Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns.

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.

T follicular regulatory cells in mice and men.

It has long been known that CD4 T cells are necessary to provide help to B cells, triggering a germinal centre (GC) reaction where affinity maturation and isotype switching occur. However, the nature of the dedicated CD4 helper T cells, known as T follicular helper (Tfh), was only recently described. Here, we review the biology and function of the recently described T follicular regulatory (Tfr) cells, another CD4 T-cell population also found within GCs but with regulatory function and characteristics. Tfr cells have been identified in mice and humans as simultaneously presenting characteristics of T follicular cells (namely CXCR5 expression) and regulatory T cells (including Foxp3 expression). These Tfr cells have been implicated in the regulation of the magnitude of the GC reaction, as well as in protection from immune-mediated pathology.

Multimodal profiling reveals tissue-directed signatures of human immune cells altered with age.

The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.

Infection with hypervirulent Mycobacterium tuberculosis triggers emergency myelopoiesis but not trained immunity.

Introduction: During infection, bone marrow (BM) hematopoiesis is reprogrammed toward myeloid cell production, a mechanism named emergency myelopoiesis. In addition to replenishing myeloid cells, emergency myelopoiesis has been linked to trained immunity, a process that allows enhanced innate immune responses to secondary challenges. Although hematopoietic alterations during tuberculosis (TB) have been described and Mycobacterium tuberculosis may colonize the BM, studies using the mouse model of infection and the laboratory reference strain M. tuberculosis H37Rv have demonstrated limited emergency myelopoiesis and trained immunity. Methods: To further address this issue, we aerosol- infected C57BL/6 mice with high doses of the hypervirulent M. tuberculosis isolate HN878 and monitored alterations to the BM. This experimental model better resembles the human blood immune signature of TB. Results and discussion: We found increased frequencies of lineage-Sca-1+cKit+ (LSK) cells and the granulocyte/macrophage progenitor (GMP) population. At the mature cell level, we observed an increase of monocytes and neutrophils in the blood and lung, likely reflecting the increased BM myeloid output. Monocytes or monocyte-derived macrophages recovered from the BM of M. tuberculosis HN878-infected mice did not show signs of trained immunity, suggesting an uncoupling of emergency myelopoiesis and trained immunity in the BM. Surprisingly, M. tuberculosis HN878-induced emergency myelopoiesis was not fully dependent on IFNγ, as mice lacking this cytokine and infected under the same conditions as wild-type mice still presented BM alterations. These data expand our understanding of the immune response to M. tuberculosis and raise awareness of pathogen strain-imposed differences to host responses.

Mycobacterium tuberculosis Infection Up-Regulates Sialyl Lewis X Expression in the Lung Epithelium.

Glycans display increasingly recognized roles in pathological contexts, however, their impact in the host-pathogen interplay in many infectious diseases remains largely unknown. This is the case for tuberculosis (TB), one of the ten most fatal diseases worldwide, caused by infection of the bacteria Mycobacterium tuberculosis. We have recently reported that perturbing the core-2 O-glycans biosynthetic pathway increases the host susceptibility to M. tuberculosis infection, by disrupting the neutrophil homeostasis and enhancing lung pathology. In the present study, we show an increased expression of the sialylated glycan structure Sialyl-Lewis X (SLeX) in the lung epithelium upon M. tuberculosis infection. This increase in SLeX glycan epitope is accompanied by an altered lung tissue transcriptomic signature, with up-regulation of genes codifying enzymes that are involved in the SLeX core-2 O-glycans biosynthetic pathway. This study provides novel insights into previously unappreciated molecular mechanisms involving glycosylation, which modulate the host response to M. tuberculosis infection, possibly contributing to shape TB disease outcome.

Interleukin-10 induces interferon-γ-dependent emergency myelopoiesis.

In emergency myelopoiesis (EM), expansion of the myeloid progenitor compartment and increased myeloid cell production are observed and often mediated by the pro-inflammatory cytokine interferon gamma (IFN-γ). Interleukin-10 (IL-10) inhibits IFN-γ secretion, but paradoxically, its therapeutic administration to humans causes hematologic changes similar to those observed in EM. In this work, we use different in vivo systems, including a humanized immune system mouse model, to show that IL-10 triggers EM, with a significant expansion of the myeloid progenitor compartment and production of myeloid cells. Hematopoietic progenitors display a prominent IFN-γ transcriptional signature, and we show that IFN-γ mediates IL-10-driven EM. We also find that IL-10, unexpectedly, reprograms CD4 and CD8 T cells toward an activation state that includes IFN-γ production by these T cell subsets in vivo. Therefore, in addition to its established anti-inflammatory properties, IL-10 can induce IFN-γ production and EM, opening additional perspectives for the design of IL-10-based immunotherapies.

Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils.

Modulation of immunity and disease by glycans is increasingly recognized. However, how host glycosylation shapes and is shaped by tuberculosis remains poorly understood. We show that deficiency in the glucosaminyl (N-acetyl) transferase 1 (Gcnt1), a key enzyme for core-2 O-glycans biosynthesis, drives susceptibility to Mycobacterium tuberculosis infection. The increased susceptibility of Gcnt1 deficient mice was characterized by extensive lung immune pathology, mechanistically related to neutrophils. Uninfected Gcnt1 deficient mice presented bone marrow, blood and lung neutrophilia, which further increased with infection. Blood neutrophilia required Gcnt1 deficiency in the hematopoietic compartment, relating with enhanced granulopoiesis, but normal cellular egress from the bone marrow. Interestingly, for the blood neutrophilia to translate into susceptibility to M. tuberculosis infection, Gnct1 deficiency in the stroma was also necessary. Complete Gcnt1 deficiency associated with increased lung expression of the neutrophil chemoattractant CXCL2. Lastly, we demonstrate that the transcript levels of various glycosyltransferase-encoding genes were altered in whole blood of active tuberculosis patients and that sialyl Lewis x, a glycan widely present in human neutrophils, was detected in the lung of tuberculosis patients. Our findings reveal a previously unappreciated link between Gcnt1, neutrophilia and susceptibility to M. tuberculosis infection, uncovering new players balancing the immune response in tuberculosis.

Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1ß production.

Genetic diversity of Mycobacterium tuberculosis affects immune responses and clinical outcomes of tuberculosis (TB). However, how bacterial diversity orchestrates immune responses to direct distinct TB severities is unknown. Here we study 681 patients with pulmonary TB and show that M. tuberculosis isolates from cases with mild disease consistently induce robust cytokine responses in macrophages across multiple donors. By contrast, bacteria from patients with severe TB do not do so. Secretion of IL-1ß is a good surrogate of the differences observed, and thus to classify strains as probable drivers of different TB severities. Furthermore, we demonstrate that M. tuberculosis isolates that induce low levels of IL-1ß production can evade macrophage cytosolic surveillance systems, including cGAS and the inflammasome. Isolates exhibiting this evasion strategy carry candidate mutations, generating sigA recognition boxes or affecting components of the ESX-1 secretion system. Therefore, we provide evidence that M. tuberculosis strains manipulate host-pathogen interactions to drive variable TB severities.

Experimental Evidence for Limited in vivo Virulence of Mycobacterium africanum.

Tuberculosis remains a public health problem and a main cause of death to humans. Both Mycobacterium tuberculosis and Mycobacterium africanum cause tuberculosis. In contrast to M. tuberculosis, which is geographically spread, M. africanum is restricted to West Africa. Differences have also been found in the growth rate and type of disease caused by M. africanum, globally suggesting an attenuation of this bacteria. In this study, we used the mouse model of infection to follow the dynamics of M. africanum infection in terms of bacterial burdens and tissue pathology, as well as the immune response triggered. Our findings support a lower virulence of M. africanum as compared to M. tuberculosis, including in mice lacking IFN-γ, a major protective cytokine in tuberculosis. Furthermore, the lung immune response triggered by M. africanum infection in wild-type animals was characterized by a discrete influx of leukocytes and a modest transcriptional upregulation of inflammatory mediators. Our findings contribute to elucidate the pathogenesis of M. africanum, supporting the hypothesis that this is an attenuated member of the tuberculosis-causing bacteria. Understanding the biology of M. africanum and how it interacts with the host to establish infection will have implications for our knowledge of TB and for the development of novel and better tools to control this devastating disease.

T follicular helper and T follicular regulatory cells have different TCR specificity.

Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity.

Human blood Tfr cells are indicators of ongoing humoral activity not fully licensed with suppressive function.

Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high-affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cells defined as CXCR5+Foxp3+ T cells remains elusive. We found that blood Tfr cells are increased in Sjögren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titers, as well as in healthy individuals upon influenza vaccination. Although blood Tfr cells correlated with humoral responses, they lack full B cell-suppressive capacity, despite being able to suppress T cell proliferation. Blood Tfr cells have a naïve-like phenotype, although they are absent from human thymus or cord blood. We found that these cells were generated in peripheral lymphoid tissues before T-B interaction, as they are maintained in B cell-deficient patients. Therefore, blood CXCR5+Foxp3+ T cells in human pathology indicate ongoing humoral activity but are not fully competent circulating Tfr cells.

Identification of Foxp3⁺ T follicular regulatory (Tfr) cells by flow cytometry.

Flow cytometry is a technology that allows multiparametric analysis of individual cells. As a consequence, it is among the most commonly used tools for the study of immune cells. It is useful both for the study of ex vivo cell populations isolated from experimental animals or human tissue and for characterizing the phenotype of cultured cells. The phenotypic analysis is based on antibodies associated to different fluorophores that specifically bind to key molecules. Genetically modified mouse strains that express a reporter gene under the control of a promoter of interest offer an important alternative for the staining of intracellular molecules without the need to permeabilize the cell membrane. In this chapter, we describe how Foxp3(+) follicular regulatory T (Tfr) cells, a population of regulatory T (Treg) cells related to T follicular helper (Tfh) cells and involved in the regulation of germinal centers (GC), can be identified by flow cytometry.