Genetic and Dietary Iron Overload Differentially Affect the Course of Salmonella Typhimurium Infection

Abstract

Genetic and dietary forms of iron overload have distinctive clinical and pathophysiological features. HFE-associated hereditary hemochromatosis is characterized by overwhelming intestinal iron absorption, parenchymal iron deposition, and macrophage iron depletion. In contrast, excessive dietary iron intake results in iron deposition in macrophages. However, the functional consequences of genetic and dietary iron overload for the control of microbes are incompletely understood. Using Hfe^+/+ and Hfe^-/- mice in combination with oral iron overload in a model of Salmonella enterica serovar Typhimurium infection, we found animals of either genotype to induce hepcidin antimicrobial peptide expression and hypoferremia following systemic infection in an Hfe-independent manner. As predicted, Hfe^-/- mice, a model of hereditary hemochromatosis, displayed reduced spleen iron content, which translated into improved control of Salmonella replication. Salmonella adapted to the iron-poor microenvironment in the spleens of Hfe^-/- mice by inducing the expression of its siderophore iron-uptake machinery. Dietary iron loading resulted in higher bacterial numbers in both WT and Hfe^-/- mice, although Hfe deficiency still resulted in better pathogen control and improved survival. This suggests that Hfe deficiency may exert protective effects in addition to the control of iron availability for intracellular bacteria. Our data show that a dynamic adaptation of iron metabolism in both immune cells and microbes shapes the host-pathogen interaction in the setting of systemic Salmonella infection. Moreover, Hfe-associated iron overload and dietary iron excess result in different outcomes in infection, indicating that tissue and cellular iron distribution determines the susceptibility to infection with specific pathogens.

Keywords: Salmonella; hepcidin; infection; iron; lipocalin; macrophage; siderophore.

Figure 1

Influence of Hfe, dietary iron challenge and Salmonella infection on systemic iron parameters. Hfe^−/− and congenic C57BL/6 WT animals (Hfe^+/+) were fed either a standard iron-adequate diet (IA) or an iron-enriched diet (IE) and infected i.p. with 500 CFU of S. Typhimurium (S. Tm.). Mock-infected controls (Ctrl.) received diluent. Serum iron (A) and ferritin (FT) levels (B) were measured after 48 h. In parallel, the expression of Hamp mRNA (C) in the liver was determined relative to the house-keeping gene Hprt by quantitative RT-PCR. Total liver iron content 48 post-infection was measured colorimetrically and normalized for wet tissue weight (D). Data were compared by means of ANOVA with Tukey's post hoc test. Values are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges. Statistical significant differences within each diet group are indicated. Additional letters represent statistically significant differences (P < 0.05) as follows: (a) Hfe^+/+ Ctrl. IA vs. Hfe^+/+ Ctrl. IE; (b) Hfe^−/− Ctrl. IA vs. Hfe^−/− Ctrl. IE; (c) Hfe^+/+ S. Tm. IA vs. Hfe^+/+ S. Tm. IE; Hfe^−/− S. Tm. IA vs. Hfe^−/− S. Tm. IE. n = 7–10 per group.

Figure 2

Influence of Hfe, dietary iron challenge and Salmonella infection on splenic iron parameters. Total spleen iron content (A) and mRNA levels of iron metabolic genes (B–G) in the spleen as determined by quantitative RT-PCR were measured after 48 h. Hamp (B), Fpn1 (C), Dmt1 (D), TfR1 (E), LcnR (F), and Lcn2 (G) mRNA levels were determined relative to the house-keeping gene Hprt at baseline and 48 h post-infection. Data were compared by means of ANOVA with Tukey's post hoc test. Values are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges and only statistically significant differences are indicated exactly as described for Figure 1. Additional letters represent statistically significant differences (P < 0.05) as follows: (a) Hfe^+/+ Ctrl. IA vs. Hfe^+/+ Ctrl. IE; (b) Hfe^−/− Ctrl. IA vs. Hfe^−/− Ctrl. IE; (c) Hfe^+/+ S. Tm. IA vs. Hfe^+/+ S. Tm. IE; Hfe^−/− S. Tm. IA vs. Hfe^−/− S. Tm. IE. n = 7–10 per group.

Figure 3

Influence of dietary iron content on the course of Salmonella Typhimurium infection in Hfe WT and Hfe^−/− mice. Survival during systemic infection with Salmonella Typhimurium was monitored over an observation period of 10 days (A). Data are depicted as Kaplan-Meier curves and were compared by log rank test. n = 9–11 per group. Statistically signficant differences are as follows: P = 0.031 for Hfe^+/+ IA vs. Hfe^−/− IA. P = 0.028 for Hfe^+/+ IA vs. Hfe^+/+ IE. P = 0.954 for Hfe^−/− IA vs. Hfe^−/− IE. P = 0.002 for Hfe^+/+ IE vs. Hfe^−/− IE. P = 0.041 for Hfe^+/+ IA vs. Hfe^−/− IE. P = 0.003 for Hfe^+/+ IE vs. Hfe^−/− IA. Bacterial loads of at least 6 animals per group were determined in livers (B) and spleens (C) of randomly selected animals on d 2 post-infection. CFU data were log-transformed and compared by means of ANOVA with Tukey's post hoc test. All statistically significant differences are indicated as lines. Values are depicted as lower quartile, median, and upper quartile (boxes), and minimum/maximum ranges and statistical significances are indicated.

Figure 4

Influence of dietary iron content on tissue damage during Salmonella Typhimurium infection in Hfe WT and Hfe^−/− mice. HE-stained sections of livers (A) and spleens (B) of WT and Hfe^−/− mice on day 4 of infection show macro-abscesses (arrow heads) in both organs of WT mice on an iron-excessive diet (IE) and scarce inflammatory foci (arrows) in Hfe^−/− mice fed an iron-adequate diet (IA) with intermediate pathology in the other two treatment/genotype groups. Scale bars: 400 μm.

Figure 5

Classical innate immune functions are Hfe-independent. The expression of TNF-α (A), IL-1ß (B), IL-6 (C), Nos2 (D), and the p47 phox subunit (E) in the spleen was determined relative to the housekeeping gene Hprt by quantitative RT-PCR. Data were analyzed and presented exactly as described in the legend to Figure 1. All statistically significant differences are indicated. Additional letters represent statistically significant differences (P <0.05) as follows: (c) Hfe^+/+ S. Tm. IA vs. Hfe^+/+ S. Tm. IE. n = 7–10 per group.

Figure 6

Salmonella adapts to the iron-restricted myeloid compartment of Hfe^−/− mice. The expression of bacterial iron metabolic genes in the spleen was measured by qPCR. Expression of iroN (A), fepA (B), cirA (C), iroC (D), feoB (E), and sitB (F) was determined relative to the housekeeping gene gyrB. Data were compared by means of ANOVA with Tukey's post hoc test. Values are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges and statistical significances are indicated. n = 14–17 per group.

Figure 7

Hfe^−/− macrophages more efficiently restrict iron from bacteria. WT and Hfe^−/− macrophages were infected with S. Typhimurium and exposed to ⁵⁹Fe for 24 h. Intracellular bacterial uptake of NTBI (A) and TBI (B), respectively, was determined in ⁵⁹Fe-transport studies. The data were compared by a two-tailed unpaired Student's t-test and are shown as mean ± S.E.M of at least 3 independent experiments.