The small intestine harbors a substantial number of commensal bacteria and is sporadically invaded by pathogens, but the response to these microorganisms is fundamentally different. We identified a discriminatory sensor by using Toll-like receptor 3 (TLR3). Double-stranded RNA (dsRNA) of one major commensal species, lactic acid bacteria (LAB), triggered interferon-β (IFN-β) production, which protected mice from experimental colitis. The LAB-induced IFN-β response was diminished by dsRNA digestion and treatment with endosomal inhibitors. Pathogenic bacteria contained less dsRNA and induced much less IFN-β than LAB, and dsRNA was not involved in pathogen-induced IFN-β induction. These results identify TLR3 as a sensor to small intestinal commensal bacteria and suggest that dsRNA in commensal bacteria contributes to anti-inflammatory and protective immune responses.

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Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)‐gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN‐γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation‐mediated hepatitis. After Con A challenge, liver of wild‐type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor‐1 (PAI‐1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ−/− mice and Ifnγ−/−Tnf−/− mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti‐TF monoclonal antibody protected against Con A–induced hepatitis, whereas Pai1−/− mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage‐depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription‐1 (STAT1) essential for IFN‐γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage‐depleted Stat1−/− mice reconstituted with WT macrophages. Exogenous IFN‐γ and TNF rendered T‐cell‐null, Con A–resistant mice deficient in recombination‐activating gene 2, highly susceptible to Con A–induced liver injury involving TF. Conclusions: Collectively, these results strongly suggest that proinflammatory signals elicited by IFN‐γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation‐mediated hepatitis. (HEPATOLOGY 2013)

A genomic locus called "region of difference 1" (RD1) in Mycobacterium tuberculosis has been shown to contribute to the generation of host protective immunity as well as to the virulence of the bacterium. To gain insight into the molecular mechanism, we investigated the difference in the cytokine-inducing ability between H37Rv and a mutant strain deficient for RD1 (⌬RD1). We found that RD1 is implicated in the production of caspase-1-dependent cytokines, interleukin-18 (IL-18) and IL-1␤, from infected macrophages. The expression of these cytokines was similarly induced after infection with H37Rv and ⌬RD1. However, the activation of caspase-1 was observed only in H37Rv-infected macrophages. The cytokine production and caspase-1 activation were induced independently of type I interferon receptor signaling events. We also found that the activation of caspase-1 was markedly inhibited with increasing concentrations of extracellular KCl. Furthermore, the production of IL-18 and IL-1␤ and caspase-1 activation were induced independently of a P2X7 purinergic receptor, and the inability of ⌬RD1 in caspase-1 activation was compensated for by nigericin, an agent inducing the potassium ion efflux. Based on these results, we concluded that RD1 participates in caspase-1-dependent cytokine production via induction of the potassium ion efflux in infected macrophages.

Pneumolysin is a pore-forming cytolysin known as a major virulence determinant of Streptococcus pneumoniae. This protein toxin has also been shown to activate the Toll-like receptor 4 (TLR4) signaling pathway. In this study, a mutant S. pneumoniae strain deficient in pneumolysin (⌬ply) and a recombinant pneumolysin protein (rPLY) were constructed. Upon infection of macrophages in vitro, the ability to induce the production of interleukin-1␣ (IL-1␣), IL-1␤, and IL-18 was severely impaired in the ⌬ply mutant, whereas there was no marked difference in the induction of tumor necrosis factor alpha (TNF-␣) and IL-12p40 between the wild type and the ⌬ply mutant of S. pneumoniae. When macrophages were stimulated with rPLY, the production of IL-1␣, IL-1␤, and IL-18 was strongly induced in a TLR4-dependent manner, whereas lipopolysaccharide, a canonical TLR4 agonist, hardly induced these cytokines. In contrast, lipopolysaccharide was more potent than rPLY in inducing the production of TNF-␣, IL-6, and IL-12p40, the cytokines requiring no caspase activation. Activation of caspase-1 was observed in macrophages stimulated with rPLY but not in those stimulated with lipopolysaccharide, and the level of activation was higher in macrophages infected with wild-type S. pneumoniae than in those infected with the ⌬ply mutant. These results clearly indicate that pneumolysin plays a key role in the host response to S. pneumoniae, particularly in the induction of caspase-1-dependent cytokines.

Hyper-coagulation, hypothermia, systemic inflammatory responses and shock are major clinical manifestations of endotoxin shock syndrome in human. As previously reported, mice primed with heat-killed Propionibacterium acnes are highly susceptible to the action of LPS to induce tumour necrosis factor (TNF)-alpha and to that of TNF-alpha to trigger lethal shock. Here we investigated the mechanisms underlying the P. acnes-induced sensitization to LPS and TNF-alpha and the development of individual symptoms after subsequent challenge with LPS or TNF-alpha. Propionibacterium acnes-primed wild-type (WT) mice, but not naive mice, exhibited hyper-coagulation with elevated levels of thrombin-antithrombin complexes and anti-fibrinolytic plasminogen activator inhibitor 1 in their plasma, hypothermia, systemic inflammatory responses and high mortality rate after LPS or TNF-alpha challenge. Propionibacterium acnes treatment reportedly induces both T(h)1 and T(h)17 cell development. Propionibacterium acnes-primed Il12p40(-/-) and Ifngamma(-/-) mice, while not Il17A(-/-) mice, evaded all these symptoms/signs upon LPS or TNF-alpha challenge, indicating essential requirement of IL-12-IFN-gamma axis for the sensitization to LPS and TNF-alpha. Furthermore, IFN-gamma blockade just before LPS challenge could prevent P. acnes-primed WT mice from endotoxin shock syndrome. These results demonstrated requirement of IFN-gamma to the development of endotoxin shock and suggested it as a potent therapeutic target for the treatment of septic shock.

The molecular mechanisms of Fas (CD95/Apo-1)-mediated apoptosis are increasingly understood. However, the role of Fas-mediated production of proinflammatory cytokines such as IL-18 and IL-1β in bacterial infection is unclear. We demonstrate the importance of Fas-mediated signaling in IL-18/IL-1β production postinfection with Listeria monocytogenes without the contribution of caspase-1 inflammasome. IL-18/IL-1β production in L. monocytogenes–infected peritoneal exudate cells from Fas-deficient mice was lower than those from wild type mice, indicating that Fas signaling contributes to cytokine production. L. monocytogenes infection induced Fas ligand expression on NK cells, which stimulates Fas expressed on the infected macrophages, leading to the production of IL-18/IL-1β. This was independent of caspase-1, caspase-11, and nucleotide-binding domain and leucine-rich repeat–containing receptors (NLRs) such as Nlrp3 and Nlrc4, but dependent on apoptosis-associated speck-like protein containing a caspase recruitment domain. Wild type cells exhibited caspase-8 activation, whereas Fas-deficient cells did not. L. monocytogenes–induced caspase-8 activation was abrogated by inhibitor for intracellular reactive oxygen species, N-acetyl-L-cysteine. L. monocytogenes–infected macrophages produced type-I IFNs such as IFN-β1, which was required for Il18 gene expression. Thus, Fas signaling regulates innate inflammatory cytokine production in L. monocytogenes infection.

CD4 Th cells play crucial roles in orchestrating immune responses against pathogenic microbes, after differentiating into effector subsets. Recent research has revealed the importance of IFN-γ and IL-17 double-producing CD4 Th cells, termed Th17/Th1 cells, in the induction of autoimmune and inflammatory diseases. In addition, Th17/Th1 cells are involved in the regulation of infection caused by the intracellular bacterium in humans. However, the precise mechanism of Th17/Th1 induction during pathogen infection is unclear. In this study, we showed that the inflammasome and Fas-dependent IL-1β induces Th17/Th1 cells in mice, in response to infection with the pathogenic intracellular bacterium In the spleens of infected wild-type mice, Th17/Th1 cells were induced, and expressed T-bet and Rorγt. In mice, which lack the adaptor molecule of the inflammasome (apoptosis-associated speck-like protein containing a caspase recruitment domain), Th17/Th1 induction was abolished. In addition, the Fas-mediated IL-1β production was required for Th17/Th1 induction during bacterial infection: Th17/Th1 induction was abolished in mice, whereas supplementation with recombinant IL-1β restored Th17/Th1 induction via IL-1 receptor 1 (IL-1R1), and rescued the mortality of mice infected with IL-1R1, but not apoptosis-associated speck-like protein containing a caspase recruitment domain or Fas on T cells, was required for Th17/Th1 induction, indicating that IL-1β stimulates IL-1R1 on T cells for Th17/Th1 induction. These results indicate that IL-1β, produced by the inflammasome and Fas-dependent mechanisms, contributes cooperatively to the Th17/Th1 induction during bacterial infection. This study provides a deeper understanding of the molecular mechanisms underlying Th17/Th1 induction during pathogenic microbial infections in vivo.