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M. Nairz et al.
acinar cells, leading to organ damage. Conversely, mono- cytes and macrophages are iron-deficient in type I HH.3-5
An allelic frequency of approximately 5-10% renders the HFE C282Y missense mutation the most common genetic defect in individuals of Northwestern European ancestry. It has been hypothesized that the mutation may protect from iron-deficiency and/or infections; thus con- ferring an evolutionary advantage to healthy heterozy- gous carriers.6,7 Several mechanisms by which the HFE protein controls systemic iron balance have been pro- posed: early studies have shown that HFE, in association with b2-microglobulin, directly interacts with transferrin receptor-1 (TFR1) on the cell surface8 and lowers its affin- ity for transferrin-bound iron (TBI). Once the iron regula- tory hormone hepcidin had been discovered, it became apparent that the HFE C282Y mutation causes systemic hepcidin deficiency and its consequences.9 The HFE muta- tion disrupts iron-inducible BMP/SMAD (for bone mor- phogenetic protein/suppressor of mothers against decapentaplegic) signaling and prevents appropriate hep- cidin transcription.1,2 The relative lack of hepcidin then causes unrestricted dietary iron absorption by the duode- num and increased iron export from iron-recycling macrophages due to the stabilization of the iron exporter ferroportin (FPN)-1.10,11 As a result, iron accumulates in parenchymal cells where it causes tissue damage by toxic radical formation.12
HFE is an MHC-I like protein, but so far it has remained unclear whether it plays a role in immune function and/or host-pathogen interaction. HFE-deficient monocytes and macrophages are iron poor.3,4,13 Possible explanations include reduced TBI uptake, increased iron export via FPN1 as a consequence of decreased hepcidin levels or increased synthesis of the siderophore-iron binding pep- tide lipocalin (LCN)-2.3,5,13
For almost all bacteria, iron is essential as it stimulates growth and thus impacts on the course and outcome of many infectious diseases.14 However, iron requirements, iron uptake strategies and proliferation kinetics may great- ly vary between bacterial species, possibly explaining species-specific effects on infection outcomes.15 In mice, constitutive Hfe deficiency partially protects from S. enter- ica Typhimurium (S. Tm.) infection.13 By contrast, Hfe deficient mice are more susceptible to Mycobacterium avium infection.16 Furthermore, human monocyte-derived macrophages from patients with HH limit iron availability for intracellular Mycobacterium tuberculosis, resulting in an improved control of infection.17 On the other hand, indi- viduals with HH type I are highly susceptible to infection with Yersinia species, whose virulence is iron-dependent, as documented by case reports of human subjects and by mouse models.18,19 These diverse outcomes are counterin- tuitive given that all three pathogens, Salmonella, Mycobacterium and Yersinia, share a predominately intracel- lular lifestyle pointing to the importance of cell- and tis- sue-specific iron distribution for susceptibility to these infections.20
Because Hfe exerts contrasting effects in different infec- tious diseases, we asked whether Hfe plays a cell type- specific role during infection and whether this is linked to alterations of tissue iron distribution or associated with iron-independent effects of Hfe. We herein demonstrate that macrophage-specific deletion of Hfe (LysMCre+ Hfefl/fl) recapitulates the antibacterial phenotype of constitutive Hfe-/- mice in response to S. Tm. infection. By contrast,
exclusive deletion of Hfe in hepatocytes (AlfpCre+ Hfefl/fl) is associated with an adverse outcome of S. Tm. infection. These contrasting cell type-specific effects of Hfe-deficien- cy correlate with bacterial iron availability and anti-micro- bial effector immune functions. Our findings support the idea that Hfe controls iron concentrations in the microen- vironment thus differentially affecting immune effector mechanisms and bacterial growth in intra- and extracellu- lar compartments.
Methods
Salmonella infection in vivo
All infection experiments were performed according to
the guidelines of the Medical University of Innsbruck and the Austrian Ministry for Science and Education based on the Austrian Animal Testing Act of 1988 (approvals BMWFW-66.011/0074-C/GT/2007, 66.011/0154- II/3b/2010 and 66.011/0031-WF/V/3b/2015). Male mice were used at 12-16 weeks of age and infected by intraperi- toneal (i.p.) injection with 500 colony forming units (CFU) of S. Tm. diluted in 200 mL of phosphate buffered saline (PBS). Unless otherwise specified, S. Tm. Wild-type (WT), strain ATCC 14028s was used for the experiments. Where appropriate, mice were fed an iron adequate control diet (C1000 from Altromin containing 180 mg per g) or an iron- enriched diet (C1038 from Altromin supplemented with 25 mg carbonyl iron per g). After 3 weeks, mice were infected by i.p. injection with 500 CFU of S. Tm. diluted in 200 mL of PBS as detailed in the Online Supplementary Methods.
In vitro experiments
The isolation of bone marrow-derived macrophages
(BMDM) was performed as detailed in the Online Supplementary Methods.
RNA extraction and quantitative real-time polymerase chain reaction
Preparation of total RNA, reverse transcription and quantification of mRNA expression by quantitative Taqman real-time polymerase chain reaction (qRT-PCR) was performed as described.21 Results were first normal- ized using the housekeeping gene Hprt and then divided by the means of the control group (WT Hfe+/+ or Cre– mice as appropriate) to obtain expression data that is relative to the respective control group. Sequences of primers and probes are listed in the Online Supplementary Methods.
Measurement of iron and protein concentrations
Measurement of tissue iron concentrations has been described in detail.22 The serum iron concentration was quantified using the QuantiChrom Iron Assay Kit (BioAssay Systems). Intracellular iron concentrations were determined in adherent bone marrow macrophages by atomic absorption spectrometry as described.23 The quan- tification of protein levels in sera and tissues is detailed in the Online Supplementary Methods.
Statistical analysis
Statistical analysis was carried out using a GraphPad Prism statistical package and Microsoft Excel. We deter- mined significance by unpaired two-tailed Student’s t-test or Mann-Whitney test to assess data, where only two
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