V. Siow1,2, E. A. Novak1, G. J. Vincent1, K. Cunningham1,2, K. P. Mollen1 1Children’s Hospital Of Pittsburgh,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA
Introduction: The role of mitochondrial dysfunction in the pathogenesis of inflammatory bowel disease (IBD) is just beginning to be understood. Our lab and others have shown evidence of mitochondrial stress and a disruption in mitochondrial function within the intestinal epithelium of humans with IBD as well as mice undergoing experimental colitis. We now hypothesize that mitochondrial stress leads to a release of the damage-associated molecular pattern (DAMP) mitochondrial DNA (mtDNA), which in turn contributes to immune activation and the potentiation of inflammation.
Methods: Serum samples were obtained from patients with severe active IBD and from control subjects according to University and IRB protocol. MtDNA content was determined using qRT-PCR. Mice were subjected to 3% Dextran Sodium Sulfate (DSS) for 7 days. The mitochondria within the intestinal epithelium of control mice vs. DSS-subjected mice were analyzed via electron microscopy. Total DNA was isolated from murine intestinal crypts in control and DSS-subjected mice. This DNA was then utilized to determine the levels of mtDNA relative to nuclear DNA via qPCR and the ΔΔCt method. Intestinal epithelial HT29 cells were treated with mtDNA at 37°C. RNA was then isolated, cDNA synthesized, and qPCR setup to determine the expression of cytokines in control vs. mtDNA-treated cells.
Results: For the first time, we show evidence of mtDNA release in the bloodstream of humans with severe active IBD. Mice undergoing experimental colitis exhibit dramatically altered mitochondrial structure as well as a decrease in overall mtDNA within the intestinal epithelium as compared to control mice. Although total mtDNA content was decreased, there was evidence of free mtDNA within the cytosol of intestinal epithelial cells of mice subjected to experimental colitis. Additionally, HT29 cells treated with mtDNA exhibited an increase in the expression of pro-inflammatory cytokines, including TNF-α as well as TLR9 expression.
Conclusion: Here we investigate the role of mtDNA in intestinal inflammation. Our previous studies demonstrate a role for mitochondrial dysfunction in the pathogenesis of colitis. However, it remains unclear how this dysfunction leads to inflammatory change. We now demonstrate that the amount of circulating mtDNA in the blood of patients with IBD is increased as compared to healthy control patients, suggesting that a release of mtDNA from dysfunctional mitochondria occurs during disease. We also show that intestinal epithelial cells treated with mtDNA demonstrate an increase in expression of pro-inflammatory cytokines. Our studies suggest that mtDNA may contribute to local and systemic inflammation in patients with IBD. Strategies aimed at limiting mitochondrial dysfunction or sequestering cell-free mtDNA may lead to new therapeutic approaches to combat disease.