Thus, Treg activity may still contribute to protection in IL-10?/?NOD2?/? mice. We have shown that T cells from selleck screening library IL-10?/?NOD2?/? mice produced significantly more IL-2 (Fig. 2B). As IL-2 is a key growth factor for Treg cells (46), we investigated whether loss of NOD2 altered the number of Treg cells in the spleen. The percentage of CD4+ CD25+FOXP3+ cells was not significantly higher in the spleen of IL-10?/?NOD2?/? mice compared with IL-10?/? mice both before inflammation and in mice with colitis (Fig. 2E). Macrophages from IL-10?/?NOD2?/? have a diminished response to LPS Macrophages contribute to colitis in IL-10?/? mice by having an enhanced response to bacterial stimuli (19), resulting in the production of proinflammatory cytokines such as IL-12, which in turn favors the differentiation of Th1 cells (47).

NOD2 expression was initially demonstrated in monocytes (48) and subsequently shown to be a key mediator of macrophage responses to bacterial stimuli (4). The key roles of IL-10 and NOD2 on macrophage function prompted us to investigate macrophage responses to bacterial stimulus in IL-10?/? and IL-10?/?NOD2?/? mice. CD11b-positive cells were purified from the spleens and peritoneum of 18-wk-old WT, NOD2?/?, IL-10?/?, and IL-10?/?NOD2?/? mice. Isolated cells were then stimulated with LPS, and proinflammatory cytokine production was measured. In agreement with previous studies using bone marrow�Cderived macrophages (4), there was no significant difference in response to LPS between peritoneal and spleen macrophages from WT and NOD2?/? mice (Fig. 3).

Also, in accordance with previous findings (19), macrophages from colitic IL-10?/? mice have enhanced responsiveness to LPS compared with WT mice, as demonstrated by significantly elevated levels of TNF-��, IL-12p40, and IL-6 (Fig. 3). There was also a significant increase in IL-6 release from unstimulated IL-10?/? peritoneal macrophages, demonstrating the potent regulatory effect that IL-10 has on the basal activity of peritoneal cells (Fig. 3C). Loss of NOD2 in IL-10?/? mice reduced the proinflammatory response of macrophages to LPS when compared with macrophages isolated from IL-10?/? mice with intact NOD2 signaling. Macrophages GSK-3 isolated from the spleen of IL-10?/?NOD2?/? mice produced significantly less IL-12p40, TNF-��, and IL-6 compared with cells from IL-10?/? mice (Fig. 3). This phenotype was not restricted to spleen macrophages as peritoneal macrophages from IL-10?/?NOD2?/? mice also produced significantly less TNF-�� and IL-12p40 when stimulated with LPS (Fig. 3A, ,3B).3B). However, there was no difference in IL-6 production, possibly reflecting different in vivo phenotypes and activated states of spleen and peritoneal macrophages (Fig. 3C).