Define mechanisms by which host-microbe metabolite exchange regulates cellular and system iron homeostasis. - PROJECT SUMMARY Intestinal iron absorption is the major mechanism of iron delivery in mammals. Cellular or systemic iron deficiency can lead to dysregulation of metabolic pathways and anemic disorders. However, excess iron such as in hemochromatosis can lead to tissue injury and cell death. Therefore, systemic and cellular iron levels are tightly regulated. Due to the reactive nature of iron, iron is stored in ferritin, a protein capable of binding up to 4500 iron atoms. In conditions of high iron demand or deficiency, ferritin is degraded, releasing iron for cellular and systemic utilization. Ferritin is a critical protein that controls cellular iron, intestinal iron absorption, and oxidative metabolic pathways. Moreover, ferritin is essential in nutritional immunity, sequestering iron from microbial pathogens that require it for growth. Notably, in germ-free (GF) or antibiotic treated (Abx) mice, we observed a significant decrease of ferritin expression both locally in the intestine and in peripheral tissues. These findings indicate a dynamic regulatory role of host cells in modulating their iron storage pathways in response to commensal bacteria. We demonstrated that ferritin levels were restored in germ-free (GF) or antibiotic treated (Abx) mice upon treatment with microbial metabolites. This data suggests a metabolic exchange between commensals and epithelial cells that drives the ferritin response. Specifically, a synthetic community composed of 11 gram-negative and gram-positive commensals successfully restored intestinal ferritin expression in germ- free mice. Despite assessing several canonical mechanisms of ferritin regulation, including mRNA levels, autophagic degradation of ferritin (ferritinophagy), and hypoxic pathways, no changes were observed. We found altered activity of iron regulatory proteins (IRPs) in germ-free mice compared to wild type. IRPs are RNA binding proteins that regulate ferritin translation. I hypothesize microbial metabolites suppress mRNA binding activity of IRPs leading to an increase in host ferritin expression. However, studying microbial and intestinal epithelial cell interactions in vitro presents challenges due to the anoxic conditions required to grow anaerobes. Our ongoing work aims to elucidate the precise mechanism of microbial regulation of host ferritin levels and identify microbes and metabolites capable of inducing ferritin expression. I will test this hypothesis through two aims. Aim 1 will identify microbe(s) and their metabolites capable of inducing ferritin expression. This will be done using an asymmetric co-culture system that allows us to culture anaerobic bacteria adjacent to primary intestinal epithelial cells in an environment mimicking the intestine. Aim 2 will determine the cellular mechanism of microbial regulation of host ferritin. I will utilize conditional knockout mice for IRPs to determine if ferritin translation is regulated by microbes. Understanding the mechanism of microbial integration into host ferritin regulation is crucial for defining precise mechanisms by which microbiota regulate intestinal iron absorption, iron related disorders and in microbial pathogenesis.
