Putrescine supplementation limits the expansion of pks+ Escherichia coli and tumor development in the colon.

Escherichia coli that harbor the polyketide synthase (pks) genomic island produce colibactin and are associated with sporadic colorectal cancer development (CRC). Given the considerable prevalence of pks+ bacteria in healthy individuals, we sought to identify strategies to limit the growth and expansion of pks+ E. coli. We found that culture supernatants of the probiotic strain E. coli Nissle 1917 were able to inhibit the growth of the murine pathogenic strain pks+ E. coli NC101 (EcNC101). We performed a non-targeted analysis of the metabolome in supernatants from several E. coli strains and identified putrescine as a potential postbiotic capable of suppressing EcNC101 growth in vitro. The effect of putrescine supplementation was then evaluated in the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of CRC in mice colonized with EcNC101. Putrescine supplementation inhibited the growth of pks+ E. coli; reduced the number and size of colonic tumors; and downmodulated the release of inflammatory cytokines in the colonic lumen. Additionally, putrescine supplementation led to shifts in the composition and function of gut microbiota, characterized by an increase of the Firmicutes/Bacteroidetes ratio and enhanced acetate production. The effect of putrescine was further confirmed in vitro using a pks+ E. coli strain isolated from a CRC patient. These results suggest that probiotic-derived metabolites can be used as an alternative to live bacteria in individuals at risk of developing CRC due to the presence of pks+ bacteria in their colon.

Modulating Gut Microbiota Prevents Anastomotic Leak to Reduce Local Implantation and Dissemination of Colorectal Cancer Cells after Surgery.

PURPOSE: Anastomotic leak (AL) is a major complication in colorectal cancer surgery and consists of the leakage of intestinal content through a poorly healed colonic wound. Colorectal cancer recurrence after surgery is a major determinant of survival. We hypothesize that AL may allow cancer cells to escape the gut and lead to cancer recurrence and that improving anastomotic healing may prevent local implantation and metastatic dissemination of cancer cells. EXPERIMENTAL DESIGN: We investigated the association between AL and postoperative outcomes in patients with colorectal cancer. Using mouse models of poor anastomotic healing, we assessed the processes of local implantation and dissemination of cancer cells. The effect of dietary supplementation with inulin and 5-aminosalicylate (5-ASA), which activate PPAR-γ in the gut, on local anastomotic tumors was assessed in mice undergoing colonic surgery. Inulin and 5-ASA were also assessed in a mouse model of liver metastasis. RESULTS: Patients experiencing AL displayed lower overall and oncologic survival than non-AL patients. Poor anastomotic healing in mice led to larger anastomotic and peritoneal tumors. The microbiota of patients with AL displays a lower capacity to activate the antineoplastic PPAR-γ in the gut. Modulation of gut microbiota using dietary inulin and 5-ASA reinforced the gut barrier and prevented anastomotic tumors and metastatic spread in mice. CONCLUSIONS: Our findings reinforce the hypothesis that preventing AL is paramount to improving oncologic outcomes after colorectal cancer surgery. Furthermore, they pave the way toward dietary targeting of PPAR-γ as a novel way to enhance healing and diminish cancer recurrence.

Inulin impacts tumorigenesis promotion by colibactin-producing Escherichia coli in ApcMin/+ mice.

Introduction: The prebiotic inulin has previously shown both protective and tumor-promoting effects in colorectal cancer (CRC). These inconsistencies may be due to the gut microbial composition as several bacteria have been associated with CRC. Specifically, polyketide synthase-positive (pks+) Escherichia coli promotes carcinogenesis and facilitates CRC progression through the production of colibactin, a genotoxin that induces double-strand DNA breaks (DSBs). We investigated whether colibactin-producing Escherichia coli changed the protection conferred by inulin against tumor growth and progression using the ApcMin/+ mouse model of CRC. Methods: Mice received a 2% dextran sodium sulfate (DSS) solution followed by oral gavage with the murine pks + E. coli strain NC101 (EcNC101) and were fed a diet supplemented with 10% cellulose as control or 10% inulin for 4 weeks. Results: Inulin supplementation led to increase EcNC101 colonization compared to mice receiving the control diet. The increased colonization of EcNC101 resulted in more DSBs, tumor burden, and tumor progression in ApcMin/+ mice. The tumorigenic effect of EcN101 in ApcMin/+ mice mediated by inulin was dependent on colibactin production. Pasteurized E. coli Nissle 1917 (EcN), a probiotic, suppressed the inulin-driven EcNC101 expansion and impacted tumor progression. Discussion: Our results suggest that the presence of pks + E. coli influences the outcome of inulin supplementation in CRC and that microbiota-targeted interventions may mitigate this effect. Given the prevalence of pks + E. coli in both healthy and CRC populations and the importance of a fiber-rich diet, inulin supplementation in individuals colonized with pks + bacteria should be considered with caution.

Gut microbiota influence anastomotic healing in colorectal cancer surgery through modulation of mucosal proinflammatory cytokines.

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.

Prevalence of pks + bacteria and enterotoxigenic Bacteroides fragilis in patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most diagnosed cancer and the second most common cause of cancer deaths worldwide. CRC patients present with an increase in pathogens in their gut microbiota, such as polyketide synthase-positive bacteria (pks +) and enterotoxigenic Bacteroides fragilis (ETBF). The pks + Escherichia coli promotes carcinogenesis and facilitates CRC progression through the production of colibactin, a genotoxin that induces double-strand DNA breaks (DSBs). ETBF is a procarcinogenic bacterium producing the B. fragilis toxin (bft) that promotes colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. METHODS: Fecal samples were collected from healthy controls (N = 62) and CRC patients (N = 94) from the province of Québec (Canada), and a bacterial DNA extraction was performed. Fecal DNA samples were then examined for the presence of the pks island gene and bft using conventional qualitative PCR. RESULTS: We found that a high proportion of healthy controls are colonized by pks + bacteria (42%) and that these levels were similar in CRC patients (46%). bft was detected in 21% of healthy controls and 32% of CRC patients, while double colonization by both pks + bacteria and ETBF occurred in 8% of the healthy controls and 13% of the CRC patients. Most importantly, we found that early-onset CRC (< 50 years) patients were significantly less colonized with pks + bacteria (20%) compared to late-onset CRC patients (52%). CONCLUSIONS: Healthy controls had similar levels of pks + bacteria and ETBF colonization as CRC patients, and their elevated levels may place both groups at greater risk of developing CRC. Colonization with pks + bacteria was less prevalent in early-compared to late-onset CRC.

The amino acid sensor GCN2 controls red blood cell clearance and iron metabolism through regulation of liver macrophages.

GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that controls messenger RNA translation in response to amino acid availability and ribosome stalling. Here, we show that GCN2 controls erythrocyte clearance and iron recycling during stress. Our data highlight the importance of liver macrophages as the primary cell type mediating these effects. During different stress conditions, such as hemolysis, amino acid deficiency or hypoxia, GCN2 knockout (GCN2-/-) mice displayed resistance to anemia compared with wild-type (GCN2+/+) mice. GCN2-/- liver macrophages exhibited defective erythrophagocytosis and lysosome maturation. Molecular analysis of GCN2-/- cells demonstrated that the ATF4-NRF2 pathway is a critical downstream mediator of GCN2 in regulating red blood cell clearance and iron recycling.

Oral iron supplementation after antibiotic exposure induces a deleterious recovery of the gut microbiota.

BACKGROUND: Oral iron supplementation is commonly prescribed for anemia and may play an important role in the gut microbiota recovery of anemic individuals who received antibiotic treatment. This study aims to investigate the effects of iron supplementation on gut microbiota recovery after antibiotics exposure. RESULTS: Mice were subjected to oral antibiotic treatment with neomycin and metronidazole and were fed diets with different concentrations of iron. The composition of the gut microbiota was followed throughout treatment by 16S rRNA sequencing of DNA extracted from fecal samples. Gut microbiota functions were inferred using PICRUSt2, and short-chain fatty acid concentration in fecal samples was assessed by liquid-chromatography mass spectrometry. Iron supplementation after antibiotic exposure shifted the gut microbiota composition towards a Bacteroidetes phylum-dominant composition. At the genus level, the iron-supplemented diet induced an increase in the abundance of Parasutterella and Bacteroides, and a decrease of Bilophila and Akkermansia. Parasutterella excrementihominis, Bacteroides vulgatus, and Alistipes finegoldii, were more abundant with the iron excess diet. Iron-induced shifts in microbiota composition were accompanied by functional modifications, including an enhancement of the biosynthesis of primary bile acids, nitrogen metabolism, cyanoamino acid metabolism and pentose phosphate pathways. Recovery after antibiotic treatment increased propionate levels independent of luminal iron levels, whereas butyrate levels were diminished by excess iron. CONCLUSIONS: Oral iron supplementation after antibiotic therapy in mice may lead to deleterious changes in the recovery of the gut microbiota. Our results have implications on the use of oral iron supplementation after antibiotic exposure and justify further studies on alternative treatments for anemia in these settings.

Improvement of colonic healing and surgical recovery with perioperative supplementation of inulin and galacto-oligosaccharides.

BACKGROUND AND AIMS: Anastomotic leak (AL) is a major complication in colorectal surgery. Recent evidence suggests that the gut microbiota may affect healing and may cause or prevent AL. Butyrate is a beneficial short-chain fatty acid (SCFA) that is produced as a result of bacterial fermentation of dietary oligosaccharides and has been described as beneficial in the maintenance of colonic health. To assess the impact of oligosaccharides on colonic anastomotic healing in mice, we propose to modulate the microbiota with oligosaccharides to increase butyrate production via enhancement of butyrate-producing bacteria and, consequently, improve anastomotic healing in mice. METHODS: Animal experiments were conducted in mice that were subjected to diets supplemented with inulin, galacto-oligosaccharides (GOS) or cellulose, as a control, for two weeks before undergoing a surgical colonic anastomosis. Macroscopic and histological assessment of the anastomosis was performed. Extent of epithelial proliferation was assessed by Ki-67 immunohistochemistry. Gelatin zymography was used to evaluate the extent of matrix metalloproteinase (MMP) hydrolytic activity. RESULTS: Inulin and GOS diets were associated with increased butyrate production and better anastomotic healing. Histological analysis revealed an enhanced mucosal continuity, and this was associated with an increased re-epithelialization of the wound as determined by increased epithelial proliferation. Collagen concentration in peri-anastomotic tissue was higher with inulin and GOS diets and MMP activity, a marker of collagen degradation, was lower with both oligosaccharides. Inulin and GOS diets were further associated with lower bacterial translocation. CONCLUSIONS: Dietary supplementation with inulin and GOS may improve anastomotic healing and reinforce the gut barrier in mice.

Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains.

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.

The role of butyrate in surgical and oncological outcomes in colorectal cancer.

Butyrate is a short-chain fatty acid produced by colonic gut bacteria as a result of fermentation of dietary fibers. In the colon, butyrate is a major energy substrate and contributes to the nutritional support and proliferation of a healthy mucosa. It also promotes the intestinal barrier function by enhancing mucus production and tight junctions. In addition to its pro-proliferative effect in healthy colonocytes, butyrate inhibits the proliferation of cancer cells. The antineoplastic effect of butyrate is associated with the inhibitory effect of butyrate on histone deacetylase (HDAC) enzymes, which promote carcinogenesis. Due to the metabolic shift of cancer cells toward glycolysis, unused butyrate accumulates and inhibits procarcinogenic HDACs. In addition, recent studies suggest that butyrate may improve the healing of colonic tissue after surgery in animal models, specifically at the site of reconnection of colonic ends, anastomosis, after surgical resection. Here, we review current evidence on the impact of butyrate on epithelial integrity and colorectal cancer and present current knowledge on data that support its potential applications in surgical practice.

Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis.

Iron homeostasis is an essential biological process that ensures the tissue distribution of iron for various cellular processes. As the major producer of hepcidin, the liver is central to the regulation of iron metabolism. The liver is also home to many immune cells, which upon activation may greatly impact iron metabolism. Here, we focus on the role of invariant natural killer T (iNKT) cells, a subset of T lymphocytes that, in mice, is most abundant in the liver. Activation of iNKT cells with the prototypical glycosphingolipid antigen, α-galactosylceramide, resulted in immune cell proliferation and biphasic changes in iron metabolism. This involved an early phase characterized by hypoferremia, hepcidin induction and ferroportin suppression, and a second phase associated with strong suppression of hepcidin despite elevated levels of circulating and tissue iron. We further show that these changes in iron metabolism are fully dependent on iNKT cell activation. Finally, we demonstrate that the biphasic regulation of hepcidin is independent of NK and Kupffer cells, and is initially driven by the STAT3 inflammatory pathway, whereas the second phase is regulated by repression of the BMP/SMAD signaling pathway. These findings indicate that iNKT activation and the resulting cell proliferation influence iron homeostasis.

Current evidence on the relation between gut microbiota and intestinal anastomotic leak in colorectal surgery.

BACKGROUND: Anastomotic leak (AL) is a major complication in colorectal surgery. It worsens morbidity, mortality and oncological outcomes in colorectal cancer. Some evidence suggests a potential effect of the intestinal microbiome on wound healing. This review aims to provide a comprehensive review on historical and current evidence regarding the relation between the gastrointestinal microbiota and AL in colorectal surgery, and the potential microbiota-modifying effect of some perioperative commonly used measures. DATA SOURCES: A comprehensive search was conducted in Pubmed, Medline and Embase for historical and current clinical and animal studies addressing perioperative intestinal microbiota evaluation, intestinal healing and AL. CONCLUSIONS: Evidence on microbes' role in AL is mainly derived from animal experiments. The microbiota's composition and implications are poorly understood in surgical patients. Elaborate microbiota sequencing is required in colorectal surgery to identify potentially beneficial microbial profiles that could lead to specific perioperative microbiome-altering measures and improve surgical and oncological outcomes.

Curcumin induces mild anemia in a DSS-induced colitis mouse model maintained on an iron-sufficient diet.

Anemia is frequently encountered in patients with inflammatory bowel disease (IBD), decreasing the quality of life and significantly worsening the prognosis of the disease. The pathogenesis of anemia in IBD is multifactorial and results mainly from intestinal blood loss in inflamed mucosa and impaired dietary iron absorption. Multiple studies have proposed the use of the polyphenolic compound curcumin to counteract IBD pathogenesis since it has significant preventive and therapeutic properties as an anti-inflammatory agent and very low toxicity, even at high dosages. However, curcumin has been shown to possess properties consistent with those of an iron-chelator, such as the ability to modulate proteins of iron metabolism and decrease spleen and liver iron content. Thus, this property may further contribute to the development and severity of anemia of inflammation and iron deficiency in IBD. Herein, we evaluate the effects of curcumin on systemic iron balance in the dextran sodium sulfate (DSS) model of colitis in C57Bl/6 and BALB/c mouse strains that were fed an iron-sufficient diet. In these conditions, curcumin supplementation caused mild anemia, lowered iron stores, worsened colitis and significantly decreased overall survival, independent of the mouse strain. These findings suggest that curcumin usage as an anti-inflammatory supplement should be accompanied by monitoring of erythroid parameters to avoid exacerbation of iron deficiency anemia in IBD.

MyD88 Regulates the Expression of SMAD4 and the Iron Regulatory Hormone Hepcidin.

The myeloid differentiation primary response gene 88 (MyD88) is an adaptive protein that is essential for the induction of inflammatory cytokines through almost all the Toll-like receptors (TLRs). TLRs recognize molecular patterns present in microorganisms called pathogen-associated molecular patterns. Therefore, MyD88 plays an important role in innate immunity since its activation triggers the first line of defense against microorganisms. Herein, we describe the first reported role of MyD88 in an interconnection between innate immunity and the iron-sensing pathway (BMP/SMAD4). We found that direct interaction of MyD88 with SMAD4 protein activated hepcidin expression. The iron regulatory hormone hepcidin is indispensable for the intestinal regulation of iron absorption and iron recycling by macrophages. We show that MyD88 induces hepcidin expression in a manner dependent on the proximal BMP responsive element on the hepcidin gene (HAMP) promoter. We identified the Toll/interleukin-1 receptor (TIR) domain of MyD88 as the domain of interaction with SMAD4. Furthermore, we show that BMP6 stimulation, which activates SMAD6 expression, also induces MyD88 proteosomal degradation as a negative feedback mechanism to limit hepcidin induction. Finally, we report that the MyD88 gain-of-function L265P mutation, frequently encountered in B-cell lymphomas such as Waldenström's macroglobulinemia, enhances hepcidin expression and iron accumulation in B cell lines. Our results reveal a new potential role for MyD88 in the SMAD signaling pathway and iron homeostasis regulation.

MyD88 Adaptor Protein Is Required for Appropriate Hepcidin Induction in Response to Dietary Iron Overload in Mice.

Iron homeostasis is tightly regulated to provide virtually all cells in the body, particularly red blood cells, with this essential element while defending against its toxicity. The peptide hormone hepcidin is central to the control of the amount of iron absorbed from the diet and iron recycling from macrophages. Previously, we have shown that hepcidin induction in macrophages following Toll-like receptor (TLR) stimulation depends on the presence of myeloid differentiation primary response gene 88 (MyD88). In this study, we analyzed the regulation of iron metabolism in MyD88-/- mice to further investigate MyD88 involvement in iron sensing and hepcidin induction. We show that mice lacking MyD88 accumulate significantly more iron in their livers than wild-type counterparts in response to dietary iron loading as they are unable to appropriately control hepcidin levels. The defect was associated with inappropriately low levels of Smad4 protein and Smad1/5/8 phosphorylation in liver samples found in the MyD88-/- mice compared to wild-type mice. In conclusion, our results reveal a previously unknown link between MyD88 and iron homeostasis, and provide new insights into the regulation of hepcidin through the iron-sensing pathway.

Dietary Heme Induces Gut Dysbiosis, Aggravates Colitis, and Potentiates the Development of Adenomas in Mice.

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 α-diversity, a reduction of Firmicutes and an increase of Proteobacteria, particularly Enterobacteriaceae. These changes were similar to shifts seen in dextran sodium sulfate (DSS)-treated mice to induce colitis. In addition, dietary heme, but not systemically delivered heme, contributed to the exacerbation of DSS-induced colitis and facilitated adenoma formation in the azoxymethane/DSS colorectal cancer (CRC) mouse model. Using inferred metagenomics, 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 levels was found in mice fed the heme supplemented diet compared to mice fed the control 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 toward favoring 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 approaches aimed at lowering gastrointestinal heme levels through heme chelation or degradation using probiotics and nutritional interventions.

The hemochromatosis protein HFE 20 years later: An emerging role in antigen presentation and in the immune system.

INTRODUCTION: Since its discovery, the hemochromatosis protein HFE has been primarily defined by its role in iron metabolism and homeostasis, and its involvement in the genetic disease termed hereditary hemochromatosis (HH). While HH patients are typically afflicted by dysregulated iron levels, many are also affected by several immune defects and increased incidence of autoimmune diseases that have thereby implicated HFE in the immune response. Growing evidence has supported an immunological role for HFE with recent studies describing HFE specifically as it relates to MHC I antigen presentation. METHODS/RESULTS: Here, we present a comprehensive overview of the relationship between iron metabolism, HFE, and the immune system to better understand the origin and cause of immune defects in HH patients. We further describe the role of HFE in MHC I antigen presentation and its potential to impair autoimmune responses in homeostatic conditions, a mechanism which may be exploited by tumors to evade immune surveillance. CONCLUSION: Overall, this increased understanding of the role of HFE in the immune response sets the stage for better treatment and management of HH and other iron-related diseases, as well as of the immune defects related to this condition.

Iron Supplements Modulate Colon Microbiota Composition and Potentiate the Protective Effects of Probiotics in Dextran Sodium Sulfate-induced Colitis.

BACKGROUND: Iron is an important nutrient for both the host and colonizing bacteria. Oral iron supplementation may impact the composition of the microbiota and can be particularly damaging to patients suffering from inflammatory bowel disease (IBD). However, patients with IBD may require iron supplementation to treat their anemia. METHODS: We fed mice with diets supplemented with ferrous sulfate at different doses (5, 50, and 500 mg of iron/kg chow) and with different iron formulations (ferrous sulfate, ferrous bisglycinate and ferric ethylenediaminetetraacetic acid [FEDTA]), and analyzed the effects on the composition of the gut microbiota by 16S ribosomal RNA gene sequencing. Using the dextran sodium sulfate (DSS)-induced colitis mouse model, we investigated the effects of iron supplementation in colitis severity, as well as the use of the probiotic Escherichia coli Nissle 1917 (EcN) in combination with iron supplementation. RESULTS: Iron supplementation at different doses induced shifts in the gut microbial communities and inferred metabolic pathways. However, depending on the iron formulation used in the diets, iron supplementation during dextran sodium sulfate-induced colitis was either beneficial (ferrous bisglycinate) or highly detrimental (FEDTA). Finally, the beneficial effect of the probiotic EcN in the dextran sodium sulfate-induced colitis model was potentiated by oral iron supplementation with ferrous sulfate. CONCLUSIONS: These results show that the iron formulations used to treat iron deficiency influence the gut microbiota and colitis in mice and suggest that distinct iron compounds may be of particular relevance to patients with IBD. In addition, the beneficial action of probiotics in IBD may be enhanced by oral iron supplementation.