Modelling Systemic Iron Regulation during Dietary Iron Overload and Acute Inflammation: Role of Hepcidin-Independent Mechanisms

<div><p>Systemic iron levels must be maintained in physiological concentrations to prevent diseases associated with iron deficiency or iron overload. A key role in this process plays ferroportin, the only known mammalian transmembrane iron exporter, which releases iron from duodenal enterocytes, hepatocytes, or iron-recycling macrophages into the blood stream. Ferroportin expression is tightly controlled by transcriptional and post-transcriptional mechanisms in response to hypoxia, iron deficiency, heme iron and inflammatory cues by cell-autonomous and systemic mechanisms. At the systemic level, the iron-regulatory hormone hepcidin is released from the liver in response to these cues, binds to ferroportin and triggers its degradation. The relative importance of individual ferroportin control mechanisms and their interplay at the systemic level is incompletely understood. Here, we built a mathematical model of systemic iron regulation. It incorporates the dynamics of organ iron pools as well as regulation by the hepcidin/ferroportin system. We calibrated and validated the model with time-resolved measurements of iron responses in mice challenged with dietary iron overload and/or inflammation. The model demonstrates that inflammation mainly reduces the amount of iron in the blood stream by reducing intracellular ferroportin transcription, and not by hepcidin-dependent ferroportin protein destabilization. In contrast, ferroportin regulation by hepcidin is the predominant mechanism of iron homeostasis in response to changing iron diets for a big range of dietary iron contents. The model further reveals that additional homeostasis mechanisms must be taken into account at very high dietary iron levels, including the saturation of intestinal uptake of nutritional iron and the uptake of circulating, non-transferrin-bound iron, into liver. Taken together, our model quantitatively describes systemic iron metabolism and generated experimentally testable predictions for additional ferroportin-independent homeostasis mechanisms.</p></div

Experimental data used for model validation.

<p>The table summarizes the experimental perturbations, time scales and measured quantities for the different datasets used.</p

Model correctly predicts responses to perturbations in the SMAD4-hepcidin-pathway as well as development of anemia under chronic inflammation.

<p>A-Model quantitatively predicts experimentally measured responses for 2 months old C326S knock-in mice expressing a hepcidin-resistant FPN mutant or or SMAD4-knockout mice. The model simulations are shown as blue bars and the corresponding data from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.ref037" target="_blank">37</a>] and [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.ref053" target="_blank">53</a>] as red bars, respectively. Fold changes are referred to the wildtype levels. The model error bars are calculated from the predictions of the 30 best fitting parameter sets (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#pcbi.1005322.s001" target="_blank">S1 Text</a>). B-Model prediction for body iron pools when ferroportin regulation by hepcidin is out of action in one of the indicated organs. Shown are model simulations whithout experimental validation. C-Model qualitatively reproduces the development of anemia of inflammation upon chronic elevation of body LPS. Simulation of plasma, RBC and liver iron evolution when the inflammatory Il6/STAT pathway is permanently activated by a persistent LPS stimulus (0.17 <i>μg</i>/g body weight). Shown are model simulations without a quantitative comparison to experiments.</p

Iron overload as a consequence of an iron enriched diet leads to the preferential iron accumulation in the liver, which is quantitatively reproduced by the model considering NTBI uptake and liver ferritin storage.

<p>A-The measured distribution of iron between the organs is reproduced by the model fits for both normal and an iron enriched diet. The iron content of all compartments increases in mice maintained for 4 weeks on an iron rich diet, with most iron accumulating in the liver (arrows). B-Measured liver iron content under conditions of dietary iron overload and in HAMP-KO mice as well as best fit for the full model and models lacking NTBI uptake or liver ferritin storage, or both.</p

Experimental data used for model calibration.

<p>The table summarizes the experimental perturbations, time scales and measured quantities for the different datasets used.</p

LPS-induced dynamics of iron-related parameters under normal/enriched iron diet is well reproduced/predicted by the model.

<p>A-Serum iron, B-Liver Iron, C-Liver hepcidin, D-Spleen iron, E-Liver BMP6, F-Liver Fpn mRNA, G-Spleen Fpn protein, H-Liver pSTAT, I-Liver pSMAD, J-Duodenum iron, K-Red blood cells iron, L-Liver Fpn protein. 4–6 weeks old male C57BL/6-mice were administrated a normal diet, containing 200 ppm iron (blue), or a high iron diet, supplemented by 2% carbonyl iron containing about 20000 ppm iron (red). After 4 weeks, mice were injected with 1 <i>μg</i> LPS/g body weight and sacrificed 6/18/48 hours after the injection. Experimental data are given as means with standard deviation of 4–6 replicates and the model simulation for the best fitting parameter set is represented by curves (solid lines: fitted time courses, dashed lines: predicted time courses). Data represented be empty circles were used in fitting as a part of the calibration data set (LPS response for normal diet and the iron parameters after 4 weeks of high iron diet before injection of LPS). The LPS response for high iron diet data (filled circles) was used to test the model predictions. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005322#sec014" target="_blank">Materials and Methods</a> for the description of the experiment.</p

Hepcidin-mediated control and dietary uptake saturation are critical parameters for serum iron homeostasis under dietary iron overload.

<p>Simulation of steady state serum iron levels as a function of the dietary iron content for different model variants. For comparison, a linear increase of serum iron levels with increased dietary iron content is depicted.</p

Hepcidin-mediated Fpn control and inhibition of Fpn transcription contribute to the acute inflammatory response.

<p>A-Experimental data (means with standard deviation) and simulated data (lines) for serum iron content following peritoneal injection of LPS for mice maintained on a standard or iron-enriched diet. Comparison of the full model with models where either hepcidin-mediated ferroportin degradation or inflammation-mediated ferroportin mRNA reduction are removed. The simulations correspond to the best fitting parameter set. B-LPS-induced changes of liver ferroportin mRNA and protein levels relative to the normal diet steady state.</p