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.

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The ability to gain entry and resist the antimicrobial intracellular environment of mammalian cells is an essential virulence property of Mycobacterium tuberculosis. A purified recombinant protein expressed by a 1362 bp locus (mce1) in the M. tuberculosis genome promoted uptake into HeLa cells of polystyrene latex microspheres coated with the protein. N‐terminus deletion constructs of Mce1 identified a domain located between amino acid positions 106 and 163 that was needed for this cell uptake activity. Mce1 contained hydrophobic stretches at the N‐terminus predictive of a signal sequence, and colloidal gold immunoelectron microscopy indicated that the corresponding native protein is expressed on the surface of the M. tuberculosis organism. The complete M. tuberculosis genome sequence revealed that it contained four homologues of mce (mce1, mce2, mce3, mce4) and that they were all located within operons composed of genes arranged similarly at different locations in the chromosome. Recombinant Mce2, which had the highest level of identity (67%) to Mce1, was unable to promote the association of microspheres with HeLa cells. Although the exact function of Mce1 is still unknown, it appears to serve as an effector molecule expressed on the surface of M. tuberculosis that is capable of eliciting plasma membrane perturbations in non‐phagocytic mammalian cells.

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.

isolated from Chromobacterium violaceum No. 968, were studied in animals. FR901228 (ip) prolonged the life of mice bearing such murine ascitic tumors as P388 and L1210 leukemias and B16 melanoma, and inhibited (iv) the growth of murine solid tumors (Colon 38 carcinoma, M5076 reticulum cell sarcoma and Meth A fibrosarcoma) and human solid tumors (Lu-65 and LC-6 lung carcinomas, and SC-6 stomach adenocarcinoma) implanted in normal and nude mice, respectively. Its antitumor activity was especially potent against murine Meth A fibrosarcoma and human SC-6 stomach adenocarcinoma which were refractory to mitomycin C or cisplatin. FR901228also was more effective against mitomycin C-, cyclophosphamide-, vincristine-and 5-fluorouracil-resistant P388 leukemias than against non-resistant P388 in mice. These results suggest that FR901228will be a new type of drug for the treatment of cancer.

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

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