Distinct Roles for Nod2 Protein and Autocrine Interleukin-1β in Muramyl Dipeptide-induced Mitogen-activated Protein Kinase Activation and Cytokine Secretion in Human Macrophages

Elucidating factors regulating Crohn's disease-associated nucleotide-binding oligomerization domain 2 (Nod2) responses is critical to understanding the mechanisms of intestinal immune homeostasis. Stimulation of primary monocyte-derived macrophages by muramyl dipeptide (MDP), a component of bacterial peptidoglycan and specific Nod2 ligand, produces cytokines, including IL-1 beta. We found that IL-1 beta blockade profoundly inhibits MDP-induced cytokine production in human monocyte-derived macrophages, demonstrating a key role for IL-1 beta autocrine secretion in Nod2-mediated responses. Importantly, although MAPK activation has previously been attributed directly to Nod2 signaling, we determined that the IL-1 beta autocrine loop is responsible for the majority of MDP-induced MAPK activation. Because the critical effects of IL-1 beta autocrine secretion on MAPK activation are observed as early as 10 min after Nod2 stimulation, we hypothesized that secretion of IL-1 beta from preexisting intracellular pro-IL-1 beta stores is necessary for optimal MDP-mediated cytokine induction. Consistently, we detected IL-1 beta secretion within 10 min of MDP treatment. Moreover, caspase-1 inhibition significantly attenuates MDP-mediated early MAPK activation. Importantly, selective JNK/p38 activation is sufficient to rescue the decreased cytokine secretion during Nod2 stimulation in the absence of autocrine IL-1 beta. Finally, we found that the IL-1 beta autocrine loop significantly enhances responses by a broad range of pattern recognition receptors. Taken together, MDP stimulation activates Nod2 to process and release preexisting pro-IL-1 beta stores in a caspase-1-dependent fashion; this secreted IL-1 beta, in turn, contributes to the majority of MDP-initiated MAPK activation and leads to subsequent cytokine secretion. Our findings clarify mechanisms of IL-1 beta contributions to Nod2 responses and elucidate the dominant role of IL-1 beta in MDP-initiated MAPK and cytokine secretion.