Cell-specific expression of Hfe determines the outcome of Salmonella enterica serovar Typhimurium infection in mice

Abstract

Mutations in HFE cause hereditary hemochromatosis type I hallmarked by increased iron absorption, iron accumulation in hepatocytes and iron deficiency in myeloid cells. HFE encodes an MHC-I like molecule, but its function in immune responses to infection remains incompletely understood. Here, we investigated putative roles of Hfe in myeloid cells and hepatocytes, separately, upon infection with Salmonella Typhimurium, an intracellular bacterium with iron-dependent virulence. We found that constitutive and macrophage-specific deletion of Hfe protected infected mice. The propagation of Salmonella in macrophages was reduced due to limited intramacrophage iron availability for bacterial growth and increased expression of the anti-microbial enzyme nitric oxide synthase-2. By contrast, mice with hepatocyte-specific deletion of Hfe succumbed earlier to Salmonella infection because of unrestricted extracellular bacterial replication associated with high iron availability in the serum and impaired expression of essential host defense molecules such as interleukin-6, interferon-γ and nitric oxide synthase-2. Wild-type mice subjected to dietary iron overload phenocopied hepatocyte-specific Hfe deficiency suggesting that increased iron availability in the serum is deleterious in Salmonella infection and underlies impaired host immune responses. Moreover, the macrophage-specific effect is dominant over hepatocyte-specific Hfe-depletion, as Hfe knock-out mice have increased survival despite the higher parenchymal iron load associated with systemic loss of Hfe. We conclude that cell-specific expression of Hfe in hepatocytes and macrophages differentially affects the course of infections with specific pathogens by determining bacterial iron access and the efficacy of anti-microbial immune effector pathways. This may explain the high frequency and evolutionary conservation of human HFE mutations.

Figure 1.

Cell-type specific effect of Hfe deletion on the course of systemic Salmonella infection. Survival (A, E and I) and bacterial load in spleen (B, F and J), liver (C, G and K) and serum (D, H and L) of Hfe^-/- (A-D) mice and mice lacking Hfe in hepatocytes (AlfpCre⁺ Hfe^fl/fl in E-H) or macrophages (LysMCre⁺ Hfe^fl/fl in I-L), respectively, compared to matched controls. Mice were infected with 500 colony forming units (CFU) of S. enterica serovar Typhimurim by intraperitoneal injection and monitored for 14 days (336 hours ). Data represent two independent experiments. Statistics: survival data between control and mutant mice were compared using the Log-rank (Mantel-Cox) Test. n=18 for Hfe^+/+, n=16 for Hfe^-/-, n=13 for AlfpCre^- Hfe^fl/fl, n=9 for AlfpCre⁺ Hfe^fl/fl, n=16 for LysMCre^- Hfe^fl/fl, n=15 for LysMCre⁺ Hfe^fl/fl. Log CFU data of tissue bacterial load of randomly selected mice euthanized after 72 hours of Salmonella infection were compared using student t-test. CFU data of serum bacterial load were compared by Mann-Whitney test. n=12 for Hfe^+/+, n=12 for Hfe^-/-, n=20 for AlfpCre^- Hfe^fl/fl, n=14 for AlfpCre+ Hfefl/fl, n=10 for LysMCre^- Hfe^fl/fl, n=10 for LysMCre⁺ Hfe^fl/fl.

Figure 2.

Reduced iron content in spleen and bone marrow macrophages in the absence of Hfe. Spleen sections of Hfe^-/- mice (A), AlfpCre⁺ Hfe^fl/fl mice (D) and LysMCre⁺ Hfe^fl/fl mice (G) infected for 72 hours with Salmonella were stained by Prussian blue to analyze iron distribution. Scale bars: 200 mM. Iron content in infected spleen (B, E and H) and bone marrow macrophages (C, F and I) was measured and normalized for protein content. Data were compared by Mann-Whitney test. n=12 for Hfe^+/+, n=12 for Hfe^-/-, n=13-20 for AlfpCre^– Hfe^fl/fl, n=11-14 for AlfpCre⁺ Hfe^fl/fl, n=10 for LysMCre^– Hfe^fl/fl, n=10 for LysMCre⁺ Hfe^fl/fl.

Figure 3.

Serum iron and hepcidin-1 levels are differentially affected by Hfe. Serum iron (A to C), hepcidin-1 (D and F) and Lcn2 (G to I) concentrations of the mice infected for 72 hours were compared by Mann-Whitney test. n=9-12 for Hfe^+/+, n=9-12 for Hfe^-/-, 20 for AlfpCre^– Hfe^fl/fl, n=11-13 for AlfpCre⁺ Hfe^fl/fl, n=10-15 for LysMCre^– Hfe^fl/fl, n=10-15 for LysMCre⁺ Hfe^fl/fl.

Figure 4.

Bacterial proliferation is affected by Hfe. RPMI was spiked with 10% sera of naïve mice of the indicated genotypes. Spiked RPMI was inoculated with wild-type (WT) S. enterica Typhimurium (S. Tm.) and its isogenic derivatives mutated in one of three or all three iron uptake systems (entC, sitABCD, feo). Where applicable, deferasirox (DFX), ferrous sulfate (FeSO) and recombinant murine Lcn2 (rmuLcn2) was added. Liquid cultures were assessed for extracellular bacterial proliferation (G to I) using the optical density at 600 nm (OD600). **P<0.01, ***P<0.001 for the comparison between mouse genotypes, ^#P<0.05, ^##P<0.01 and ^###P<0.001 for the comparison to solvent (Ctrl) or the S. Tm. WT strain as applicable. n=4-6 independent experiments.

Figure 5.

Cell-type specific effects of Hfe on protein expression in the spleen. Spleen homogenates were prepared to quantify the expression of iron and immune relevant proteins by enzyme-linked immunosorbent assay. Protein levels of Fpn1 (A), H-Ft (B), Tfr1 (C), Lcn2 (D), IFN-g (E) and Nos2 (F), normalized for total protein content, are depicted as means ± standard deviations. Statistically significant differences as calculated by unpaired, two-sided student t-test are indicated. n=10 for Hfe^+/+, n=12 for Hfe^-/-, n=20 for AlfpCre^– Hfe^fl/fl, n=14 for AlfpCre⁺ Hfe^fl/fl, n=15 for LysMCre^– Hfe^fl/fl, n=15 for LysMCre⁺ Hfe^fl/fl.

Figure 6.

Elevated iron levels correlate with reduced IFN-g production and increased bacterial numbers in the serum. Serum complement factor C3 (A), IL-6 (B) and IFN-γ (C) concentrations were analyzed in AlfpCre⁺ Hfe^fl/fl mice infected for 72 hours with Salmonella. n=19-20 for AlfpCre^– Hfe^fl/fl, n=8-14 for AlfpCre⁺ Hfe^fl/fl. sIndependently, wild-type (WT) mice were fed an iron-adequate (IA) or iron-enriched diet (IO) 3 weeks prior to and during S. enterica Typhimurium (S. Tm.) infection. Serum bacterial load (D), IL-6 (E) and IFN-g (F) concentrations were determined. Statistically significant differences as calculated by Mann-Whitney test are indicated. n= 8-9 for IA, n=8 for IO.