THE ROLE OF ARYL HYDROCARBON RECEPTOR SIGNALING IN POSTOPERATIVE INTESTINAL HEALING

The mammalian tissues express a huge number of circular RNAs (circRNAs) with an active role in gene regulation. circRNAs are covalently closed at the 5’ and 3’ ends, unusually stable, and are highly distributed in the cytoplasm. We recently reported that ~300 circRNAs, including Cdr1as, are differentially expressed in damaged intestinal mucosa in septic mice, but functions of most of these circRNAs in the intestinal epithelium remain unknown. In this study, we elucidated the in vivo function of the circRNA Cdr1as in the intestinal epithelium and identified Cdr1as as a repressor of mucosal regeneration. Methods: Cdr1as knockout (Cdr1as-/-) mice were generated by CRISPR-Cas9. Intestinal mucosa was collected from mice after injury and from patients with IBD and sepsis. Colonic mucosal injury was induced by 3% dextran sulfate sodium (DSS) in drinking water, while small intestinal mucosal injury was produced by cecal ligation and puncture or mesenteric ischemia/reperfusion (I/R). Primary enterocytes were isolated from the small intestine of mice and organoids were derived. Caco-2 cells were used for an in vitro intestinal epithelial injury model. Levels of Cdr1as were elevated in vitro and ex vivo by transfection with a lentiviral vector that expressed Cdr1as. Results: Colitis and septic stress increased the levels of intestinal mucosal Cdr1as in mice and human intestinal mucosa from patients with IBD and sepsis also exhibited increased Cdr1as abundance. After excluding the off-target genes, three founders of Cdr1as-/- mice were selected and shown to bear with a specific ablation of the Cdr1as locus. Loss of the Cdr1as locus from the mouse genome enhanced renewal of the intestinal mucosa, promoted injury-induced epithelial regeneration after exposure to I/R, and protected the mucosa against DSS-induced colitis. Cdr1as-/- mice also displayed deregulated expression of >40 microRNAs in the intestinal epithelium, including a significant decrease in miR-195 content. Increasing the levels of Cdr1as inhibited intestinal epithelial repair after wounding in vitro and repressed growth of intestinal organoids ex vivo but this inhibition was abolished by miR-195 silencing. Expression of miR-195 was posttranscriptionally misregulated in Cdr1as-/- mice by altering the stability of the miR-195 precursor (pre-miR-195) and its biogenesis. The half-life of pre-miR-195 increased in cells overexpressing Cdr1as, whereas the levels of Dicer complex proteins (essential for miRNA processing in the cytoplasm) decreased in the Cdr1as-deficient intestinal epithelium. Conclusions: These findings indicate that Cdr1as downregulates mucosal regeneration after injury and impairs epithelial defense via interaction with miR-195 and highlight a novel role of increased Cdr1as in the pathogenesis of chronic and unhealed wounds and disrupted renewal of the intestinal mucosa.

Enteric infections affect over 1.7 billion individuals annually. Microbial sensing and bacterial clearance by the host defense/immune system are important to maintain homeostasis. Alterations of this process are often associated with chronic inflammation. Previously, we have shown that the engulfment and cell motility protein 1 (ELMO1), belonging to the host engulfment pathway, also acts as a cytosolic microbial sensor and is involved in bacterial internalization and clearance. ELMO1 binds to the Brain Angiogenesis Inhibitor 1 (BAI1), which recognizes bacterial lipopolysaccharide and activates Rac1 to induce pro-inflammatory cytokine production. ELMO1 induces a differential immune response by binding to bacterial effectors with a signature WxxxE motif. Interestingly, WxxxE effectors are present only in pathogens but not in commensals. We hypothesized that ELMO1 interacts with other host signaling pathways to control the fate of the bacteria and chronic inflammation. Using Salmonella as a model of enteric pathogens, we found that the WxxxE effector SifA secreted by Salmonella binds to ELMO1 and impacts bacterial clearance and inflammation. ELMO1 also controls bacteria clearance through interaction with another microbial sensor, NOD2. ELMO1 with mutant NOD2, which have been linked to Crohn’s disease are associated with defective bacterial clearance and inflammatory responses. Similarly, single nucleotide polymorphisms of ELMO1 have been linked with several inflammatory diseases, including intestinal bowel diseases (IBDs). We demonstrated that ELMO1 expression is elevated in the colonic epithelium of Inflammatory Bowel Disease (IBD) patients, and also positively correlated with the elevated expression of pro-inflammatory cytokines. Furthermore, in the murine model of colitis, ELMO1 promotes the severity of colitis in DSS-treated mice. Organoid-derived monolayers from WT and ELMO1 KO mice and IBD patients also showed that ELMO1-mediated bacterial sensing is important for the release of early pro-inflammatory cytokines. ELMO1 also maintains the integrity of the Salmonella Containing Vacuole. SifA is reported to bind host endo-lysosomal proteins, including members of the Rab GTPase family, which allows Salmonella to subvert the host endo-lysosomal signaling and survive inside cells. Using immunoprecipitation and GST pulldown assay in control and ELMO1-depleted macrophages challenged with wild-type and Salmonella lacking SifA, we found that ELMO1 interacts with endo-lysosomal proteins Rab5, Rab9, and Rab14 and the interaction is impaired after infection. Our biochemical data and cell-based functional assays elucidate a new role of ELMO1 in manipulating the endos-lysosomal pathways and activation of the inflammatory response, thus regulating the fate of bacteria.

Mitochondria generate ATP as cellular energy and can also function as signaling organelles. Recent studies reveal that mitochondrial impairment is involved in the pathogenesis of IBD and other mucosal pathologies by lowering intestinal epithelial defense. Mitochondrial dysfunction by deleting prohibitin-1 (PHB1) disrupts intestinal epithelial integrity and leads to murine ileitis. HuR is a RNA-binding protein and is highly expressed in the intestinal epithelium. HuR acts as a key biological regulator of gut mucosal homeostasis by controlling stability and translation of the target mRNAs. Conditional deletion of HuR in intestinal epithelial cells inhibits epithelial renewal and delays mucosal healing after acute injury. HuR can physically interact with several microRNAs including miR-195 to jointly regulate gene expression synergistically or antagonistically. The current study was to test the hypothesis that HuR regulates mitochondrial function via interaction with miR-195. Methods: Studies were conducted in conditional HuR knockout (IE-HuR^-/-) mice, primarily cultured intestinal organoids, and Caco-2 cells. The functions of HuR and Phb1 were examined by their gene silencing or overexpression in vitro. Levels of mitochondrial proteins were determined by Western blotting analysis, while mitochondrial respiration was measured by seahorse analysis. The interactions of Phb1 mRNA with HuR or miR-195 were examined by RNP immunoprecipitation/qPCR analysis. Results: Intestinal epithelial tissue-specific HuR deletion in mice caused mitochondrial defects as indicated by a significant decrease in the levels of several mitochondrial proteins, including PHB1, Hsp60, GRSF1, VDAC, cyto-C, and PDHG, in the small intestinal mucosa of IE-HuR^-/- mice relative to control littermates. HuR deletion also inhibited growth of the small intestinal mucosa in vivo and ex vivo. Decreasing the levels of HuR by transfection of Caco-2 cells with specific siRNA targeting HuR also decreased the levels of these mitochondrial proteins and resulted in mitochondrial dysfunction. HuR silencing decreased mitochondrial respiration and reduced MitoTracker green but it increased superoxide production. This mitochondrial dysfunction in HuR-silenced cells was almost totally prevented by ectopically expressing PHB1 or Hsp60. The mechanistic study demonstrated that HuR did not directly bind to the Phb1 mRNA, but it interacted with miR-195 and formed a HuR/miR-195 complex, thus abolishing miR-195-induced inhibition of PHB1 expression. Conclusions: These results indicate that 1) HuR is essential for maintaining mitochondrial function in the intestinal epithelium; 2) HuR regulates PHB1 expression via interaction with miR-195; and 3) HuR promotes intestinal epithelial renewal at least partially through mitochondrial activation.

Intestinal anastomotic leakage (AL) is a complication occurring in up to 28% of colorectal surgical patients associated with a high mortality rate¹, yet its pathophysiology remains unclear. The Aryl hydrocarbon receptor (AhR) plays an essential role in regulating intestinal homeostasis upon activation with tryptophan(Trp)-derived agonists generated by microbiota^2-4. AhR activation in immune cells regulates the production of interleukin-22 (IL22) involved in mucosal healing. Inflammatory lesions were shown to be associated with altered Trp metabolism leading to reduced levels of AhR agonists and its downstream target IL22. We aimed to study AhR activation and IL22 production in the prevention of AL.
We studied the role of Trp metabolites and AhR in models of anastomotic healing (AH). AhR^-/- and WT mice underwent an intestinal anastomosis following validated protocols⁵ and post-operative recovery was monitored at 6, 24, and 120 hours using anastomotic complication score (ACS), with ACS≥3 representing AL⁵. To evaluate the importance of IL22 in AH, a neutralizing anti-IL22 antibody was injected intraperitoneal pre and post-operative. To alter the production of AhR agonists, WT mice were fed for 33 days with high/low Trp diet. To recover AhR activity, indole-3-carbinol (I3C) was gavaged prior and post-surgery. Finally, Trp metabolite levels were measured in preoperative fecal samples collected from patients undergoing colorectal resection (REVEAL study⁶) from which 34 healed and 19 developed AL.
We first showed increased AL symptoms at 120 hours (ACS:4-5) in AhR^-/- mice suggesting a key role of AhR in AH (ACS:0-2). The symptoms (ACS:3, p<0.0001) were already observed 24 hours post-surgery in AhR^-/- mice, indicating molecular involvement of AhR in the early phases of AH. Furthermore, we showed an AhR-dependent IL22 upregulation 6 and 24 hours after anastomosis. WT mice treated with the anti-IL22 antibody showed increased AL complications (aIL22; ACS:3 vs. ctrl; ACS:1, p=0.0004) confirming the essential role of IL22. Moreover, reduced AhR activation due to low AhR agonists levels, induced in low Trp diet-fed mice, promoted AL (low Trp; ACS:3 vs. high Trp; ACS:1, p<0.0001), indicating the role of AhR activation in AH. Finally, AL induced with low Trp diet was prevented by supplementation of I3C (I3C; ACS:1 vs. ctrl; ACS:3, p<0.0001). Notably, lower fecal levels (p=0.07) of indole-3-acetic acid (IAA) were also measured in AL patients compared to AH-matched patients, confirming our murine observations that altered Trp metabolism precedes AL in patients’ preoperative fecal microbiome.
Our results suggest that: (1) AhR deficiency and altered AhR-activity led to colonic AL; (2) supplementation of AhR agonists restored AH, (3) neutralization of IL22 leads to AL. These data indicate a possible beneficial mechanism of AhR/IL22 pathway activation in intestinal AH.