AUPHOS, A NOVEL DRUG THAT RESTORES INTESTINAL HOMEOSTATIS BY CORRECTING DYSBIOSIS IN INFLAMMATORY BOWEL DISEASES

BACKGROUND: Enterochromaffin cells (ECs) are specialized gastrointestinal (GI) epithelial cells that transduce luminal forces and chemicals into release of signal molecules including serotonin (5-HT) to alter GI function including mucosal ion transport. To determine the role of ECs in ion transport, direct activation of ECs is required to localize the downstream effects to ECs versus other cell types.
AIM: Determine whether optogenetic activation of ECs modulates intestinal ion transport.
METHODS: Full-thickness ileum preparations of EC^ReaChR (Tph1^Cre-ERT/+::Rosa26^{LSL-ReaChR-mCitrine/+}) mice treated with tamoxifen (50mg/kg p.o.) four consecutive days prior to tissue harvest were either cryosectioned and immunostained for 5-HT or mounted in 4mL Ussing chambers with Kreb’s solution (NKS). The mucosal side was intermittently stimulated using a fiber optic cable (625-nm LED, Prizmatix) at 2Hz, 40% duty cycle for 20 cycles. Veratridine (30µM), a sodium channel opener, and Substance P (0.3 µM), a neurokinin receptor agonist, were also used to stimulate I_sc. TTX (1µM) was used to block neurotransmission and ondansetron (1µM) and GR113808 (30nM) were used to block serotonin receptors. Responses were measured as change in short-circuit current (ΔI_sc) = I_sc_peak − I_sc_baseline (µA/cm²) with differences detected with Wilcoxson ranked sum tests.
RESULTS: 57% of 5-HT-immunoreactive (ir) cells expressed mCitrine and 96% of mCitrine+ cells were 5-HT-ir illustrating the recombination efficiency and specificity of EC^ReaChR expression. Light stimulation increased I_sc in the ileum from EC^ReaChR mice but did not change I_sc from negative control mice that lacked either Tph1^Cre-ERT or Rosa26^{LSL-ReaChR-mCitrine} transgenes suggesting that light selectively stimulated ReaChR expressed in ECs. Removal of Cl^- from NKS, blocked light-induced I_sc (79 ± 20 µA/cm², NKS, 3.2 ± 0.6 µA/cm², Cl^- free, n=17, P<0.001). While TTX blocked the neurogenic stimuli of veratridine (37 ± 6 µA/cm², veratridine, 3 ± 1 µA/cm², veratridine+TTX, n = 11-23, P<0.001) and Substance P (42 ± 13 µA/cm², Substance P, 9 ± 3 µA/cm² Substance P+TTX, n = 6, P<0.05), TTX reduced but did not fully block light-induced I_sc (73 ± 20 µA/cm² control, 21 ± 5 TTX µA/cm², n=18, P<0.0001). 5-HT receptor antagonists (5HTRa) had no effect on light-induced I_sc (23 ± 6 µA/cm², control, 17 ± 3, 5HTRa, n = 8, P>0.05). However, when TTX and 5HTRa were delivered together, the light induced I_sc was completely blocked (19 ± 5 µA/cm², control, 1.3 ± 0.4 µA/cm², TTX+5HTRa, n = 9, P<0.01).
CONCLUSIONS: Optogenetic stimulation of ECs represents a novel approach to understand mechanisms of EC-initiated ion transport. Optogenetic stimulation of ECs induce mucosal chloride secretion via a combination of neurogenic and paracrine signaling from ECs to secretory epithelial cells.
Supported by NIH AT010875, DK123549, DK129315 and NS118790

Background: Intricate connections exit between dysbiotic microbiome dominated by facultative anaerobes and inflammatory bowel diseases (IBD). Data from Baumler and colleagues support a model where mitochondrial (mito) dysfunction causes disease-associated dysbiosis by increasing oxygen (O2) availability to the microbiome. Here, we posit that a novel compound (AuPhos) restores mito respiration in intestinal epithelial cells (IECs) thereby reducing O2 availability to the microbiome to promote a healthy anaerobic environment (e.g. firmicute-bloom). Methods: The effect of AuPhos on the microbiome was tested in acute (2% DSS-7d) colitis (C57/BL/6) and in germ-free IL10 KO reconstituted with human IBD stool to induce IBD-associated dysbiosis (Hu-IL10 KO; Rousta et al., Nutrients, 2021). Mice were treated with AuPhos (25mg/kg) or vehicle (q3d; n=8/group), and colon and stool samples collected at different time-points. Blinded histological scoring (d32) on Hu-IL10-/- mice was performed and fecal lipocalin-2 (LCN2) levels (d0-d32) determined. Microbial DNA was isolated from stool samples followed by 16S rRNA metagenomic sequencing. Differentially abundant bacterial species and functional potentials of bacterial communities were assessed by Linear Discriminant Analysis (LDA) and PICRUST2, respectively. AuPhos-induced hypoxia in IECs was assessed by Hypoxyprobe-1 staining in sections from pimonidazole HCl-infused DSS-mice. Immunohistochemistry of mito complex I (NDUFB6) was performed on colon sections from Hu-IL10 KO mice. Results: While Hu-IL10 KO mice showed marked histologic inflammation, transmural colitis scores and fecal LCN2 levels in AuPhos-fed Hu-IL10 KO mice significantly decreased (Veh. vs AuPhos; *p<0.05) at d32. Metagenomic (16S) analysis of stool samples from DSS-colitis and Hu-IL10-/- mice showed reduction in relative abundance of (O2 consuming) Proteobacteria, which includes facultatively anaerobic Enterobacteriaceae family, with concomitant increase in obligate anaerobes e.g. Firmicutes including Clostridia (Faecalibacterium prausnitzii, Roseburia sp.), Bifidobacterium, etc. in AuPhos-fed mice. PICRUST2 and LDA revealed that AuPhos decreased bacterial LPS biosynthetic pathway and increased overall fatty acid biosynthesis pathways in AuPhos-fed DSS-colitis mice. Interestingly, hypoxyprobe staining showed AuPhos-induced hypoxia in luminal surface IECs, facilitating obligate anaerobe-promoting environment in the gut. Concomitantly, AuPhos significantly (3-fold; *p<0.05) increased NDUFB6 expression in colonic IECs of Hu-IL10 KO mice compared to vehicle controls. Conclusion: These finding suggest that AuPhos is a “first-in-class” oral therapeutic that corrects microbial dysbiosis in IBD by enhancing mitochondrial respiration and oxygen utilization (i.e. inducing IEC hypoxia) thereby promoting a healthy microbiome dominated by obligate anaerobes.