NK cell receptors. For resisters, these poly-cytotoxic T cells over-represented activating NK cell receptors and were present at 15-fold higher numbers in alveoli compared to LTBI. Altogether, our results showed that alveolar lymphocytosis, with increased numbers of alveolar IFNG -expressing cells and CD8 + poly-cytotoxic T cells, as well as activated AM were strongly associated with protection from persistent Mtb infection in PLWH.

Suppressive myeloid cells in SARS-CoV-2 and Mycobacterium tuberculosis co-infection.

Epidemiologic data show that both current and previous tuberculosis (TB) increase the risk of in-hospital mortality from coronavirus disease-2019 (COVID-19), and there is a similar trend for poor outcomes from Mycobacterium tuberculosis (Mtb) infection after recent SARS-CoV-2. A shared dysregulation of immunity explains the dual risk posed by co-infection, but the specific mechanisms are being explored. While initial attention focused on T cell immunity, more comprehensive analyses revealed a dysfunctional innate immune response in COVID-19, characterized by reduced numbers of dendritic cells, NK cells and a redistribution of mononuclear phagocytes towards intermediate myeloid subsets. During hyper- or chronic inflammatory processes, activation signals from molecules such as growth factors and alarmins lead to the expansion of an immature population of myeloid cells called myeloid-deprived suppressor cells (MDSC). These cells enter a state of pathological activation, lose their ability to rapidly clear pathogens, and instead become broadly immunosuppressive. MDSC are enriched in the peripheral blood of patients with severe COVID-19; associated with mortality; and with higher levels of inflammatory cytokines. In TB, MDSC have been implicated in loss of control of Mtb in the granuloma and ineffective innate and T cell immunity to the pathogen. Considering that innate immune sensing serves as first line of both anti-bacterial and anti-viral defence mechanisms, we propose MDSC as a crucial mechanism for the adverse clinical trajectories of TB-COVID-19 coinfection.

Immunologic and vascular biomarkers of mortality in critical COVID-19 in a South African cohort.

Introduction: Biomarkers predicting mortality among critical Coronavirus disease 2019 (COVID-19) patients provide insight into the underlying pathophysiology of fatal disease and assist with triaging of cases in overburdened settings. However, data describing these biomarkers in Sub-Saharan African populations are sparse. Methods: We collected serum samples and corresponding clinical data from 87 patients with critical COVID-19 on day 1 of admission to the intensive care unit (ICU) of a tertiary hospital in Cape Town, South Africa, during the second wave of the COVID-19 pandemic. A second sample from the same patients was collected on day 7 of ICU admission. Patients were followed up until in-hospital death or hospital discharge. A custom-designed 52 biomarker panel was performed on the Luminex® platform. Data were analyzed for any association between biomarkers and mortality based on pre-determined functional groups, and individual analytes. Results: Of 87 patients, 55 (63.2%) died and 32 (36.8%) survived. We found a dysregulated cytokine response in patients who died, with elevated levels of type-1 and type-2 cytokines, chemokines, and acute phase reactants, as well as reduced levels of regulatory T cell cytokines. Interleukin (IL)-15 and IL-18 were elevated in those who died, and levels reduced over time in those who survived. Procalcitonin (PCT), C-reactive protein, Endothelin-1 and vascular cell adhesion molecule-1 were elevated in those who died. Discussion: These results show the pattern of dysregulation in critical COVID-19 in a Sub-Saharan African cohort. They suggest that fatal COVID-19 involved excessive activation of cytotoxic cells and the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome. Furthermore, superinfection and endothelial dysfunction with thrombosis might have contributed to mortality. HIV infection did not affect the outcome. A clinically relevant biosignature including PCT, pH and lymphocyte percentage on differential count, had an 84.8% sensitivity for mortality, and outperformed the Luminex-derived biosignature.

Optimising the yield from bronchoalveolar lavage on human participants in infectious disease immunology research.

Bronchoalveolar lavage (BAL) is becoming a common procedure for research into infectious disease immunology. Little is known about the clinical factors which influence the main outcomes of the procedure. In research participants who underwent BAL according to guidelines, the BAL volume yield, and cell yield, concentration, viability, pellet colour and differential count were analysed for association with important participant characteristics such as active tuberculosis (TB) disease, TB exposure, HIV infection and recent SARS-CoV-2 infection. In 337 participants, BAL volume and BAL cell count were correlated in those with active TB disease, and current smokers. The right middle lobe yielded the highest volume. BAL cell and volume yields were lower in older participants, who also had more neutrophils. Current smokers yielded lower volumes and higher numbers of all cell types, and usually had a black pellet. Active TB disease was associated with higher cell yields, but this declined at the end of treatment. HIV infection was associated with more bloody pellets, and recent SARS-CoV-2 infection with a higher proportion of lymphocytes. These results allow researchers to optimise their participant and end assay selection for projects involving lung immune cells.

An Archetypical Model for Engrafting Bacteroides fragilis into Conventional Mice Following Reproducible Antibiotic Conditioning of the Gut Microbiota.

Bacteroides fragilis is a commonly investigated commensal bacterium for its protective role in host diseases. Here, we aimed to develop a reproducible antibiotic-based model for conditioning the gut microbiota and engrafting B. fragilis into a conventional murine host. Initially, we selected different combinations of antibiotics, including metronidazole, imipenem, and clindamycin, and investigated their efficacy in depleting the mouse Bacteroides population. We performed 16S rRNA sequencing of DNA isolated from fecal samples at different time points. The α-diversity was similar in mice treated with metronidazole (MET) and differed only at weeks 1 (p = 0.001) and 3 (p = 0.009) during metronidazole/imipenem (MI) treatment. Bacteroides compositions, during the MET and MI exposures, were similar to the pre-antibiotic exposure states. Clindamycin supplementation added to MET or MI regimens eliminated the Bacteroides population. We next repeated metronidazole/clindamycin (MC) treatment in two additional independent experiments, followed by a B. fragilis transplant. MC consistently and reproducibly eliminated the Bacteroides population. The depleted Bacteroides did not recover in a convalescence period of six weeks post-MC treatment. Finally, B. fragilis was enriched for ten days following engraftment into Bacteroides-depleted mice. Our model has potential use in gut microbiota studies that selectively investigate Bacteroides' role in diseases of interest.

Optimising the yield from bronchoalveolar lavage on human participants in infectious disease immunology research.

Bronchoalveolar lavage (BAL) is becoming a common procedure for research into infectious disease immunology. Little is known about the clinical factors which influence the main outcomes of the procedure. In research participants who underwent BAL according to guidelines, the BAL volume yield, and cell yield, concentration, viability, pellet colour and differential count were analysed for association with important participant characteristics such as active tuberculosis (TB) disease, TB exposure, HIV infection and recent SARS-CoV-2 infection. In 337 participants, BAL volume and BAL cell count were correlated in those with active TB disease, and current smokers. The right middle lobe yielded the highest volume. BAL cell and volume yields were lower in older participants, who also had more neutrophils. Current smokers yielded lower volumes and higher numbers of all cell types, and usually had a black pellet. Active TB disease was associated with higher cell yields, and higher proportions of granulocytes, but this declined at the end of treatment. HIV infection was associated with lower cell yields and more bloody pellets, and recent SARS-CoV-2 infection with a higher proportion of lymphocytes. These results allow researchers to optimise their participant and end assay selection for projects involving lung immune cells.

Neutrophil degranulation, NETosis and platelet degranulation pathway genes are co-induced in whole blood up to six months before tuberculosis diagnosis.

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

Sildenafil, a Type-5 Phosphodiesterase Inhibitor, Fails to Reverse Myeloid-Derived Suppressor Cell-Mediated T Cell Suppression in Cells Isolated From Tuberculosis Patients.

Successful TB treatment is hampered by increasing resistance to the two most effective first-line anti-TB drugs, namely isoniazid and rifampicin, thus innovative therapies focused on host processes, termed host-directed therapies (HDTs), are promising novel approaches for increasing treatment efficacy without inducing drug resistance. We assessed the ability of Sildenafil, a type-5 phosphodiesterase inhibitor, as a repurposed compound, to serve as HDT target, by counteracting the suppressive effects of myeloid-derived suppressor cells (MDSC) obtained from active TB cases on T-cell responsiveness. We confirm that MDSC suppress non-specific T-cell activation. We also show that Sildenafil treatment fails to reverse the MDSC-mediated suppression of T-cell functions measured here, namely activation and proliferation. The impact of Sildenafil treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

Cascade Immune Mechanisms of Protection against Mycobacterium tuberculosis (IMPAc-TB): study protocol for the Household Contact Study in the Western Cape, South Africa.

BACKGROUND: Natural immunity against Mycobacterium tuberculosis exists, and > 90% of those infected remain disease-free. Innate and adaptive immune responses required to mediate such protection against tuberculosis (TB) are, however, poorly understood. METHODS: This is an analytical study exploring protective and non-protective pathways of immunity against Mycobacterium tuberculosis. Adults without HIV infection are recruited at community healthcare clinics in high TB incidence areas of the Western Cape Province, South Africa. Data regarding participants' medical, social and medication usage will be collected, and clinical examinations and point-of-care tests documented. Reference tests for TB (chest radiographs and sputum tests for GeneXpert MTB/RIF Ultra®, Auramine smear and liquid cultures) and investigations to classify infection states [interferon-gamma release assay (IGRA) and SARS-CoV-2 polymerase chain reaction (PCR) nasopharyngeal swab and IgG], are done on all participants who meet the inclusion criteria. 18F-Fluorodeoxyglucose positron emission tomography combined with computerized tomography will be done on all close contacts (contacts) and healthy control (controls) participants. Participants are divided into 12 study groups representing a spectrum of TB clinical phenotypes and prior SARS-CoV-2 infection based on their TB status, exposure history, results of IGRA test at baseline and 3 months, SARS-CoV-2 serology, and PCR results, and for contacts and controls, PET-CT imaging findings indicative of sub-clinical TB lesions. Samples for experimental assays include whole blood for isolation of peripheral blood mononuclear cells and blood in PAXgene® tubes for RNA isolation. All SARS-CoV-2 PCR negative study participants undergo bronchoscopy for collecting bronchoalveolar lavage samples. DISCUSSION: The paired blood and BAL samples will be used for comprehensive analyses of the tissue-specific and systemic immunity that will include e.g., cytometry by time-of-flight analyses, RNA-sequencing, multiplex immunoassays, epigenetic analysis, and mechanistic studies of control of infection by Mycobacterium tuberculosis. Results will be integrated with those from mice and non-human primate studies to provide a comprehensive analysis of protective pathways in natural and vaccine-induced immunity against Mycobacterium tuberculosis.

Immunometabolism of Myeloid-Derived Suppressor Cells: Implications for Mycobacterium tuberculosis Infection and Insights from Tumor Biology.

The field of immunometabolism seeks to decipher the complex interplay between the immune system and the associated metabolic pathways. The role of small molecules that can target specific metabolic pathways and subsequently alter the immune landscape provides a desirable platform for new therapeutic interventions. Immunotherapeutic targeting of suppressive cell populations, such as myeloid-derived suppressor cells (MDSC), by small molecules has shown promise in pathologies such as cancer and support testing of similar host-directed therapeutic approaches in MDSC-inducing conditions such as tuberculosis (TB). MDSC exhibit a remarkable ability to suppress T-cell responses in those with TB disease. In tumors, MDSC exhibit considerable plasticity and can undergo metabolic reprogramming from glycolysis to fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) to facilitate their immunosuppressive functions. In this review we look at the role of MDSC during M. tb infection and how their metabolic reprogramming aids in the exacerbation of active disease and highlight the possible MDSC-targeted metabolic pathways utilized during M. tb infection, suggesting ways to manipulate these cells in search of novel insights for anti-TB therapies.

Targeted Gene Expression Profiling of Human Myeloid Cells From Blood and Lung Compartments of Patients With Tuberculosis and Other Lung Diseases.

Myeloid-derived suppressor cells (MDSC) have been identified in the peripheral blood and granulomas of patients with active TB disease, but their phenotype-, function-, and immunosuppressive mechanism- spectrum remains unclear. Importantly, the frequency and signaling pathways of MDSC at the site of disease is unknown with no indication how this compares to MDSC identified in peripheral blood or to those of related myeloid counterparts such as alveolar macrophages and monocytes. Most phenotypic and functional markers have been described in oncological studies but have not yet been validated in TB. Using a panel of 43 genes selected from pathways previously shown to contribute to tumor-derived MDSC, we set out to evaluate if the expression of these additional functional markers and properties may also be relevant to TB-derived MDSC. Differential expression was investigated between MDSC, alveolar macrophages and monocytes enriched from bronchoalveolar lavage fluid and peripheral blood of patients with active TB, patients with other lung diseases (OLD). Results demonstrated that anatomical compartments may drive compartment-specific immunological responses and subsequent MDSC immunosuppressive functions, demonstrated by the observation that MDSC and/or monocytes from PB alone can discriminate, via hierarchical clustering, between patients with active TB disease and OLD. Our data show that the gene expression patterns of MDSC in peripheral blood and bronchoalveolar lavage fluid do not cluster according to disease states (TB vs OLD). This suggests that MDSC from TB patients may display similar gene expression profiles to those found for MDSC in cancer, but this needs to be validated in a larger cohort. These are important observations for TB research and may provide direction for future studies aimed at repurposing and validating cancer immunotherapies for use in TB.

The Intestinal Commensal, Bacteroides fragilis, Modulates Host Responses to Viral Infection and Therapy: Lessons for Exploration during Mycobacterium tuberculosis Infection.

The gut microbiota has emerged as a critical player in host health. Bacteroides fragilis is a prominent member of the gut microbiota within the phyla Bacteroidetes. This commensal bacterium produces unique capsular polysaccharides processed by antigen-presenting cells and activates CD4+ T cells to secrete inflammatory cytokines. Indeed, due to their immunomodulatory functions, B. fragilis and its capsular polysaccharide-A (PSA) are arguably the most explored single commensal microbiota/symbiotic factor. B. fragilis/PSA has been shown to protect against colitis, encephalomyelitis, colorectal cancer, pulmonary inflammation, and asthma. Here, we review recent data on the immunomodulatory role of B. fragilis/PSA during viral infections and therapy, B. fragilis PSA's dual ability to mediate pro-and anti-inflammatory processes, and the potential for exploring this unique characteristic during intracellular bacterial infections such as with Mycobacterium tuberculosis. We also discuss the protective roles of single commensal-derived probiotic species, including B. fragilis in lung inflammation and respiratory infections that may provide essential cues for possible exploration of microbiota based/augmented therapies in tuberculosis (TB). Available data on the relationship between B. fragilis/PSA, the immune system, and disease suggest clinical relevance for developing B. fragilis into a next-generation probiotic or, possibly, the engineering of PSA into a potent carbohydrate-based vaccine.

Evaluation of autophagy mediators in myeloid-derived suppressor cells during human tuberculosis.

Myeloid-derived suppressor cells (MDSC) are induced during active TB disease to restore immune homeostasis but instead exacerbate disease outcome due to chronic inflammation. Autophagy, in conventional phagocytes, ensures successful clearance of M.tb. However, autophagy has been demonstrated to induce prolonged MDSC survival. Here we investigate the relationship between autophagy mediators and MDSC in the context of active TB disease and during anti-TB therapy. We demonstrate a significant increase in MDSC frequencies in untreated active TB cases with these MDSC expressing TLR4 and significantly more mTOR and IL-6 than healthy controls, with mTOR levels decreasing during anti-TB therapy. Finally, we show that HMGB1 serum concentrations decrease in parallel with mTOR. These findings suggest a complex interplay between MDSC and autophagic mediators, potentially dependent on cellular localisation and M.tb infection state.

Establishment of a Patient-Derived, Magnetic Levitation-Based, Three-Dimensional Spheroid Granuloma Model for Human Tuberculosis.

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M. tuberculosis) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization, and activation are driven not only by coexisting cell types and multicellular interactions but also by M. tuberculosis-mediated changes involving metabolic heterogeneity, epigenetic reprogramming, and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility, and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma and the difficulty in translating findings from animal models to human disease. Here, we describe a novel biomimetic in vitro three-dimensional (3D) human lung spheroid granuloma model, resembling early "innate" and "adaptive" stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles, and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these spheroid granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions. IMPORTANCE TB is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M. tuberculosis infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M. tuberculosis are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in two dimensions on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro spheroid granuloma model which allows researchers to study features of granuloma-forming diseases in a 3D structural environment resembling in vivo granuloma architecture and cellular orientation.

Inhaled particulate matter affects immune responsiveness of human lung phagocytes to mycobacteria.

The influence of smoke-derived or air pollution-derived cytoplasmic particulate matter (PM) can be detrimental and can lead to failed lung immunity. We investigated mycobacterial uptake, intracellular replication, and soluble immune-mediator responses of human bronchoalveolar lavage cells (BALCs) loaded with/without PM, to infection with mycobacterial strains. We observed that only BALCs containing PM display an ex vivo phenotypic profile dominated by spontaneous interleukin (IL)-10 production. PM-loaded BALCs retained the ability to phagocytose both Mycobacterium bovis Bacille Calmette Guérin (BCG) and Mycobacterium tuberculosis (M.tb) ΔleuDΔpanCD at equal efficacy as clear non-PM-loaded BALCs. However, immune responsiveness, such as the production of IL-6 (P = 0.015) and tumor necrosis factor-α (TNF)-α (P = 0.0172) immediately post M. bovis BCG infection, were dramatically lower in black BALCs loaded with PM versus clear non-PM-loaded BALCs. By 24 h post infection, differential immune responses to M. bovis BCG between black versus clear BALC waned, and instead, production of IL-6 (P = 0.03) and IL-1α (P = 0.04) by black BALCs was lower versus clear BALCs following M.tb ΔleuDΔpanCD infection. Considering that TNF-α and IL-6 are characterized as critical to host protection against mycobacteria, our findings suggest that BALCs loaded with inhaled PM, display lower levels of antimycobacterial mediators and that the response magnitude differs according to infective mycobacterial strain. Even though this did not translate into altered mycobacterial killing at early time points post infection, the long-term impact of such changes remains to be established.

Targeting of myeloid-derived suppressor cells by all-trans retinoic acid as host-directed therapy for human tuberculosis.

Conventional anti-tuberculosis (TB) therapies comprise lengthy antibiotic treatment regimens, exacerbated by multi-drug resistant and extensively drug resistant mycobacterial strains. We assessed the ability of all-trans retinoic acid (ATRA), as repurposed compound serving as host-directed therapy (HDT), to counteract the suppressive effects of myeloid-derived suppressor cells (MDSCs) obtained from active TB cases (untreated or during week one of treatment) on T-cell responsiveness. We show for the first time that MDSCs suppress non-specific T-cell activation and production of interleukin (IL)-2, IL-4, IL-13 and GM-CSF via contact-dependent mechanisms. ATRA treatment decreases MDSC frequency, but fails to mature MDSCs to non-suppressive, terminally differentiated myeloid cells and does not restore T-cell function or cytokine production in the presence of MDSCs. The impact of ATRA treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

Mycobacterium tuberculosis-stimulated whole blood culture to detect host biosignatures for tuberculosis treatment response.

Host markers to monitor the response to tuberculosis (TB) therapy hold some promise. We evaluated the changes in concentration of Mycobacterium tuberculosis (M.tb)-induced soluble biomarkers during early treatment for predicting short- and long-term treatment outcomes. Whole blood samples from 30 cured and 12 relapsed TB patients from diagnosis, week 1, 2, and 4 of treatment were cultured in the presence of live M.tb for seven days and patients followed up for 24 weeks after the end of treatment. 57 markers were measured in unstimulated and antigen-stimulated culture supernatants using Luminex assays. Top performing multi-variable models at diagnosis using unstimulated values predicted outcome at 24 months after treatment completion with a sensitivity of 75.0% (95% CI, 42.8-94.5%) and specificity of 72.4% (95% CI, 52.8-87.3%) in leave-one-out cross validation. Month two treatment responder classification was correctly predicted with a sensitivity of 79.2% (95% CI, 57.8-92.9%) and specificity of 92.3% (95% CI, 64.0-99.8%). This study provides evidence of the early M.tb-specific treatment response in TB patients but shows that the observed unstimulated marker models are not outperformed by stimulated marker models. Performance of unstimulated predictive host marker signatures is promising and requires validation in larger studies.

Higher SARS-CoV-2 seroprevalence in workers with lower socioeconomic status in Cape Town, South Africa.

BACKGROUND: Inequality is rife throughout South Africa. The first wave of COVID-19 may have affected people in lower socioeconomic groups worse than the affluent. The SARS-CoV-2 seroprevalence and the specificity of anti-SARS-CoV-2 antibody tests in South Africa is not known. METHODS: We tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott SARS-CoV-2 IgG assay. We assessed the association between antibody positivity and COVID-19 symptom status, medical history, and sociodemographic variables. We tested 137 serum samples from healthy controls collected in Cape Town prior to the COVID-19 pandemic, to confirm the specificity of the assay in the local population. RESULTS: Of the 405 volunteers tested one month after the first peak of the epidemic in Cape Town, 96(23.7%) were SARS-CoV-2 IgG positive. Of those who tested positive, 46(47.9%) reported no symptoms of COVID-19 in the previous 6 months. Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The specificity of the assay was 98.54%(95%CI 94.82%-99.82%) in the pre-COVID controls. CONCLUSIONS: There is a high background seroprevalence in Cape Town, particularly in people of lower socioeconomic status. Almost half of cases are asymptomatic, and therefore undiagnosed by local testing strategies. These results cannot be explained by low assay specificity.

Monocytic myeloid-derived suppressor cells reflect tuberculosis severity and are influenced by cyclooxygenase-2 inhibitors.

Myeloid-derived suppressor cells (MDSCs) increase in tuberculosis (TB) and may be targets for host-directed therapy (HDT). In this study, we use flow cytometry to analyze the effects of cyclooxygenase-2 inhibitors (COX-2i) on monocytic (M)-MDSCs in blood from TB patients attending a clinical trial of COX-2i. The effects of COX-2i on M-MDSCs and mycobacterial uptake were also studied by an in vitro mycobacterial infection model. We found that M-MDSC frequencies correlated with TB disease severity. Reduced M-MDSC (P = 0.05) and IDO (P = 0.03) expression was observed in the COX-2i group. We show that peripheral blood-derived M-MDSCs successfully internalized Mycobacterium bovis and that in vitro mycobacterial infection increased COX-2 (P = 0.002), PD-L1 (P = 0.01), and Arginase-1 (P = 0.002) expression in M-MDSCs. Soluble IL-1ß, IL-10, and S100A9 were reduced in COX-2i-treated M-MDSCs cultures (P < 0.05). We show novel data that COX-2i had limited effect in vivo but reduced M-MDSC cytokine production in vitro. The relevance of COX-2i in a HDT strategy needs to be further explored.