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.