Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

Background: The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. Methods: The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. Results: We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4 = 1.2, L4 = 1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Conclusion: Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

CD38 Expression by Antigen-Specific CD4 T Cells Is Significantly Restored 5 Months After Treatment Initiation Independently of Sputum Bacterial Load at the Time of Tuberculosis Diagnosis.

T cell activation markers (TAM) expressed by antigen-specific T cells constitute promising candidates to attest the presence of an active infection by Mycobacterium tuberculosis (Mtb). Reciprocally, their modulation may be used to assess antibiotic treatment efficacy and eventually attest disease resolution. We hypothesized that the phenotype of Mtb-specific T cells may be quantitatively impacted by the load of bacteria present in a patient. We recruited 105 Tanzanian adult tuberculosis (TB) patients and obtained blood before and after 5 months of antibiotic treatment. We studied relationships between patients' clinical characteristics of disease severity and microbiological as well as molecular proxies of bacterial load in sputum at the time of diagnosis. Besides, we measured by flow cytometry the expression of CD38 or CD27 on CD4+ T cells producing interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) in response to a synthetic peptide pool covering the sequences of Mtb antigens ESAT-6, CFP-10, and TB10.4. Reflecting the difficulty to extrapolate bacterial burden from a single end-point read-out, we observed statistically significant but weak correlations between Xpert MTB/RIF, molecular bacterial load assay and time to culture positivity. Unlike CD27, the resolution of CD38 expression by antigen-specific T cells was observed readily following 5 months of antibiotic therapy. However, the intensity of CD38-TAM signals measured at diagnosis did not significantly correlate with Mtb 16S RNA or rpoB DNA detected in patients' sputa. Altogether, our data support CD38-TAM as an accurate marker of infection resolution independently of sputum bacterial load.

Extracellular Matrix-Induced GM-CSF and Hypoxia Promote Immune Control of Mycobacterium tuberculosis in Human In Vitro Granulomas.

Several in vitro cellular models have been developed with the aim to reproduce and dissect human granulomatous responses, the hallmark of tuberculosis (TB) immunopathogenesis. In that context, we compared two- (2D) versus three-dimensional (3D) granuloma models resulting from infection of human peripheral blood mononuclear cells with M. tuberculosis (Mtb) in the absence or presence of a collagen-based extracellular matrix (ECM). Granuloma formation was found to be significantly enhanced in the 2D model. This feature was associated with an earlier chemokine production and lymphocyte activation, but also a significantly increased bacterial burden. Remarkably, the reduction in Mtb burden in the 3D model correlated with an increase in GM-CSF production. GM-CSF, which is known to promote macrophage survival, was found to be inherently induced by the ECM. We observed that only 3D in vitro granulomas led to the accumulation of lipid inclusions within Mtb. Our data suggest that a hypoxic environment within the ECM could be responsible for this dormant-like Mtb phenotype. Furthermore, exposure to a TNF-α antagonist reverted Mtb dormancy, thereby mimicking the reactivation of TB observed in rheumatic patients receiving this therapy. To conclude, we showed that only in vitro granulomas generated in the presence of an ECM could recapitulate some clinically relevant features of granulomatous responses in TB. As such, this model constitutes a highly valuable tool to study the interplay between immunity and Mtb stress responses as well as to evaluate novel treatment strategies.

Case-control diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood.

CD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24 h turnaround time. We recruited 479 GeneXpert positive cases and 108 symptomatic but GeneXpert negative controls from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing IFN-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus. Significantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84-0.91). The assay performance was not significantly affected by HIV status. To conclude, we successfully implemented TAM-TB immunoassay routine testing with a 24 h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test.

TNF-α antagonists differentially induce TGF-ß1-dependent resuscitation of dormant-like Mycobacterium tuberculosis.

TNF-α- as well as non-TNF-α-targeting biologics are prescribed to treat a variety of immune-mediated inflammatory disorders. The well-documented risk of tuberculosis progression associated with anti-TNF-α treatment highlighted the central role of TNF-α for the maintenance of protective immunity, although the rate of tuberculosis detected among patients varies with the nature of the drug. Using a human, in-vitro granuloma model, we reproduce the increased reactivation rate of tuberculosis following exposure to Adalimumab compared to Etanercept, two TNF-α-neutralizing biologics. We show that Adalimumab, because of its bivalence, specifically induces TGF-ß1-dependent Mycobacterium tuberculosis (Mtb) resuscitation which can be prevented by concomitant TGF-ß1 neutralization. Moreover, our data suggest an additional role of lymphotoxin-α-neutralized by Etanercept but not Adalimumab-in the control of latent tuberculosis infection. Furthermore, we show that, while Secukinumab, an anti-IL-17A antibody, does not revert Mtb dormancy, the anti-IL-12-p40 antibody Ustekinumab and the recombinant IL-1RA Anakinra promote Mtb resuscitation, in line with the importance of these pathways in tuberculosis immunity.

Generating Three-dimensional Human Granulomas in vitro to Study Mycobacterium tuberculosis-Host Interaction.

Granulomas are organized multicellular structures that constitute the hallmark of an infection by the human pathogen Mycobacterium tuberculosis (Mtb). A better understanding of the complex host-Mtb interactions within the granuloma's environment may lead to new therapeutic or preventive tools to improve the control of the tuberculosis pandemic. To date, several in vitro models that are able to mimic human nascent granulomas have been reported. Here we describe a protocol in which Mtb-infected human peripheral blood mononuclear cells (PBMCs) are embedded within a collagen matrix leading to the formation of three-dimensional micro-granulomas. Subsequently, PBMCs and Mtb can be retrieved allowing multiparametric readouts from both the host and the pathogen. In addition to the incorporation of a physiological extracellular matrix, this model has the singular advantage of recapitulating dormant-like Mtb features, as well as reproducing Mtb resuscitation observed under immunomodulatory treatments, which have not been reported in other published protocols to generate in vitro granulomas.

Mycobacterial Phenolic Glycolipids Selectively Disable TRIF-Dependent TLR4 Signaling in Macrophages.

Phenolic glycolipids (PGLs) are cell wall components of a subset of pathogenic mycobacteria, with immunomodulatory properties. Here, we show that in addition, PGLs exert antibactericidal activity by limiting the production of nitric oxide synthase (iNOS) in mycobacteria-infected macrophages. PGL-mediated downregulation of iNOS was complement receptor 3-dependent and comparably induced by bacterial and purified PGLs. Using Mycobacterium leprae PGL-1 as a model, we found that PGLs dampen the toll-like receptor (TLR)4 signaling pathway, with macrophage exposure to PGLs leading to significant reduction in TIR-domain-containing adapter-inducing interferon-ß (TRIF) protein level. PGL-driven decrease in TRIF operated posttranscriptionally and independently of Src-family tyrosine kinases, lysosomal and proteasomal degradation. It resulted in the defective production of TRIF-dependent IFN-ß and CXCL10 in TLR4-stimulated macrophages, in addition to iNOS. Our results unravel a mechanism by which PGLs hijack both the bactericidal and inflammatory responses of host macrophages. Moreover, they identify TRIF as a critical node in the crosstalk between CR3 and TLR4.

ESX-1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis.

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT-6). This small protein, which is secreted by the type VII secretion system ESX-1 (T7SS/ESX-1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock-out or knock-in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX-1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.

Playing hide-and-seek with host macrophages through the use of mycobacterial cell envelope phthiocerol dimycocerosates and phenolic glycolipids.

Mycobacterial pathogens, including Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), have evolved a remarkable ability to evade the immune system in order to survive and to colonize the host. Among the most important evasion strategies is the capacity of these bacilli to parasitize host macrophages, since these are major effector cells against intracellular pathogens that can be used as long-term cellular reservoirs. Mycobacterial pathogens employ an array of virulence factors that manipulate macrophage function to survive and establish infection. Until recently, however, the role of mycobacterial cell envelope lipids as virulence factors in macrophage subversion has remained elusive. Here, we will address exclusively the proposed role for phthiocerol dimycocerosates (DIM) in the modulation of the resident macrophage response and that of phenolic glycolipids (PGL) in the regulation of the recruitment and phenotype of incoming macrophage precursors to the site of infection. We will provide a unique perspective of potential additional functions for these lipids, and highlight obstacles and opportunities to further understand their role in the pathogenesis of TB and other mycobacterial diseases.

Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host-pathogen interactions.

Several specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence.

Attenuated Mycobacterium tuberculosis SO2 vaccine candidate is unable to induce cell death.

It has been proposed that Mycobacterium tuberculosis virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the M. tuberculosis phoP mutant SO2, we sought to evaluate its potential to induce host cell death. The parental M. tuberculosis MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models in vitro and in vivo. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages in vitro nor during lung infection in vivo. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during M. tuberculosis infection.