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.

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.

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.

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.