Its Mechanism in Humans and Mice Anti-Inflammatory Activity of PYNOD and

Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat–containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/ NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associatedspeck-like proteincontainingacaspaserecruitmentdomain(ASC)inreconstitutionexperimentsusingHEK293 cells. In this study, we investigated the molecular mechanism of PYNOD’s anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1–mediated IL-1 b processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1–mediated IL-1 b secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF- k B activation and apoptosis and to reduce ASC’s ability to promote caspase-1–mediated IL-1 b production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1 b processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1– mediated IL-1 b processing without inhibiting caspase-4 (mouse caspase-11)–mediated caspase-1 processing. Furthermore, PY-NOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo. The Journal of Immunology , 2010, 184: 000–000. cultured in a 96-well plate for 2 h, and the nonadherent cells were then removed. To induce cytokine production, adherent macrophages were cul- tured in RPMI 1640 medium supplemented with 10% FCS, penicillin, and streptomycin, with or without LPS (1 m g/ml, from Escherichia coli 055: B5; Sigma-Aldrich, St. Louis, MO) overnight. In some experiments, LPS-primed macrophages were pretreated with inhibitors, such as z-LEED– fluoromethylketone (fmk) (MBL, Nagoya, Japan), z-VAD–fmk, and Ac-YVAD–chloromethylketone (cmk) (Calbiochem, La Jolla, CA), or DMSO as the solvent control for 1 h. The cells were washed with fresh culture medium that lacked antibiotics and then infected with Salmonella typhi- murium (ATCC14028s)atamultiplicityofinfection(moi)of10,20,or50or stimulated with R837 (10 m g/ml; InvivoGen,San Diego, CA) for 12 h. After 1 h of S. typhimurium infection, gentamycin (final concentration of 50 m g/ ml) was added to kill the extracellular bacteria. Whole 4-h PECs (1 3 10 5 cells/well) were cultured in a 96-well plate, stimulated, infected, or both as described above, except that the cells were cultured in antibiotic-free me- dium from the beginning and were not washed after the LPS pretreatment.