Dietary heme can be used by colonic bacteria equipped with heme-uptake systems as a growth factor and thereby impact on the microbial community structure. The impact of heme on the gut microbiota composition may be particularly pertinent in chronic inflammation such as in inflammatory bowel disease (IBD), where a strong association with gut dysbiosis has been consistently reported. In this study we investigated the influence of dietary heme on the gut microbiota and inferred metagenomic composition, and on chemically-induced colitis and colitis-associated adenoma development in mice. Using 16S rRNA gene sequencing, we found that mice fed a diet supplemented with heme significantly altered their microbiota composition, characterized by a decrease in -diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes where similar to shifts seen in dextran sodium sulfate (DSS) -treated mice to induce colitis. In addition, dietary heme contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the Azoxymethane/DSS colorectal mouse model. Using inferred metagenomics (PICRUSt), we found that the microbiota alterations elicited by dietary heme resulted in non-beneficial functional shifts, which were also characteristic of DSS-induced colitis. Furthermore, a reduction in fecal butyrate was found in mice fed the heme supplemented diet compared to mice fed the standard diet. Iron metabolism genes known to contribute to heme release from red blood cells, heme uptake, and heme exporter proteins, were significantly enriched, indicating a shift towards favouring the growth of bacteria able to uptake heme and protect against its toxicity. In conclusion, our data suggest that luminal heme, originating from dietary components or gastrointestinal bleeding in IBD and, to lesser extent in CRC, directly contributes to microbiota dysbiosis. Thus, luminal heme levels may further exacerbate colitis through the modulation of the gut microbiota and its metagenomic functional composition. Our data may have implications in the development of novel targets for therapeutic intervention aimed at lowering gastrointestinal heme levels through heme chelation or degradation using probiotics and nutritional interventions.

Objective: Colorectal cancer (CRC) is the third most diagnosed cancer, and requires surgical resection and reconnection, or anastomosis, of the remaining bowel to re-establish intestinal continuity. Anastomotic leak (AL) is a major complication that increases mortality and cancer recurrence. Our objective is to assess the causal role of gut microbiota in anastomotic healing. Design: The causal role of gut microbiota was assessed in a murine AL model receiving faecal microbiota transplantation (FMT) from patients with CRC collected before surgery and who later developed or not, AL. Anastomotic healing and gut barrier integrity were assessed after surgery. Bacterial candidates implicated in anastomotic healing were identified using 16S rRNA gene sequencing and were isolated from faecal samples to be tested both in vitro and in vivo. Results: Mice receiving FMT from patients that developed AL displayed poor anastomotic healing. Profiling of gut microbiota of patients and mice after FMT revealed correlations between healing parameters and the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementation with A. onderdonkii resulted in a higher rate of leaks in mice, while gavage with P. goldsteinii improved healing by exerting an anti-inflammatory effect. Patients with AL and mice receiving FMT from AL patients presented upregulation of mucosal MIP-1α, MIP-2, MCP-1 and IL-17A/F before surgery. Retrospective analysis revealed that patients with AL present higher circulating neutrophil and monocyte counts before surgery. Conclusion: Gut microbiota plays an important role in surgical colonic healing in patients with CRC. The impact of these findings may extend to a vast array of invasive gastrointestinal procedures.

Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Hepcidin is stimulated when iron stores increase and during inflammation and, conversely, is inhibited by hypoxia and augmented erythropoiesis. In many pathologic situations, such as in the anemia of chronic disease (ACD) and iron-loading anemias, several of these factors may be present concomitantly and may generate opposing signaling to regulate hepcidin expression. Here, we address the question of dominance among the regulators of hepcidin expression. We show that erythropoiesis drive, stimulated by erythropoietin but not hypoxia, down-regulates hepcidin in a dose-dependent manner, even in the presence of lipopolysaccharide (LPS) or dietary iron-loading, which may act additively. These effects are mediated through down-regulation of phosporylation of Stat3 triggered by LPS and of Smad1/5/8 induced by iron. In conclusion, hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli rather than by an absolute hierarchy among signaling pathways. Our findings also suggest that erythropoietic drive can inhibit both inflammatory and iron-sensing pathways, at least in part, via the suppression of STAT3 and SMAD4 signaling in vivo.

Hepcidin is a negative regulator of iron absorption produced mainly by the liver in response to changes in iron stores and inflammation, and its levels have been shown to regulate the intestinal basolateral iron transporter ferroportin1 ( Fp1). Hereditary hemochromatosis patients and Hfe-deficient mice show inappropriate expression of hepcidin but, in apparent contradiction, still retain the ability to regulate iron absorption in response to alterations of iron metabolism. To further understand the molecular relationships among Hfe, hepcidin, and Fp1, we investigated hepcidin and Fp1 regulation in Hfe-deficient mice ( Hfe^−/−and β2m^−/−) in response to iron deprivation, iron loading, and acute inflammation. We found that whereas basal hepcidin levels were manifestly dependent on the presence of Hfe and on the mouse background, all Hfe-deficient mice were still able to regulate hepcidin in situations of altered iron homeostasis. In the liver, Fp1 was modulated in opposite directions by iron and LPS, and its regulation in Hfe-deficient mice was similar to that observed in wild-type mice. In addition, we found that iron-deprived mice were able to mount a robust response after LPS challenge and that Toll-like receptor 4 (TLR-4)-deficient mice fail to regulate hepcidin expression in response to LPS. In conclusion, these results suggest that although Hfe is necessary for the establishment of hepcidin basal levels, it is dispensable for hepcidin regulation through both the iron-sensing and inflammatory pathways, and hepatic Fp1 regulation is largely independent of hepcidin and Hfe. The inflammatory pathway overrides the iron-sensing pathway and is TLR-4 dependent.

Background: Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. Methods: Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. Results: Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. Conclusion: Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.