Modulation of mitochondrial respiration to treat colitis

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The importance of mucosal healing in inflammatory bowel disease (IBD) derives from clinical data that demonstrate it to be a predictor of remission. Unfortunately, many IBD patients remain refractory or lose responsiveness to therapies leading to persistent mucosal ulceration. Prospective pediatric studies show that reduced mitochondrial gene expression predict unfavorable outcomes in ulcerative colitis (UC). Consistently, increased expression of mitochondrial respiratory chain genes has been shown to predict favorable Crohn’s disease (CD) outcomes. Based on these results and our own data, we hypothesize that a critical driver of chronic ulceration in IBD is the failure of intestinal epithelial cells (IEC) to increase mitochondrial respiration. In studies leading up to this submission we found that mitochondrial deficiency caused poor ulcer healing due to inadequate crypt fissioning occurring at ulcer edges (Progress Report). These findings led us to develop a novel gold (Au)- based drug based on the current safe and effective gold therapy for Rheumatoid arthritis (Auranofin). This new drug, AuPhos, localizes to mitochondria where it increases mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) production. Induction of low levels of “endogenous” mtROS are known to promote healthy adaptive responses in cells including anti-oxidant enzyme activities (Background). In preliminary studies, we found that oral AuPhos 1) enhances mucosal repair in DSS colitis, 2) increases IEC mitochondrial oxygen consumption rate (OCR) and oxidative phosphorylation (OXPHOS), 3) mtROS and H2O2 production in IEC as well as 4) phosphoinositides-3 kinase (PI3K) and nuclear factor erythroid 2-related factor (Nrf2) signaling with 5) improved expression of proteins that induce mitochondrial biogenesis [peroxisome proliferator activated receptor-γ coactivator 1 alpha (PGC1α) and transcription factor A, mitochondrial (TFAM). Studies proposed in Aim 1 interrogate mitochondrial function under baseline conditions to allow assessment of AuPhos-induced changes in mitochondrial mass, activity (mtROS) and OCR, ATP turnover, electron transport chain (ETC) activity, induction of adaptive responses [anti-oxidant catalase, MnSOD (superoxide dismutase) and glutathione (reduced/oxidized)] as well as assess changes in tissue oxidant stress (4HNE, protein carbonylation, nitrotyrosine IHC). Aim 2 will build upon these studies by evaluating AuPhos effects in TNF-stimulated small bowel (SB) and colonic organoids from veterans (normal and IBD). Studies in 3D organoids will allow for mechanistic testing of AuPhos-induced crypt budding and IEC gene expression. Results of Aim 2 will instruct studies in Aim 3A that study therapeutic effects of AuPhos on IEC gene expression and crypt fissioning that heal ulcers in DSS colitis. In Aim 3B, scRNAseq analysis of human biopsies incubated with AuPhos will allow us to examine induction of mitochondrial gene expression in discrete IEC clusters. Proposed studies are driven by a novel hypothesis that restoration of IEC mitochondrial respiration is a key step in mucosal healing. Together, these “preclinical” studies with a VA-sponsored new drug (AuPhos) have the potential of transitioning this agent to the therapy of IBD in veterans.