Eosinophils are part of the granulocyte response in tuberculosis and promote host resistance in mice

Host resistance to Mycobacterium tuberculosis (Mtb) infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. An influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, using complementary genetic models of eosinophil deficiency, we demonstrate that in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice

IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection

IL-21 is produced predominantly by activated CD4(+) T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (gamma(c)) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8(+) T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4(+) and CD8(+) T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R(-/-) mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R(-/-) T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.This work was supported by National Institutes of Health Grants R21 AI100766, R01 AI106725, and P01 AI073748

Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.publishedVersio

Monosodium urate crystals promote innate anti-mycobacterial immunity and improve BCG efficacy as a vaccine against tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination

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-?, 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.Fundação para a Ciência e Tecnologia, Portugal and cofunded by Programa Operacional Regional do Norte (ON.2–O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through the Fundo Europeu de Desenvolvimento Regional (FEDER). Project grants: PTDC/SAU-MII/101977/2008 (to AGC) and PTDC/BIA-BCM/102776/2008 (to MS). LMT was supported by FCT Grant SFRH/BPD/77399/20

Mycobacterium tuberculosis clinical isolates of the Beijing and East-African Indian lineage induce fundamentally different host responses in mice compared to H37Rv

Substantial differences exist in virulence among Mycobacterium tuberculosis strains in preclinical TB models. In this study we show how virulence affects host responses in mice during the first four weeks of infection with a mycobacterial strain belonging to the Beijing, East-African-Indian or Euro-American lineage. BALB/c mice were infected with clinical isolates of the Beijing-1585 strain or the East-African Indian (EAI)-1627 strain and host responses were compared to mice infected with the non-clinical H37Rv strain of the Euro-American lineage. We found that H37Rv induced a 'classical' T-cell influx with high IFN-γ levels, while Beijing-1585 and EAI-1627 induced an influx of B-cells into the lungs together with elevated pulmonary IL-4 protein levels. Myeloid cells in the lungs appeared functionally impaired upon infection with Beijing-1585 and EAI-1627 with reduced iNOS and IL-12 expression levels compared to H37Rv infection. This impairment might be related to significantly reduced expression in the bone marrow of IFN-γ, TNF-α and IFN-β in mice infected with Beijing-1585 and EAI-1627, which could be detected from the third day post infection onwards. Our findings suggest that increased virulence of two clinical isolates compared to H37Rv is associated with a fundamentally different systemic immune response, which already can be detected early during infection

Inflammasome-dependent Pyroptosis and IL-18 Protect against Burkholderia pseudomallei Lung Infection while IL-1β Is Deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β

Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1- and Cathepsin B-Independent Necrosis

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis

BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF / ) mice and compared outcomes of disease against TNF f/f control and global TNF / mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF / mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF / mice were resistant to infection and presented with a phenotype similar to that in TNF f/f control mice. Impaired immunity in TNF / mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB