TLR4‐signaling is required for NOD2 but not NOD1 activation in hepatocytes

NACHT‐LRR receptors (NLRs) are recently identified intracellular pattern recognition receptors. The NLRs NOD1 and NOD2 respond to microbial ligands, e.g. diaminopimelic acid (DAP) and muramyl dipeptide (MDP) respectively. Hepatocytes (HC) are microbial‐responsive cells that express and activate Toll‐like receptors (TLRs). We hypothesized that HC also express and activate NOD1 and NOD2 as part of the initiation and regulation of innate immunity. Protein expression of NOD1 and NOD2 was detected in whole cell lysates of primary isolated HC from C57BL/6 mice. Other HC were treated with LPS (100ng/mL), iE‐DAP (100ng/mL; NOD1 ligand), or MDP (10μg/mL; NOD2‐ligand) for up to 60min before collection of whole cell lysates or nuclear isolation. NOD ligands were endotoxin free as determined by sensitive colorimetric endotoxin assay. MAPK (ERK, JNK, and p38) were activated and NFκB translocated to the nucleus in HC treated with iE‐DAP (NOD1 ligand), to similar levels as with LPS‐stimulation. However there was minimal activation of NFκB with MDP (NOD2 ligand) unless cells were pretreated for 24h with LPS. Activation of NFκB after MDP stimulation (NOD2 ligand) was partially TLR4‐dependent but activation of NFκB with iE‐DAP was similar in isolated WT and TLR4−/− HC. Taken together these data show expression of NOD1/NOD2 in HC, and differential regulation of their activation by TLR4. Work supported by NIH grant R01‐GM‐50441