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Contribution of phagocytosis and Nod2 to cytokine induction by Staphylococcus aureus: evidence for a regulatory role of Nod1.(Kapetanovic et al. Infect. Immun. 2007, 75, 830-837)
Toll-like receptors (TLR) are involved in the sensing of microbial-derived compounds. Nod1 and Nod2 are intracytoplasmic sensors that recognize distinct peptidoglycan-derived muropeptides. We analyzed the contribution of these receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we determined that TLR4 and TLR2 contribution was predominant in the induction of TNF and IL-10 by Gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to Gram-positive bacteria. In the absence of TLR2, the phagocytic property of macrophages was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA) and was markedly inhibited by cytochalasin D. By transfecting Raw 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-?B activation in response to HKSA. The unexpected interference of DN Nod1 in the response of macrophages to Gram-positive bacteria was confirmed with a Nod2 agonist (muramyl dipeptide) in transfection experiments in HEK293T cell. Finally, HKSA up-regulated the expression of Nod1 but not Nod2 mRNA in mouse Raw 264.7 macrophages, Our study shows the contribution of phagocytosis for cytokine production and that of Nod2 for macrophage response to HKSA. It also identifies a cross-talk between Nod1 and Nod2.
Synergistic stimulation of monocytes and dendritic cells by Toll-like receptor 4, Nod1- and Nod2-activating agonists(Fritz et al. Eur. J. Immunol 2005, 35, 2459-2470).
Muropeptides are degradation products of peptidoglycan (PGN). Interest in this family of molecules has increased recently with the discovery of Nod1 and Nod2 as sensors of PGN fragments (3, 4). One of these murapeptides, muramyl dipeptide (MDP) has been well characterized (13). MDP mediates signaling by Nod2, exerts adjuvant activity and synergizes with lipopolysaccharide to induce pro-inflammatory responses in vitro and in vivo. There are few and contradictory results on the stimulatory capacity of nod1 agonists toward myeloid cells. Thus, we examined the capacity of various nod1 (murnac-l-ala-?-d-glu-meso-diaminopimelic acid, m-tridap) and Nod2 (murnac-l-ala-d-isogln, MDP; murnac-l-ala-d-isogln-l-lys, m-trilys) agonists to stimulate primary human cells. We show that both cd14+ monocytes and cd1a+ immature dendritic cells express Nod1 and Nod2. Stimulation of primary human monocytes and dendritic cells with highly purified muropeptides (m-tridap, MDP and m-trilys) by itself induces release of pro-inflammatory mediators. Cooperatively with lps, Nod1 as well as Nod2 agonists stimulate release of both pro- and anti-inflammatory cytokines (IL-1beta, tnf, IL-6, IL-8, IL-10, IL-12p40) in these myeloid cell subsets. Nod1 and Nod2 agonists synergize with low amounts of lps to induce dendritic cell maturation, suggesting that nod protein agonists instruct the onset of adaptive immune responses, cooperatively with molecules sensed by toll-like receptors.These studies have been carried out in collaboration with the group of Dana Philpott and were specifically funded by the Institut Pasteur.