SUMMARY Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the effects of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut.

factor; HRB5rl, human hepatoma Hep3B cells stably transfected with the HIF-dependent Firefly luciferase reporter pH3SVL followed by the Renilla reporter construct pRL-SV40; JNJ-1935, JNJ-42041935; OTUB1, ovarian tumor domain-containing ubiquitin aldehyde binding protein 1; PHD, prolyl-4-hydroxylase domain; SDR, SensorDish Reader; SV40, simian virus 40; 2OG, 2-oxoglutarate. AbstractPharmacologic HIF hydroxylase inhibitors (HIs) are effective for the treatment of anemia in chronic kidney disease patients and may also be beneficial for the treatment of diseases such as chronic inflammation and ischemia-reperfusion injury. The selectivities of many HIs for HIF hydroxylases and possible off-target effects in cellulo are unclear, delaying the translation from preclinical studies to clinical trials. We developed a novel assay that discriminates between the inhibition of HIF-α prolyl-4-hydroxylase domain (PHD) enzymes and HIF-α asparagine hydroxylase factor inhibiting HIF (FIH). We characterized 15 clinical and preclinical HIs, categorizing them into pan-HIF-α hydroxylase (broad spectrum), PHD-selective, and FIHselective inhibitors, and investigated their effects on HIF-dependent transcriptional regulation, erythropoietin production, and cellular energy metabolism. While energy homeostasis was generally maintained following HI treatment, the pan-HIs led to a stronger increase in pericellular pO 2 than the PHD/FIH-selective HIs. Combined knockdown of FIH and PHD-selective inhibition did not further increase pericellular pO 2 . Hence, the additional increase in pericellular pO 2 by pan-over PHD-selective HIs likely reflects HIF hydroxylase independent off-target effects. Overall, these analyses demonstrate that HIs can lead to oxygen redistribution within the cellular microenvironment, which should be considered as a possible contributor to HI effects in the treatment of hypoxia-associated diseases. K E Y W O R D Sanemia, hypoxia, 2-oxoglutarate oxygenase inhibitor, prolyl hydroxylase inhibitor, Roxadustat