The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we show that Syk and JNK-dependent signaling pathways are required for caspase-1 activation via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or JNK abolished the formation of ASC specks without affecting interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and JNK-dependent manner, suggesting that Syk and JNK are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 residue in mouse ASC was critical for speck formation and caspase-1 activation. These results suggested that phosphorylation of ASC controls inflammasome activity through ASC speck formation.

Eradication of a given pathogen is dependent on the selective differentiation of T helper (Th) cells into Th1 or Th2 types. We show here that T cells from mice lacking the transcription factor IRF-1 fail to mount Th1 responses and instead exclusively undergo Th2 differentiation in vitro. Compromised Th1 differentiation is found to be associated with defects in multiple cell types, namely impaired production of interleukin-12 by macrophages, hyporesponsiveness of CD4+ T cells to interleukin-12, and defective development of natural killer cells. These results indicate the involvement of IRF-1 in multiple stages of the Th1 limb of the immune response.

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1β and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated speck like protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as NLR family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3−/− macrophages, while knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC−/− mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1β and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.

Although the NLRP3 inflammasome plays a pivotal role in host defense, its uncontrolled activation is associated with inflammatory disorders, suggesting that regulation of the inflammasome is important to prevent detrimental effects. Type I IFNs and long-term LPS stimulation were shown to negatively regulate NLRP3 activation. In this study, we found that endogenous NO is involved in the regulation of NLRP3 inflammasome activation by either IFN-β pretreatment or long-term LPS stimulation. Furthermore, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, markedly inhibited NLRP3 inflammasome activation, whereas the AIM2 and NLRC4 inflammasomes were only partially inhibited by SNAP. An increase in mitochondrial reactive oxygen species induced by ATP was only modestly affected by SNAP treatment. Interestingly, S-nitrosylation of NLRP3 was detected in macrophages treated with SNAP, and this modification may account for the NO-mediated mechanism controlling inflammasome activation. Taken together, these results revealed a novel role for NO in regulating the NLRP3 inflammasome.

Material Supplementary 8.DC1http://www.jimmunol.org/content/suppl/2010/06/18/jimmunol.100105

No abstract

The contribution of phagocytes to protection against LiJteria monocytogenes was analysed in outbred ddN mice. Most of the bacteria injected intravenously at a dose of 3 x lo3 to 4 x lo3 were trapped in the liver within 10 min. There was a transient 10-fold decrease in the number of bacteria by 6 h. Anti-listeria activity in the initial phase was resistant to X-irradiation but was inhibited by carrageenan, and was not influenced by immunization. The protection in this very early stage of infection seemed to be attributable to the function of fixed macrophages. Viable bacteria in the organs increased progressively but slowly from 6 h to 72 h to reach maximum numbers. Bacterial growth during this period was markedly enhanced by X-irradiation or treatment with carrageenan. Accumulation of free phagocytes seemed to suppress the bacterial growth in this phase. The number of bacteria began to decrease from day 4 and became undetectable by day 9. The suppressive effect on bacterial growth in this last phase may be dependent on immunologically activated macrophages and was reversed by X-irradiation and carrageenan. The course of local infection was similar to that of systemic infection except for the lack of initial decrease. We conclude that the course of infection with L. monocytogenes can be divided into three phases with regard to the roles of phagocytes in resistance.

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.