Protocol to phenotype and quantify mycobacteria-specific myeloid cells from human airways by mass cytometry.

Alveolar macrophages and other myeloid cells in the human airways are the primary cell types responding to respiratory pathogens. Here, we present a protocol for in vitro stimulation of cryopreserved human bronchoalveolar lavage (BAL) cells with mycobacterial antigens for phenotyping and quantifying proinflammatory cytokine responses in myeloid cells by mass cytometry. We demonstrate that the measure of markers of myeloid lineage and function is stable after freezing stained cells thereby allowing for batched analyses and/or machine downtime.

Agent-based model predicts that layered structure and 3D movement work synergistically to reduce bacterial load in 3D in vitro models of tuberculosis granuloma.

Tuberculosis (TB) remains a global public health threat. Understanding the dynamics of host-pathogen interactions within TB granulomas will assist in identifying what leads to the successful elimination of infection. In vitro TB models provide a controllable environment to study these granuloma dynamics.

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.

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

To understand natural resistance to ( ) infection, we studied people living with HIV (PLWH) in an area of high transmission. Given that alveolar leukocytes may contribute to this resistance, we performed single cell RNA-sequencing of bronchoalveolar lavage cells, unstimulated or stimulated with . 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).

Suppressive myeloid cells in SARS-CoV-2 and 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 (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.

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.

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.

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

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 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 population.

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.

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.

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.

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.

Immunometabolism of Myeloid-Derived Suppressor Cells: Implications for 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).

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.

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.

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.

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.

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.

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.

The Peripheral Blood Transcriptome Is Correlated With PET Measures of Lung Inflammation During Successful Tuberculosis Treatment.

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [F]FDG activity as a surrogate for lung inflammation.

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.

Safety and immunogenicity of the adjunct therapeutic vaccine ID93 + GLA-SE in adults who have completed treatment for tuberculosis: a randomised, double-blind, placebo-controlled, phase 2a trial.

Background: A therapeutic vaccine that prevents recurrent tuberculosis would be a major advance in the development of shorter treatment regimens. We aimed to assess the safety and immunogenicity of the ID93 + GLA-SE vaccine at various doses and injection schedules in patients with previously treated tuberculosis.

Methods: This randomised, double-blind, placebo-controlled, phase 2a trial was conducted at three clinical sites near Cape Town, South Africa.

Isolation and Functional Characterization of Myeloid-Derived Suppressor Cells in Infections Under High Containment.

The current absence of markers unique to MDSC, particularly those expanded during human infection, necessitate concurrent demonstration of their suppressive capacity to ensure unequivocal identification. This is further complicated by the array of heterogeneous markers used to characterize MDSC in various conditions and models. Standardization of phenotypic and functional characterization, as well as isolation, from infectious biological samples of patients, are critical for accurately reporting MDSC dynamics, function, organ abundance, and establishment of their therapeutic value in infectious diseases.