As macrophages in vivo are exposed to multiple TLR/NOD-like receptor selleckchem ligands, we investigated the impact of NOD2/MDP signaling on macrophage responsiveness in combination with various TLR stimulations. To control for the indirect effect of a heightened inflammatory state in mice with colitis, we isolated macrophages from the spleen of 6-wk-old IL-10?/? and IL-10?/?NOD2?/? mice with no colitis, as confirmed histologically. Preliminary dose-response experiments were undertaken to determine the optimal dose for LPS and MDP in our assay (Supplemental Fig. 1). As has been shown in other studies utilizing murine macrophages (4), 10 ng/ml LPS combined with 10 ��g/ml MDP gave maximal synergistic cytokine production in WT mice.

Confirming the findings in the preliminary dose-response experiments, stimulation of WT macrophages with MDP and LPS or the TLR9 ligand, unmethylated CpG oligonucleotide (CPG), resulted in synergistic activity in some cytokines, although, in comparison with IL-10?/? mice, overall cytokine induction was low (Fig. 4). However, separate statistical analysis of the responses of WT macrophages revealed that there was significant potentiation of LPS-induced TNF and CPG-induced TNF, IL-6, and IL-12p40 by MDP. There was no synergistic activity on WT macrophages with MDP and TLR2 (Fig. 4). MDP stimulation alone has been shown to be a relatively poor stimulator of cytokine production in macrophages (6, 11, 49). In agreement with these studies, cytokine levels produced by MDP stimulation alone were comparable to unstimulated cells, and there was no significant difference between genotypes (Fig.

4). When cells from IL-10?/? and IL-10?/?NOD2?/? mice were stimulated with TLR ligand alone, LPS induced significantly more TNF-�� and IL-6 compared with WT and NOD2?/? mice (Fig. 4A), and CPG stimulation resulted in significant increase in TNF-��, IL-12p40, and IL-6 production (Fig. 4B), whereas TLR2 stimulation with the synthetic ligand PAM3CSK4 increased TNF-�� and IL-6 production compared with cells from mice with intact IL-10 signaling (Fig. 4C). Thus, IL-10 deficiency renders macrophages intrinsically hyperresponsive to stimulation by various bacterial ligands. As with LPS stimulation, the heightened responsive state of IL-10?/? macrophages to a single bacterial stimulus was not influenced by NOD2 signaling, as there was no difference in cytokine production between IL-10?/? and IL-10?/?NOD2?/? when macrophages were stimulated with either LPS, CPG, or PAM3CSK4 alone (Fig. 4). However, Cilengitide when cells from IL-10?/? mice were stimulated with TLR ligands and MDP, there was a large synergistic effect that significantly increased TNF-��, IL-12p40, and IL-6 production compared with TLR stimulation alone (Fig. 4).