Enhanced mucosal mitochondrial function corrects dysbiosis and OXPHOS metabolism in IBD
Authors: Kapur Neeraj , Alam M. Ashfaqul , Hassan Syed Adeel , Patel Parth H , Wempe Lesley A. , Bhogoju Sarayu , Goretsky Tatiana , Kim Jong Hyun , Herzog Jeremy , Ge Yong , Awuah Samuel G. , Byndloss Mariana , Baumler Andreas J , Zadeh Mansour M. , Sartor R. Balfour , Barrett Terrence 
Published: March 2024

Background

Mitochondrial (Mito) dysfunction in IBD reduces mucosal O2 consumption and increases O2 delivery to the microbiome. Increased enteric O2 promotes blooms of facultative anaerobes (eg.Proteobacteria) and restricts obligate anaerobes (eg.Firmicutes). Dysbiotic metabolites negatively affect host metabolism and immunity. Our novel compound (AuPhos) upregulates intestinal epithelial cell (IEC) mito function, attenuates colitis and corrects dysbiosis in humanizedIl10-/-mice. We posit that AuPhos corrects IBD-associated dysbiotic metabolism.

Methods

Primary effect of AuPhos on mucosal Mito respiration and healing process was studied in ex vivo treated human colonic biopsies and piroxicam-accelerated (Px)Il10-/-mice. Secondary effect on microbiome was tested in DSS-colitis WT B6 and germ-free 129.SvEv WT orIl10-/-mice reconstituted with human IBD stool (Hu-Il10-/-). Mice were treated orally with AuPhos (10- or 25- mg/kg; q3d) or vehicle, stool samples collected for fecal lipocalin-2 (f-LCN2) assay and microbiome analyses using 16S rRNA sequencing. AuPhos effect on microbial metabolites was determined using untargeted global metabolomics. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Effect of AuPhos on enteric oxygenation was assessed byE. coli Nissle 1917 WT(aerobic respiration-proficient) andcytochrome oxidase (cydA)mutant (aerobic respiration-deficient).

Results

Metagenomic (16S) analysis revealed AuPhos reduced relative abundances ofProteobacteriaand increased blooms ofFirmicutesin uninflamed B6 WT, DSS-colitis, Hu-WT and Hu-Il10-/-mice. AuPhos also increased hypoxyprobe-1 staining in surface IECs suggesting enhanced O2 utilization. AuPhos-induced anaerobiosis was confirmed by a significant increase in cydA mutant compared to WT (O2-utlizing)E.coli. Ex vivo treatment of human biopsies with AuPhos showed significant increase in Mito mass, and complexes I and IV. Further, gene expression analysis of AuPhos-treated biopsies showed increase in stem cell markers (Lgr4, Lgr5, Lrig1), with concomitant decreases in pro-inflammatory markers (IL1β,MCP1, RankL). Histological investigation of AuPhos-fed Px-Il10-/-mice showed significantly decreased colitis score in AuPhos-treated Px-Il10-/-mice, with decrease in mRNA of pro-inflammatory cytokines and increase in Mito complexes (ND5,ATP6). AuPhos significantly altered microbial metabolites associated with SCFA synthesis, FAO, TCA cycle, tryptophan and polyamine biosynthesis pathways. AuPhos increased pyruvate, 4-hydroxybutyrate, 2-hydroxyglutarate and succinate, suggesting an upregulation of pyruvate and glutarate pathways of butyrate production. AuPhos reduced IBD-associated primary bile acids (BA) with concomitant increase in secondary BA (SBA). AuPhos treatment significantly decreased acylcarnitines and increased L-carnitine reflective of enhanced FAO. AuPhos increases TCA cycle intermediates and creatine, energy reservoir substrates indicating enhanced OxPHOS. Besides, AuPhos also upregulates tryptophan metabolism, decreases Kynurenine and its derivatives, and increases polyamine biosynthesis pathway (Putresceine and Spermine).

Conclusion

These findings indicate that AuPhos-enhanced IEC mitochondrial function reduces enteric O2 delivery, which corrects disease-associated metabolomics by restoring short-chain fatty acids, SBA, AA and IEC energy metabolism.

Graphical abstract