A. E. Ramos1, S. Khan1, E. A. Novak1, H. L. Mentrup1, K. P. Mollen1 1University Of Pittsburgh, General Surgery, Pittsburgh, PA, USA
Introduction: Mitochondrial dysfunction is a hallmark of intestinal disease in IBD. Mitochondrial DNA (mtDNA) released into the cytosol can bind and activate the DNA sensor, Cyclic GMP-AMP Synthase (cGAS). Our lab has demonstrated cGAS activation to be protective during intestinal inflammation. However, the relationship of cGAS and mitochondrial health within the intestinal epithelium is unknown. We hypothesize that cGAS binds to mtDNA in intestinal epithelial cells and maintains homeostasis by regulating mitochondrial biogenesis and respiration.
Methods: We subjected cGAS KO, villincre;cGASfl/fl, lysmcre;cGASfl/fl, and WT mice to a 7-day model of DSS colitis. We performed electron microscopy (EM), WB analysis, qPCR, high resolution respirometry, and RNA sequencing analysis of the intestinal epithelium. We calculated disease activity index scores and performed colonoscopies on villincre;cGASfl/fl mice, lysmcre;cGASfl/fl mice, and their floxed controls.
Results: Our results demonstrate cGAS KO mice to have worsened mitochondrial health at baseline compared to WT mice as evidenced by EM. This discrepancy is exacerbated when cGAS KO and WT mice are subjected to the DSS induced colitis model with cGAS KO mice demonstrating near complete mitochondrial destruction. RNA sequencing analysis of the intestinal epithelium of cGAS KO mice subjected to DSS colitis showed a significant downregulation of mitochondrial respiratory pathways. To further evaluate mitochondrial respiration, we performed high resolution respirometry and found a decrease in complex I of the electron transport chain in cGAS KO mice subjected to DSS colitis. To evaluate mitochondrial biogenesis, we performed WB analysis which showed a decrease in two proteins involved in mitochondrial biogenesis, PGC1a and TFAM, in cGAS KO mice subjected to DSS. To assess for the role of cGAS in the intestinal epithelium, we subjected intestinal specific cGAS KO mice (villincre;cGASfl/fl) and myeloid specific cGAS KO mice (lysmcre;cGASfl/fl) to a 7-day DSS colitis model. Our data show that villincre;cGASfl/fl have significantly worsened intestinal inflammation compared to their floxed controls when subjected to DSS colitis whereas a myeloid-specific deletion of cGAS (lysmcre;cGASfl/fl) had no difference. Lastly, qPCR analysis for proinflammatory cytokines demonstrated higher levels of TNF-α and IL-6 in DSS-subjected villincre;cGASfl/fl mice compared to their floxed controls. This difference was not seen in the DSS-subjected lysmcre;cGASfl/fl mice compared to their floxed controls, suggesting the protective role of cGAS to be epithelial-specific.
Conclusion: We demonstrate intestinal epithelial cGAS deficiency to lead to worsened intestinal inflammation. Furthermore, our data show cGAS deficiency to be associated with decreased mitochondrial respiration and biogenesis.