Beneficial or detrimental activity of regulatory T cells, indoleamine 2,3-dioxygenase, and heme oxygenase-1 in the lungs is influenced by the level of virulence of Mycobacterium tuberculosis strain infection.

Tuberculosis (TB) caused by the complex Mycobacterium tuberculosis (Mtb) is the main cause of death by a single bacterial agent. Last year, TB was the second leading infectious killer after SARS-CoV-2. Nevertheless, many biological and immunological aspects of TB are not completely elucidated, such as the complex process of immunoregulation mediated by regulatory T cells (Treg cells) and the enzymes indoleamine 2,3-dioxygenase (IDO) and heme oxygenase 1 (HO-1). In this study, the contribution of these immunoregulatory factors was compared in mice infected with Mtb strains with different levels of virulence. First Balb/c mice were infected by intratracheal route, with a high dose of mild virulence reference strain H37Rv or with a highly virulent clinical isolate (strain 5186). In the lungs of infected mice, the kinetics of Treg cells during the infection were determined by cytofluorometry and the expression of IDO and HO-1 by RT-PCR and immunohistochemistry. Then, the contribution of immune-regulation mediated by Treg cells, IDO and HO-1, was evaluated by treating infected animals with specific cytotoxic monoclonal antibodies for Treg cells depletion anti-CD25 (PC61 clone) or by blocking IDO and HO-1 activity using specific inhibitors (1-methyl-D,L-tryptophan or zinc protoporphyrin-IX, respectively). Mice infected with the mild virulent strain showed a progressive increment of Treg cells, showing this highest number at the beginning of the late phase of the infection (28 days), the same trend was observed in the expression of both enzymes being macrophages the cells that showed the highest immunostaining. Animals infected with the highly virulent strain showed lower survival (34 days) and higher amounts of Treg cells, as well as higher expression of IDO and HO-1 one week before. In comparison with non-treated animals, mice infected with strain H37Rv with depletion of Treg cells or treated with the enzymes blockers during late infection showed a significant decrease of bacilli loads, higher expression of IFN-g and lower IL-4 but with a similar extension of inflammatory lung consolidation determined by automated morphometry. In contrast, the depletion of Treg cells in infected mice with the highly virulent strain 5186 produced diffuse alveolar damage that was similar to severe acute viral pneumonia, lesser survival and increase of bacillary loads, while blocking of both IDO and HO-1 produced high bacillary loads and extensive pneumonia with necrosis. Thus, it seems that Treg cells, IDO and HO-1 activities are detrimental during late pulmonary TB induced by mild virulence Mtb, probably because these factors decrease immune protection mediated by the Th1 response. In contrast, Treg cells, IDO and HO-1 are beneficial when the infection is produced by a highly virulent strain, by regulation of excessive inflammation that produced alveolar damage, pulmonary necrosis, acute respiratory insufficiency, and rapid death.

Beyond the Clinic: The Activation of Diverse Cellular and Humoral Factors Shapes the Immunological Status of Patients with Active Tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), has killed nearly one billion people in the last two centuries. Nowadays, TB remains a major global health problem, ranking among the thirteen leading causes of death worldwide. Human TB infection spans different levels of stages: incipient, subclinical, latent and active TB, all of them with varying symptoms, microbiological characteristics, immune responses and pathologies profiles. After infection, Mtb interacts with diverse cells of both innate and adaptive immune compartments, playing a crucial role in the modulation and development of the pathology. Underlying TB clinical manifestations, individual immunological profiles can be identified in patients with active TB according to the strength of their immune responses to Mtb infection, defining diverse endotypes. Those different endotypes are regulated by a complex interaction of the patient's cellular metabolism, genetic background, epigenetics, and gene transcriptional regulation. Here, we review immunological categorizations of TB patients based on the activation of different cellular populations (both myeloid and lymphocytic subsets) and humoral mediators (such as cytokines and lipid mediators). The analysis of the participating factors that operate during active Mtb infection shaping the immunological status or immune endotypes of TB patients could contribute to the development of Host Directed Therapy.

Pathogenicity of Type I Interferons in Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease and rates have increased during the emergence of COVID-19, but many of the factors determining disease severity and progression remain unclear. Type I Interferons (IFNs) have diverse effector functions that regulate innate and adaptive immunity during infection with microorganisms. There is well-documented literature on type I IFNs providing host defense against viruses; however, in this review, we explore the growing body of work that indicates high levels of type I IFNs can have detrimental effects to a host fighting TB infection. We report findings that increased type I IFNs can affect alveolar macrophage and myeloid function, promote pathological neutrophil extracellular trap responses, inhibit production of protective prostaglandin 2, and promote cytosolic cyclic GMP synthase inflammation pathways, and discuss many other relevant findings.

Novel chemical entities inhibiting Mycobacterium tuberculosis growth identified by phenotypic high-throughput screening.

We performed a high-throughput phenotypic whole cell screen of Mycobacterium tuberculosis against a diverse chemical library of approximately 100,000 compounds from the AbbVie corporate collection and identified 24 chemotypes with anti-tubercular activity. We selected two series for further exploration and conducted structure-activity relationship studies with new analogs for the 4-phenyl piperidines (4PP) and phenylcyclobutane carboxamides (PCB). Strains with mutations in MmpL3 demonstrated resistance to both compound series. We isolated resistant mutants for the two series and found mutations in MmpL3. These data suggest that MmpL3 is the target, or mechanism of resistance for both series.

Evaluation of Host Protein Biomarkers by ELISA From Whole Lysed Peripheral Blood for Development of Diagnostic Tests for Active Tuberculosis.

Tuberculosis (TB) remains a significant global health crisis and the number one cause of death for an infectious disease. The health consequences in high-burden countries are significant. Barriers to TB control and eradication are in part caused by difficulties in diagnosis. Improvements in diagnosis are required for organisations like the World Health Organisation (WHO) to meet their ambitious target of reducing the incidence of TB by 50% by the year 2025, which has become hard to reach due to the COVID-19 pandemic. Development of new tests for TB are key priorities of the WHO, as defined in their 2014 report for target product profiles (TPPs). Rapid triage and biomarker-based confirmatory tests would greatly enhance the diagnostic capability for identifying and diagnosing TB-infected individuals. Protein-based test methods e.g. lateral flow devices (LFDs) have a significant advantage over other technologies with regard to assay turnaround time (minutes as opposed to hours) field-ability, ease of use by relatively untrained staff and without the need for supporting laboratory infrastructure. Here we evaluate the diagnostic performance of nine biomarkers from our previously published biomarker qPCR validation study; CALCOCO2, CD274, CD52, GBP1, IFIT3, IFITM3, SAMD9L, SNX10 and TMEM49, as protein targets assayed by ELISA. This preliminary evaluation study was conducted to quantify the level of biomarker protein expression across latent, extra-pulmonary or pulmonary TB groups and negative controls, collected across the UK and India, in whole lysed blood samples (WLB). We also investigated associative correlations between the biomarkers and assessed their suitability for ongoing diagnostic test development, using receiver operating characteristic/area under the curve (ROC) analyses, singly and in panel combinations. The top performing single biomarkers for pulmonary TB versus controls were CALCOCO2, SAMD9L, GBP1, IFITM3, IFIT3 and SNX10. TMEM49 was also significantly differentially expressed but downregulated in TB groups. CD52 expression was not highly differentially expressed across most of the groups but may provide additional patient stratification information and some limited use for incipient latent TB infection. These show therefore great potential for diagnostic test development either in minimal configuration panels for rapid triage or more complex formulations to capture the diversity of disease presentations.

Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.

Oxidative damage and delayed replication allow viable Mycobacterium tuberculosis to go undetected.

"Viable but nonculturable" states of bacteria pose challenges for environmental and clinical microbiology, but their biological mechanisms remain obscure. Mycobacterium tuberculosis (Mtb), the leading cause of death from infection until the coronavirus disease 2019 pandemic, affords a notable example of this phenotype. Mtb can enter into a "differentially detectable" (DD) state associated with phenotypic antimicrobial resistance. In this state, Mtb cells are viable but undetectable as colony-forming units. We found that Mtb cells enter the DD state when they undergo sublethal oxidative stress that damages their DNA, proteins, and lipids. In addition, their replication process is delayed, allowing time for repair. Mycobacterium bovis and its derivative, BCG, fail to enter the DD state under similar conditions. These findings have implications for tuberculosis latency, detection, relapse, treatment monitoring, and development of regimens that overcome phenotypic antimicrobial resistance.