Abstract
Reduced ATP levels and irregular mitochondrial structures have been found in gut epithelia in some IBD patients, suggesting that metabolic stress could be an etiologic factor. At the same time, Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) depletion is an important susceptibility trait for IBD. As a result, in our study, we assessed if loss of NOD2 further reduces epithelial barrier integrity under conditions of metabolic stress, instigated by the addition of the oxidative phosphorylation un-coupler, dinitrophenol (DNP). T84 (human colon cell line) cells treated with non-invasive E. coli + DNP (16 hrs) showed a significant increase in NOD2 protein. The increase in intracellular bacteria in both wild-type (WT) and NOD2 knock-down cells (NOD2 KD) was dependent on reactive oxygen species (ROS) and resulted in MAPK ERK 1/2 pathway activation; this was determined by adding an antioxidant (mitoTEMPO) or an ERK (U0126) inhibitor. While there was an increase in internalized E. coli in cells, no change was seen in the internalization of bead levels or dead E. coli suggesting that reduced killing of the bacteria was the primary cause for the increased numbers of bacteria in the NOD2 KD epithelia. This conclusion is reinforced by evidence of attenuated autophagy in NOD2 KD T84 cells.
Neutrophils are potent contributors to IBD. IL-8 is a major neutrophil chemoattractant and signaling molecule; IL-8 levels have been shown to be elevated in IBD patients. As such, we assessed whether metabolic stress induced heightened IL-8 secretion. T84 cells exposed to DNP yielded heightened IL-8 secretion after 16 hrs compared to cells treated with E. coli alone in a time-dependent manner. Secretion significantly declined after mitoTEMPO treatment. Bacteria- derived products are necessary for IL-8 secretion; no response was seen to bead co-treatment, but persisted after T84 cells were treated with dead E. coli. IL-8 secretion is dependent on endosome maturation and cytoskeletal rearrangement; administration of chloroquine or cytochalsin D both significantly reduced IL-8 levels. Finally, IL-8 secretion is MyD88 dependent, implicating TLR signaling in the epithelial cell response to metabolic stress in a commensal rich environment.
Taken together, we offer a three-hit hypothesis: lowered barrier function, as a result of metabolic dysfunction is amplified in the absence of NOD2, resulting in increased numbers of live intracellular bacteria. Bacterial presence in the intestinal epithelial cells provokes the release of the neutrophil chemotactic chemokine, IL-8, resulting in inflammation. In NOD2 depleted patients, greater levels of internalized bacteria yield a potent inflammatory effect, given that the antimicrobial effects of both macrophages and Paneth cells are compromised.
