Inducing Pyroptosis Via the Pyrin Inflammasome.

The pyrin inflammasome detects bacterial toxins and effectors that inhibit RhoA GTPases and triggers inflammatory cytokine release and a fast cell death termed pyroptosis. In addition, various endogenous molecules, drugs, synthetic molecules, or mutations can trigger pyrin inflammasome activation. The pyrin protein differs between humans and mice, and the repertoire of pyrin activators is also species-specific. Here, we present the various pyrin inflammasome activators, inhibitors, the kinetics of pyrin activation in response to the various activators, and their species specificity. In addition, we present different methods to monitor pyrin-mediated pyroptosis.

Steroid hormone catabolites activate the pyrin inflammasome through a non-canonical mechanism.

The pyrin inflammasome acts as a guard of RhoA GTPases and is central to immune defenses against RhoA-manipulating pathogens. Pyrin activation proceeds in two steps. Yet, the second step is still poorly understood. Using cells constitutively activated for the pyrin step 1, a chemical screen identifies etiocholanolone and pregnanolone, two catabolites of testosterone and progesterone, acting at low concentrations as specific step 2 activators. High concentrations of these metabolites fully and rapidly activate pyrin, in a human specific, B30.2 domain-dependent manner and without inhibiting RhoA. Mutations in MEFV, encoding pyrin, cause two distinct autoinflammatory diseases pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) and familial Mediterranean fever (FMF). Monocytes from PAAND patients, and to a lower extent from FMF patients, display increased responses to these metabolites. This study identifies an unconventional pyrin activation mechanism, indicates that endogenous steroid catabolites can drive autoinflammation, through the pyrin inflammasome, and explains the "steroid fever" described in the late 1950s upon steroid injection in humans.

Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

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.

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.

Mouse transcriptome reveals potential signatures of protection and pathogenesis in human tuberculosis.

Although mouse infection models have been extensively used to study the host response to Mycobacterium tuberculosis, their validity in revealing determinants of human tuberculosis (TB) resistance and disease progression has been heavily debated. Here, we show that the modular transcriptional signature in the blood of susceptible mice infected with a clinical isolate of M. tuberculosis resembles that of active human TB disease, with dominance of a type I interferon response and neutrophil activation and recruitment, together with a loss in B lymphocyte, natural killer and T cell effector responses. In addition, resistant but not susceptible strains of mice show increased lung B cell, natural killer and T cell effector responses in the lung upon infection. Notably, the blood signature of active disease shared by mice and humans is also evident in latent TB progressors before diagnosis, suggesting that these responses both predict and contribute to the pathogenesis of progressive M. tuberculosis infection.

Myeloid HIF-1α regulates pulmonary inflammation during experimental Mycobacterium tuberculosis infection.

The transcription factor hypoxia-inducible factor-1 alpha (HIF-1α) is a key regulator of the response and function of myeloid cells in hypoxic and inflammatory microenvironments. To define the role of HIF-1α in tuberculosis, the progression of aerosol Mycobacterium tuberculosis infection was analysed in mice deficient in HIF-1α in the myeloid lineage (mHIF-1α-/- ). We show that myeloid HIF-1α is not required for the containment of the infection, as both wild-type (WT) and mHIF-1α-/- mice mounted normal Th1 responses and maintained control of bacterial growth throughout infection. However, during chronic infection mHIF-1α-/- mice developed extensive lymphocytic inflammatory involvement of the interstitial lung tissue and died earlier than WT mice. These data support the hypothesis that HIF-1α activity coordinates the response of myeloid cells during M. tuberculosis infection to prevent excessive leucocyte recruitment and immunopathological consequences to the host.

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.

Paradigm changing evidence that alter tuberculosis perception and detection: Focus on latency.

Tuberculosis remains a devastating disease to Mankind, ranking as the ninth cause of death worldwide. Eliminating tuberculosis as proven much more difficult than once anticipated. In addition to the delay in diagnosis and drug resistance problems that compromise the efficacy of treatment, the enormous reservoir of latently infected individuals continuously feeds the epidemics. However, targeting latency with prophylactic antibiotic administration is not possible at the populational level. Together, these issues call for a better understanding of latency, as well as for a more precise identification of individuals at high risk of reactivation. For this, recent paradigm changing evidence need to be taken into account, most notably, the existence of a tuberculosis spectrum; the genetic diversity of both humans and tuberculosis-causing bacteria; and the changes in the human population that interfere with tuberculosis. Here we discuss latency in the light of these variables and how that understanding can move forward tuberculosis research and elimination.

A Nonribosomal Peptide Synthase Gene Driving Virulence in Mycobacterium tuberculosis.

Nonribosomal peptide synthases produce short peptides in a manner that is distinct from classical mRNA-dependent ribosome-mediated translation. The Mycobacterium tuberculosis genome harbors a nonribosomal peptide synthase gene, nrp, which is part of a gene cluster proposed to be involved in the biosynthesis of isonitrile lipopeptides. Orthologous clusters are found in other slow-growing pathogenic mycobacteria and actinomycetes. To probe the role of the nrp gene in infection, we generated an nrp deletion mutant in M. tuberculosis H37Rv and tested its virulence in immunocompetent (C57BL/6) mice. The nrp mutant strain displayed lower initial growth rates in the lungs and a defective dissemination to the spleens of infected mice. Mice infected with the mutant strain also survived for twice as long as those infected with wild-type M. tuberculosis and, remarkably, showed subdued pathology, despite similar bacterial loads at later stages of infection. The differences in the course of infection between wild-type and nrp mutant strains were accompanied by distinct dynamics of the immune response. Most strikingly, the nrp mutant was highly attenuated in immunodeficient (SCID-, recombination activating 2 [RAG2]-, and gamma interferon [IFN-γ]-deficient) mice, suggesting that macrophages control the nrp mutant more efficiently than they control the wild-type strain. However, in the presence of IFN-γ, both strains were equally controlled. We propose that the nrp gene and its associated cluster are drivers of virulence during the early stages of infection.IMPORTANCE Over 10 million people developed tuberculosis (TB) in 2016, and over 1.8 million individuals succumbed to the disease. These numbers make TB the ninth leading cause of death worldwide and the leading cause from a single infectious agent. Therefore, finding novel therapeutic targets in Mycobacterium tuberculosis, the pathogen that causes most cases of human TB, is critical. In this study, we reveal a novel virulence factor in M. tuberculosis, the nrp gene. The lack of nrp highly attenuates the course of M. tuberculosis infection in the mouse model, which is particularly relevant in immune-deficient hosts. This is very relevant as TB is particularly incident in immune-suppressed individuals, such as HIV patients.

Type I IFN Inhibits Alternative Macrophage Activation during Mycobacterium tuberculosis Infection and Leads to Enhanced Protection in the Absence of IFN-γ Signaling.

Tuberculosis causes ∼1.5 million deaths every year, thus remaining a leading cause of death from infectious diseases in the world. A growing body of evidence demonstrates that type I IFN plays a detrimental role in tuberculosis pathogenesis, likely by interfering with IFN-γ-dependent immunity. In this article, we reveal a novel mechanism by which type I IFN may confer protection against Mycobacterium tuberculosis infection in the absence of IFN-γ signaling. We show that production of type I IFN by M. tuberculosis-infected macrophages induced NO synthase 2 and inhibited arginase 1 gene expression. In vivo, absence of both type I and type II IFN receptors led to strikingly increased levels of arginase 1 gene expression and protein activity in infected lungs, characteristic of alternatively activated macrophages. This correlated with increased lung bacterial burden and pathology and decreased survival compared with mice deficient in either receptor. Increased expression of other genes associated with alternatively activated macrophages, as well as increased expression of Th2-associated cytokines and decreased TNF expression, were also observed. Thus, in the absence of IFN-γ signaling, type I IFN suppressed the switching of macrophages from a more protective classically activated phenotype to a more permissive alternatively activated phenotype. Together, our data support a model in which suppression of alternative macrophage activation by type I IFN during M. tuberculosis infection, in the absence of IFN-γ signaling, contributes to host protection.

Delivery of LLKKK18 loaded into self-assembling hyaluronic acid nanogel for tuberculosis treatment.

Tuberculosis (TB), a disease caused by the human pathogen Mycobacterium tuberculosis, recently joined HIV/AIDS on the top rank of deadliest infectious diseases. Low patient compliance due to the expensive, long-lasting and multi-drug standard therapies often results in treatment failure and emergence of multi-drug resistant strains. In this scope, antimicrobial peptides (AMPs) arise as promising candidates for TB treatment. Here we describe the ability of the exogenous AMP LLKKK18 to efficiently kill mycobacteria. The peptide's potential was boosted by loading into self-assembling Hyaluronic Acid (HA) nanogels. These provide increased stability, reduced cytotoxicity and degradability, while potentiating peptide targeting to main sites of infection. The nanogels were effectively internalized by macrophages and the peptide presence and co-localization with mycobacteria within host cells was confirmed. This resulted in a significant reduction of the mycobacterial load in macrophages infected in vitro with the opportunistic M. avium or the pathogenic M. tuberculosis, an effect accompanied by lowered pro-inflammatory cytokine levels (IL-6 and TNF-α). Remarkably, intra-tracheal administration of peptide-loaded nanogels significantly reduced infection levels in mice infected with M. avium or M. tuberculosis, after just 5 or 10 every other day administrations. Considering the reported low probability of resistance acquisition, these findings suggest a great potential of LLKKK18-loaded nanogels for TB therapeutics.

IL-17A Promotes Intracellular Growth of Mycobacterium by Inhibiting Apoptosis of Infected Macrophages.

The fate of infected macrophages is a critical aspect of immunity to mycobacteria. By depriving the pathogen of its intracellular niche, apoptotic death of the infected macrophage has been shown to be an important mechanism to control bacterial growth. Here, we show that IL-17 inhibits apoptosis of Mycobacterium bovis BCG- or Mycobacterium tuberculosis-infected macrophages thus hampering their ability to control bacterial growth. Mechanistically, we show that IL-17 inhibits p53, and impacts on the intrinsic apoptotic pathway, by increasing the Bcl2 and decreasing Bax expression, decreasing cytochrome c release from the mitochondria, and inhibiting caspase-3 activation. The same effect of IL-17 was observed in infected macrophages upon blockade of p53 nuclear translocation. These results reveal a previously unappreciated role for the IL-17/p53 axis in the regulation of mycobacteria-induced apoptosis and can have important implications in a broad spectrum of diseases where apoptosis of the infected cell is an important host defense mechanism.

Myeloid Sirtuin 2 Expression Does Not Impact Long-Term Mycobacterium tuberculosis Control.

Sirtuins (Sirts) regulate several cellular mechanisms through deacetylation of several transcription factors and enzymes. Recently, Sirt2 was shown to prevent the development of inflammatory processes and its expression favors acute Listeria monocytogenes infection. The impact of this molecule in the context of chronic infections remains unknown. We found that specific Sirt2 deletion in the myeloid lineage transiently increased Mycobacterium tuberculosis load in the lungs and liver of conditional mice. Sirt2 did not affect long-term infection since no significant differences were observed in the bacterial burden at days 60 and 120 post-infection. The initial increase in M. tuberculosis growth was not due to differences in inflammatory cell infiltrates in the lung, myeloid or CD4+ T cells. The transcription levels of IFN-γ, IL-17, TNF, IL-6 and NOS2 were also not affected in the lungs by Sirt2-myeloid specific deletion. Overall, our results demonstrate that Sirt2 expression has a transitory effect in M. tuberculosis infection. Thus, modulation of Sirt2 activity in vivo is not expected to affect chronic infection with M. tuberculosis.

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response.

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.