Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress

Abstract

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor (NLR) family of innate immune proteins. In addition to their role as direct bacterial sensors, it was recently proposed that NOD proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family (SERCA) that pumps Ca 2+ into the ER, resulting in pro-inflammatory signalling. Here, we confirm that thapsigargin induces pro-inflammatory signalling in epithelial cells in a NODdependent manner. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signalling, and the calcium chelator BAPTA-AM blunted thapsigargin-dependent pro-inflammatory signalling, showing NOD proteins responded to a rise in intracellular Ca 2+ . Since intracellular Ca 2+ directly affects vesicular trafficking, we tested if thapsigargin-induced NOD activation required endocytosis. Our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigarginmediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice by high-pressure liquid chromatography and mass spectrometry, and identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca 2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signalling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signalling may have been previously overlooked.

TRIM21 controls Toll‐like receptor 2 responses in bone‐marrow‐derived macrophages

“…Tonic TLR signaling occurs as the result of bacterial contaminants in serum, as previously reported. [23][24][25] By comparing the expression of TLR-induced genes at baseline, we discovered a reduced TLR2-activation (PAM3CSK4) gene signature in Trim21 À/À BMDMs (Fig. 3a).…”

NOD1 and NOD2 in inflammatory and infectious diseases

“…Furthermore, inhibitors of Ca 2+ flux partially recapitulated the effects of thapsigargin. Therefore, it was hypothesized that increases in intracellular Ca 2+ can lead to endocytosis and internalization of extracellular PGN fragments present in either media or serum to activate NOD1 or NOD2 to explain results from previous studies although this has not been definitely proven 31 …”

“…Together, these results strongly suggest a PGN‐independent pathway involving ER stress can activate NOD1 and NOD2 although the precise upstream signal sensed by NOD1 and NOD2 remains unclear. However, it has also been posited based on results from another study that ER stress per se does not activate NOD1 and NOD2; rather, cellular perturbations related to Ca 2+ flux may be the main activating stimulus as thapsigargin, which induces ER stress by inhibiting ER calcium ATPase and Ca 2+ accumulation in the ER lumen, resulted in NOD1/2‐dependent cytokine production in HCT116 colon epithelial cells 31 . On the other hand, other ER inducers not associated with changes in intracellular Ca 2+ levels, such as tunicamycin, did not induce an inflammatory response via NOD1 or NOD2 31 .…”

NOD1 mediates interleukin-18 processing in epithelial cells responding to Helicobacter pylori infection

“…3a). This may be attributed to the presence of NOD1 ligand (peptidoglycan) in the serum used for tissue culture which is able to promote low level NOD1 activation 22 . Primary epithelial cells from Casp1 −/− mice, but not those from Nlrp3 −/− nor Pycard −/ − animals, produced signi cantly less IL-18 in response to stimulation with H. pylori SS1 bacteria, when compared with WT cells ( Fig.…”

“…pylori MVs, which contain peptidoglycan 14,15 . The BHI broth, from which MVs were isolated, also induced IL-18 processing, most likely due to serum in the medium 22 . As reported 26 , we also did not detect any IL-1β production in the AGS cell line (data not shown).…”