Chronic Transfusion and Iron Overload Modify the Mouse Gut Microbiome

BACKGROUND: Although iron is an essential element in critical metabolic pathways of both pathogenic microorganisms and their hosts it is less essential in certain barrier bacteria, such as Lactobacilli. Dietary iron supplementation increases mortality and affects the microbiome in African children, decreasing the abundance of beneficial Lactobacilli. Furthermore, the method of iron repletion influences the microbiome in patients with inflammatory bowel disease.

AIM: To determine whether iron status and different methods of iron supplementation/overload affect the gut microbiome in mice.

METHODS: Cohorts of iron-deficient, iron-replete, and iron-overloaded wild-type C57BL/6 female mice (n=5 per group) were generated by dietary manipulation and by injection of iron dextran (0.3mg weekly x 6) or RBC transfusion (0.3mL at 60% hematocrit weekly x 6). The day after the last injection/transfusion, mice were sacrificed and tissues (blood, liver, spleen, and duodenum) and feces, from the cecum and rectum, were collected. Iron levels in tissues and in rectal feces were quantified by a wet ashing procedure. Commercial ELISA kits were used to quantify circulating hepcidin and ferritin levels. DNA from feces was extracted using the Fecal DNA extraction kit (Mo Bio) and sent to the Molecular Research (MRDNA) center for 16S rDNA Illumina platform sequencing and analysis. Statistical analyses were performed using LEfSe database (https://huttenhower.sph.harvard.edu/galaxy/) and GraphPad Prism.

RESULTS: Mice fed an iron-deficient diet from weaning developed iron deficiency anemia with decreased intracellular iron stores, as measured by serum ferritin and liver and spleen iron (see Table). Iron dextran injections induced iron overload in mice fed either an iron deficient or iron replete diet. Chronic transfusion induced iron overload in mice fed an iron replete diet, but led to iron repletion without overload in mice fed an iron deficient diet. The iron deficient diet decreased, whereas the iron supplemented diet increased, fecal iron significantly. Although, iron dextran injections and chronic transfusion increased hepcidin levels, they did not significantly affect fecal iron. Analysis of microbiome data showed that fecal iron modulated the relative abundance of different bacteria. The phylum Proteobacteria showed a negative trend with increasing fecal iron associated with decreasing relative abundance (R² 0.5; p<0.006), Firmicutes showed a positive trend with increasing fecal iron associated with increasing relative abundance (R² 0.3; p<0.02), whereas the phylum Bacteroidetes did not show a significant association. Within the phylum Firmicutes, fecal iron concentration was a reasonable predictor of family Lactobacillaceae abundance (R² 0.5; p=0.005), with increasing iron reducing the relative abundance; in contrast, increasing iron was associated with increased relative abundance of family Clostridiaceae (R² 0.7; p<0.0001). We next investigated whether iron dextran infusions, chronic transfusions, or oral iron supplementation modulated microbiome composition. Analysis of the families belonging to class Clostrida showed that family Clostridiaceae increased with an iron supplemented diet, iron dextran infusions, or transfusional iron overload; family Eubacteriaceae increased with iron dextran and blood transfusions, but not with the iron supplemented diet, and family Peptococcaceae only increased with iron dextran treatment. These results suggest that different methods of iron supplementation or overload affect families in the class of Clostrida differently. Finally,comparisons of the cecal and rectal microbiomes did not identify any substantial differences.

CONCLUSIONS: In this study, iron status modified the microbiome in mice. The microbiome was further modulated by different types of iron overload, especially in the class Clostrida. Similar to human studies, increasing fecal iron decreases the abundance in the gut of potentially beneficial lactobacilli. Although there are differences between mouse and human gut microbiomes, this mouse model can be used to study the effects of iron supplementation strategies and iron overload and can provide the foundation for further studies focused on the role of iron in host-pathogen interactions and immune function.

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