Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1ß.

Interleukin 1 (IL-1) is an important mediator of innate immunity but can also promote inflammatory tissue damage. During chronic infections such as tuberculosis, the beneficial antimicrobial role of IL-1 must be balanced with the need to prevent immunopathology. By exogenously controlling the replication of Mycobacterium tuberculosis in vivo, we obviated the requirement for antimicrobial immunity and discovered that both IL-1 production and infection-induced immunopathology were suppressed by lymphocyte-derived interferon-γ (IFN-γ). This effect was mediated by nitric oxide (NO), which we found specifically inhibited assembly of the NLRP3 inflammasome via thiol nitrosylation. Our data indicate that the NO produced as a result of adaptive immunity is indispensable in modulating the destructive innate inflammatory responses elicited during persistent infections.

Glycolipid Metabolite ß-Glucosylceramide Is a Neutrophil Extracellular Trap-Inducing Ligand of Mincle Released during Bacterial Infection and Inflammation.

Neutrophil extracellular traps (NETs) are implicated in host defense and inflammatory pathologies alike. A wide range of pathogen- and host-derived factors are known to induce NETs, yet the knowledge about specific receptor-ligand interactions in this response is limited. We previously reported that macrophage-inducible C-type lectin (Mincle) regulates NET formation. In this article, we identify glycosphingolipid ß-glucosylceramide (ß-GlcCer) as a specific NET-inducing ligand of Mincle. We found that purified ß-GlcCer induced NETs in mouse primary neutrophils in vitro and in vivo, and this effect was abrogated in Mincle deficiency. Cell-free ß-GlcCer accumulated in the lungs of pneumonic mice, which correlated with pulmonary NET formation in wild-type, but not in Mincle-/-, mice infected intranasally with Klebsiella pneumoniae Although leukocyte infiltration by ß-GlcCer administration in vivo did not require Mincle, NETs induced by this sphingolipid were important for bacterial clearance during Klebsiella infection. Mechanistically, ß-GlcCer did not activate reactive oxygen species formation in neutrophils but required autophagy and glycolysis for NET formation, because ATG4 inhibitor NSC185058, as well as glycolysis inhibitor 2-deoxy-d-glucose, abrogated ß-GlcCer-induced NETs. Forced autophagy activation by tamoxifen could overcome the inhibitory effect of glycolysis blockage on ß-GlcCer-mediated NET formation, suggesting that autophagy activation is sufficient to induce NETs in response to this metabolite in the absence of glycolysis. Finally, ß-GlcCer accumulated in the plasma of patients with systemic inflammatory response syndrome, and its levels correlated with the extent of systemic NET formation in these patients. Overall, our results posit ß-GlcCer as a potent NET-inducing ligand of Mincle with diagnostic and therapeutic potential in inflammatory disease settings.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Klebsiella pneumoniae infection of murine neutrophils impairs their efferocytic clearance by modulating cell death machinery.

Neutrophils are the first infiltrating cell type essential for combating pneumoseptic infections by bacterial pathogens including Klebsiella pneumoniae (KPn). Following an infection or injury, removal of apoptotic infiltrates via a highly regulated process called efferocytosis is required for restoration of homeostasis, but little is known regarding the effect of bacterial infection on this process. Here we demonstrate that KPn infection impedes the efferocytic uptake of neutrophils in-vitro and in-vivo in lungs by macrophages. This impaired efferocytosis of infected neutrophils coincides with drastic reduction in the neutrophil surface exposure of apoptosis signature phospholipid phosphatidyserine (PS); and increased activity of phospholipid transporter flippases, which maintain PS in the inner leaflet of plasma membrane. Concomitantly, pharmacological inhibition of flippase activity enhanced PS externalization and restored the efferocytosis of KPn infected neutrophils. We further show that KPn infection interferes with apoptosis activation and instead activates non-apoptotic programmed cell death via activation of necroptosis machinery in neutrophils. Accordingly, pharmacological inhibition of necroptosis by RIPK1 and RIPK3 inhibitors restored the efferocytic uptake of KPn infected neutrophils in-vitro. Importantly, treatment of KPn infected mice with necroptosis inhibitor improved the disease outcome in-vivo in preclinical mouse model of KPn pneumonia. To our knowledge, this is the first report of neutrophil efferocytosis impairment by KPn via modulation of cell death pathway, which may provide novel targets for therapeutic intervention of this infection.

Galectin-3 in M2 Macrophages Plays a Protective Role in Resolution of Neuropathology in Brain Parasitic Infection by Regulating Neutrophil Turnover.

Macrophages/microglia with M2-activation phenotype are thought to play important anti-inflammatory and tissue reparative functions in the brain, yet the molecular bases of their functions in the CNS remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti, we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3-deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in vitro Furthermore, adoptive transfer of M2 macrophages from galectin-3-sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3-/- mice. Together, these results demonstrate, for the first time, a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell-protective functions in the CNS microenvironment are undefined. The current study reports that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models in vivo and in vitro, they show that galectin-3 in M2 macrophages functions to clear neutrophils accumulated in the CNS. Importantly, galectin-3 in M2 macrophages plays a central role in the containment of neuropathology and disease severity. These results provide a direct mechanistic evidence of the protective function of M2 macrophages in the CNS.

Macrophage Galactose-Type Lectin-1 Deficiency Is Associated with Increased Neutrophilia and Hyperinflammation in Gram-Negative Pneumonia.

C-type lectin receptors (CLRs), the carbohydrate-recognizing molecules, orchestrate host immune response in homeostasis and in inflammation. In the present study we examined the function of macrophage galactose-type lectin-1 (MGL1), a mammalian CLR, in pneumonic sepsis, a deadly immune disorder frequently associated with a nonresolving hyperinflammation. In a murine model of pneumonic sepsis using pulmonary infection with Klebsiella pneumoniae, the expression of MGL1 was upregulated in the lungs of K. pneumoniae-infected mice, and the deficiency of this CLR in MGL1(-/-) mice resulted in significantly increased mortality to infection than in the MGL1-sufficient wild-type mice, despite a similar bacterial burden. The phagocytic cells from MGL1(-/-) mice did not exhibit any defects in bacterial uptake and intracellular killing and were fully competent in neutrophil extracellular trap formation, a recently identified extracellular killing modality of neutrophils. Instead, the increased susceptibility of MGL1(-/-) mice seemed to correlate with severe lung pathology, indicating that MGL1 is required for resolution of pulmonary inflammation. Indeed, the MGL1(-/-) mice exhibited a hyperinflammatory response, massive pulmonary neutrophilia, and an increase in neutrophil-associated immune mediators. Concomitantly, MGL1-deficient neutrophils exhibited an increased influx in pneumonic lungs of K. pneumoniae-infected mice. Taken together, these results show a previously undetermined role of MGL1 in controlling neutrophilia during pneumonic infection, thus playing an important role in resolution of inflammation. To our knowledge, this is the first study depicting a protective function of MGL1 in an acute pneumonic bacterial infection.

Mincle-Mediated Neutrophil Extracellular Trap Formation by Regulation of Autophagy.

BACKGROUND: Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance, mechanistic understanding of NET formation is poor. In this study, we examined the mechanism by which Mincle, a C-type lectin receptor, regulates NET formation. METHODS: NET formation, reactive oxygen species, autophagy activation and intracellular signaling pathways were analyzed in Mincle-sufficient and -deficient neutrophils stimulated in vitro with various stimuli and in vivo during Klebsiella infection. RESULTS: We found that Mincle mediates NET formation in response to several activation stimuli in vitro and in vivo during pneumoseptic infection with Klebsiella pneumoniae, indicating its regulatory role in NET formation. Mechanistically, we show that attenuated NET formation in Mincle-/- neutrophils correlates with an impaired autophagy activation in vitro and in vivo, whereas reactive oxygen species (ROS) formation in these neutrophils remained intact. The requirement of autophagy in Mincle-mediated NET formation was further supported by exogenous treatment with autophagy inducer tamoxifen, which rescued the NET formation defect in Mincle-/- neutrophils. CONCLUSIONS: Our findings identify a previously unrecognized role of Mincle as a regulator of autophagy, which mediates NET formation without affecting ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs while preserving ROS production, an important innate immune defense.

Protective role of Mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis and extracellular trap formation.

BACKGROUND: Nosocomial infections with Klebsiella pneumoniae are a frequent cause of Gram-negative bacterial sepsis. To understand the functioning of host innate immune components in this disorder, we examined a previously uninvestigated role of the C-type lectin receptor Mincle in pneumonic sepsis caused by K. pneumoniae. METHODS: Disease progression in wild-type and Mincle(-/-) mice undergoing pulmonary infection with K. pneumoniae was compared. RESULTS: Whereas the wild-type mice infected with a sublethal dose of bacteria could resolve the infection with bacterial clearance and regulated host response, the Mincle(-/-) mice were highly susceptible with a progressive increase in bacterial burden, despite their ability to mount an inflammatory response that turned to an exaggerated hyperinflammation with the onset of severe pneumonia. This correlated with severe lung pathology with a massive accumulation of neutrophils in their lungs. Importantly, Mincle(-/-) neutrophils displayed a defective ability to phagocytize nonopsonic bacteria and an impaired ability to form extracellular traps (NETs), an important neutrophil function against invading pathogens, including K. pneumoniae. CONCLUSION: Our results demonstrate protective role of Mincle in host defense against K. pneumoniae pneumonia by coordinating bacterial clearance mechanisms of neutrophils. A novel role for Mincle in the regulation of neutrophil NET formation may have implications in chronic disease conditions characterized by deregulated NET formation.

Inhibition of store-operated calcium entry in microglia by helminth factors: implications for immune suppression in neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a disease of the central nervous system (CNS) caused by the cestode Taenia solium. The infection exhibits a long asymptomatic phase, typically lasting 3 to 5 years, before the onset of the symptomatic phase. The severity of the symptoms is thought to be associated with the intensity of the inflammatory response elicited by the degenerating parasite. In contrast, the asymptomatic phase shows an absence of brain inflammation, which is presumably due to immunosuppressive effects of the live parasites. However, the host factors and/or pathways involved in inhibiting inflammation remain largely unknown. Recently, using an animal model of NCC in which mice were intracranially inoculated with a related helminth parasite, Mesocestoides corti, we reported that Toll-like receptor (TLR)-associated signaling contributes to the development of the inflammatory response. As microglia shape the initial innate immune response in the CNS, we hypothesized that the negative regulation of a TLR-induced inflammatory pathway in microglia may be a novel helminth-associated immunosuppressive mechanism in NCC. METHODS AND RESULTS: Here we report that helminth soluble factors (HSFs) from Mesocestoides corti inhibited TLR ligation-induced production of inflammatory cytokines in primary microglia. This was correlated with an inhibition of TLR-initiated upregulation of both phosphorylation and acetylation of the nuclear factor κB (NF-κB) p65 subunit, as well as phosphorylation of JNK and ERK1/2. As Ca2+ influx due to store-operated Ca2+ entry (SOCE) has been implicated in induction of downstream signaling, we tested the inhibitory effect of HSFs on agonist-induced Ca2+ influx and specific Ca2+ channel activation. We discovered that HSFs abolished the lipopolysaccharide (LPS)- or thapsigargin (Tg)-induced increase in intracellular Ca2+ accumulation by blocking the ER store release and SOCE. Moreover, electrophysiological recordings demonstrated HSF-mediated inhibition of LPS- or Tg-induced SOCE currents through both TRPC1 and ORAI1 Ca2+ channels on plasma membrane. This was correlated with a decrease in the TRPC1-STIM1 and ORAI1-STIM1 clustering at the plasma membrane that is essential for sustained Ca2+ entry through these channels. CONCLUSION: Inhibition of TRPC1 and ORAI1 Ca2+ channel-mediated activation of NF-κB and MAPK pathways in microglia is likely a novel helminth-induced immunosuppressive mechanism that controls initiation of inflammatory response in the CNS.

Differential requirement of Formyl Peptide Receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection.

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. This study investigates the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed using a TB model of hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion showed no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1 -/- mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased bacterial persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1 -/- neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

18 F-FDG Brain PET/MRI in Amyotrophic Lateral Sclerosis- Frontotemporal Spectrum Disorder (ALS-FTSD).

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disorder involving both upper and lower motor neurons. Interestingly, 15 to 41% of patients with ALS have concomitant frontotemporal dementia (FTD). Approximately, 50% of patients with ALS can copresent with a broader set of neuropsychological pathologies that do not meet FTD diagnostic criteria. This association resulted in revised and expanded criteria establishing the ALS-frontotemporal spectrum disorder (FTSD). In this case report, we review background information, epidemiology, pathophysiology, and structural and molecular imaging features of ALS-FTSD.

Metformin-induced activation of Ca2+ signaling prevents immune infiltration/pathology in Sjogren's syndrome-prone mouse models.

Immune cell infiltration and glandular dysfunction are the hallmarks of autoimmune diseases such as primary Sjogren's syndrome (pSS), however, the mechanism(s) is unknown. Our data show that metformin-treatment induces Ca2+ signaling that restores saliva secretion and prevents immune cell infiltration in the salivary glands of IL14α-transgenic mice (IL14α), which is a model for pSS. Mechanistically, we show that loss of Ca2+ signaling is a major contributing factor, which is restored by metformin treatment, in IL14α mice. Furthermore, the loss of Ca2+ signaling leads to ER stress in salivary glands. Finally, restoration of metformin-induced Ca2+ signaling inhibited the release of alarmins and prevented the activation of ER stress that was essential for immune cell infiltration. These results suggest that loss of metformin-mediated activation of Ca2+ signaling prevents ER stress, which inhibited the release of alarmins that induces immune cell infiltration leading to salivary gland dysfunction observed in pSS.

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. METHODS: Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed to identify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. RESULTS: We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. CONCLUSIONS: These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages.

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Increased disease severity of parasite-infected TLR2-/- mice is correlated with decreased central nervous system inflammation and reduced numbers of cells with alternatively activated macrophage phenotypes in a murine model of neurocysticercosis.

In a murine model for neurocysticercosis (NCC), intracranial inoculation of the helminth parasite Mesocestoides corti induces multiple Toll-like receptors (TLRs), among which TLR2 is upregulated first and to a relatively high extent. Here, we report that TLR2(-/-) mice displayed significantly increased susceptibility to parasite infection accompanied by increased numbers of parasites in the brain parenchyma compared to infection in wild-type (WT) mice. This coincided with an increased display of microglial nodule formations and greater neuropathology than in the WT. Parasite-infected TLR2(-/-) brains exhibited a scarcity of lymphocytic cuffing and displayed reduced numbers of infiltrating leukocytes. Fluorescence-activated cell sorter (FACS) analyses revealed significantly lower numbers of CD11b(+) myeloid cells, γδ T cells, αß T cells, and B cells in the brains of parasite-infected TLR2(-/-) mice. This correlated with significantly reduced levels of inflammatory mediators, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), CCL2, CCL3, and interleukin-6 (IL-6) in the central nervous system (CNS) of TLR2(-/-) mice. As TLR2 has been implicated in immune regulation of helminth infections and as alternatively activated macrophages (AAMs) are thought to play a profound regulatory role in such infections, induction of AAMs in infected TLR2(-/-) mice was compared with that in WT mice. Parasite-infected WT brains showed larger numbers of macrophages/microglia (CD11b(+) cells) expressing AAM-associated molecules such as YM1, Fizz1 (found in inflammatory zone-1 antigen), and arginase 1 than TLR2(-/-) brains, consistent with a protective role of AAMs during infection. Importantly, these results demonstrate that TLR2-associated responses modulate the disease severity of murine NCC.

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu.

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.

Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome.

Interleukin-1beta (IL-1beta) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1beta secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1beta. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1beta secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase-1 activation largely by perturbing host cell membranes. Using a high-throughput shRNA-based screen we found that numerous NOD-like receptors (NLRs) and CARD domain-containing proteins (CARDs) were important for IL-1beta secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase-1 form an infection-inducible inflammasome complex that is essential for IL-1beta secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT-6, and the subsequent IL-1beta response is regulated by a number of NLR/CARD proteins.

Granulocytes act as a niche for Mycobacterium tuberculosis growth.

Granulocyte recruitment to the pulmonary compartment is a hallmark of progressive tuberculosis (TB). This process is well-documented to promote immunopathology, but can also enhance the replication of the pathogen. Both the specific granulocytes responsible for increasing mycobacterial burden and the underlying mechanisms remain obscure. We report that the known immunomodulatory effects of these cells, such as suppression of protective T-cell responses, play a limited role in altering host control of mycobacterial replication in susceptible mice. Instead, we find that the adaptive immune response preferentially restricts the burden of bacteria within monocytes and macrophages compared to granulocytes. Specifically, mycobacteria within inflammatory lesions are preferentially found within long-lived granulocytes that express intermediate levels of the Ly6G marker and low levels of antimicrobial genes. These cells progressively accumulate in the lung and correlate with bacterial load and disease severity, and the ablation of Ly6G-expressing cells lowers mycobacterial burden. These observations suggest a model in which dysregulated granulocytic influx promotes disease by creating a permissive intracellular niche for mycobacterial growth and persistence.