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

J Biol Chem. 2019 May 31;294(22):9007-9015. doi: 10.1074/jbc.RA119.007997. Epub 2019 Apr 17.

Abstract

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. 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 signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.

Keywords: NOD1; NOD2; Nod-like receptor (NLR); calcium intracellular release; cell biology; cell signaling; endoplasmic reticulum stress (ER stress); innate immunity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcimycin / chemistry
  • Calcimycin / pharmacology
  • Calcium / chemistry
  • Calcium / metabolism
  • Chemokine CXCL1 / metabolism
  • Cytokines / metabolism*
  • Endoplasmic Reticulum Stress* / drug effects
  • Gene Knockout Techniques
  • HCT116 Cells
  • Humans
  • Interleukin-8 / metabolism
  • Nod1 Signaling Adaptor Protein / deficiency
  • Nod1 Signaling Adaptor Protein / genetics
  • Nod1 Signaling Adaptor Protein / metabolism*
  • Nod2 Signaling Adaptor Protein / deficiency
  • Nod2 Signaling Adaptor Protein / genetics
  • Nod2 Signaling Adaptor Protein / metabolism*
  • Peptidoglycan / blood*
  • Signal Transduction / drug effects
  • Thapsigargin / pharmacology

Substances

  • Chemokine CXCL1
  • Cytokines
  • Interleukin-8
  • Nod1 Signaling Adaptor Protein
  • Nod2 Signaling Adaptor Protein
  • Peptidoglycan
  • Calcimycin
  • Thapsigargin
  • Calcium

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