Nix is Upregulated in the Colonic Epithelium in a Murine Model of Infectious Colitis

Author(s):
Anna Ramos; Elizabeth Novak; Heather Mentrup ; David Fletcher ; Meredith Flanagan; Kevin Mollen

Background:

Disruption of the intestinal epithelium is a hallmark of colitis. Maintenance of this barrier is an energy-dependent process that relies on mitochondrial viability. Mitochondrial quality control mechanisms, such as mitophagy, are thereby integral to ensure cell homeostasis. Multiple mitophagy pathways exist, including the Pink1/Parkin and Nix pathways. We have previously demonstrated that Nix-mediated mitophagy is upregulated in the intestinal epithelium during human inflammatory bowel disease and chemically induced murine colitis. Our preliminary data utilizing a transgenic reporter mouse with a pH-dependent fluorescent probe directed to the mitochondrial matrix (mt-Keima) also suggests an increase in mitophagy. It remains unclear, however, if this homeostatic response regulates mitochondrial quality in infectious colitis.

Hypothesis:

Mitophagy is upregulated in the colonic epithelium in infectious colitis and may represent a homeostatic response to mitochondrial damage.

Methods:

Wild-type (C57Bl/6J), male, 6-week-old mice were randomly assigned to a Citrobacter-infected or Sham-infected group. On day 0, Citrobacter-infected mice were infected with Citrobacter rodentium (1 × 10⁹ CFUs) via oral gavage, and Sham-infected mice were gavaged with 1x dPBS. Disease activity index (DAI) scores, comprising of weight loss, stool consistency, and fecal blood, were calculated daily. On day 8, mice were euthanized, and colon lengths were measured. Colons were either preserved for histology or colonic epithelial scrapings were collected for protein isolation. Expression of mitophagy proteins was compared using western blot analysis.

Results:

Citrobacter-infected mice had significantly higher DAI scores, greater weight loss, and shorter colon lengths compared to Sham-infected mice, indicating worsened colonic inflammation. H&E staining of colonic tissue demonstrated greater hyperplasia and infiltration of immune cells into the lamina propria in Citrobacter-infected mice. Nix protein expression was significantly increased in the colonic scrapings collected from Citrobacter-infected mice compared to those from Sham-infected mice (1.6 ± 0.09 vs. 1.0 ± 0.33, respectively; p < 0.005). There was no significant difference in Parkin or Pink1 protein expression.

Conclusions:

Nix is significantly increased in the colonic epithelium in mice subjected to C. rodentium-induced infectious colitis, suggesting an increase in Nix-mediated mitophagy similar to that seen in inflammatory colitis. Further work utilizing mt-Keima mice is needed to quantify alterations in mitophagic flux in the setting of infectious colitis.