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M. Nairz et al.
tocyte-specific Hfe deficiency. This suggests that the tran- scriptional induction of Nos2 in macrophages may be affected by a paracrine Hfe-dependent pathway. In con- trast, when myeloid cells express Hfe and serum iron lev- els are high because of hepatocyte-specific Hfe deficiency, the induction of Nos2 was severely impaired in the spleen. Iron inhibits Nos2 transcription45 but this regulation fails to explain the reduced Nos2 mRNA and protein levels in the spleens of AlpfCre+ Hfefl/fl mice, which are relatively iron-poor in steady-state and iron-adequate in S. Tm. Infection.25 We propose that an iron-mediated immune- deregulation secondary to the low levels of Ifn-g mRNA in spleens of these mice is a possible explanation because IFN-g is a major inducer of Nos2.46 In addition, RNS coun- teract Salmonella’s virulence and IFN- promotes Salmonella degradation in mouse macrophages.47 These mechanisms are also of central importance for immunity in human sub- jects because monogenetic defects in the IFN-g pathway result in increased susceptibility to non-typhoid Salmonella and atypical mycobacteria.48 However, additional studies are required to characterize the regulatory networks that appear to link Hfe to IFN-g and Nos2 levels.
Additionally, the observed differences in the expression of immune genes between spleen and liver argue for the involvement of other types of immune, non-parenchymal or stromal cells. In this context, it will be particularly inter- esting to study the effects of Hfe depletion in lymphocyte subsets in the context of bacteremia because an effect of Hfe on T-cell differentiation has been proposed in different models.49
Salmonella can acquire iron via different pathways. Its major siderophores, enterobactin and salmochelins, bind ferric iron with extremely high affinity, thus initiating its uptake via siderophore receptors. Independently of siderophores, ionic iron is acquired via feo, sitABCD and a less well characterized low affinity iron uptake system.15 It is interesting to note that the entC sit feo triple mutant did grow better in medium spiked with sera of hepato- cyte-specific Hfe-deficient mice than in sera of other mice. The growth of the triple mutant was significantly impaired as compared to Salmonella with only single dele- tions of iron uptake systems, entC, sit or feo, respectively (Figure 3). This suggests that the growth attenuation of this strain was partially conserved in high iron conditions. Moreover, the addition of high concentrations of recombi- nant murine Lcn2 to spiked sera of AlfpCre+ Hfefl/fl mice and corresponding controls inhibited bacterial growth in liquid cultures but did not abolish the differences between the two genotypes of mice whereas the iron chelator DFX did. These findings suggest that Salmonella is able to circum- vent Lcn2’s growth inhibiting effects by iron uptake mech- anisms that act independent of enterobactin and are thus resistant to Lcn2. Salmochelins, which are glycosylated
enterobactin derivatives to which Lcn2 cannot bind, may one of the ways by which Salmonella resists the immune response.27 However, further studies are required to under- stand the mechanisms of Salmonella’s metabolic adapta- tion to iron withdrawal by the host’s immune response.
In conclusion, our study exclusively reports on Hfe’s role in infection to demonstrate a pivotal extra-hepatic function of Hfe and to highlight the importance of intracel- lular iron levels within macrophages for the control of infections with Salmonella. We found that the lack of Hfe in macrophages increased host resistance to this particular pathogen secondary to reduced intracellular iron availabil- ity and increased Nos2 expression. Importantly, this effect was dominant over possible growth-promoting effects that increased serum iron levels may impose. In contrast, serum iron levels determined both, IFN-g levels and extra- cellular bacterial replication and increased bacteremia pre- ceding early mortality in mice lacking Hfe exclusively in hepatocytes. Our data suggest that the high penetrance of HFE mutations may originate from the immune modula- tory effects of HFE enabling a better control of infections with intracellular pathogens.
Disclosures
The authors declare that there is no conflict of interest.
Contributions
MN and CM planned and conducted experiments, acquired and analyzed data and drafted the manuscript; MV-S, A-MM, AS, DH, AH, LvR, RS, CWH, HT and PLM performed exper- iments;MM and GW conceived and designed the study, obtained funding and wrote the manuscript.
Acknowledgments
The authors would like to thank Sylvia Berger, Ines Brosch, Sabine Engl, Ines Glatz and Markus Seifert for excellent techni- cal support. We also would like to thank Ferric C. Fang, Departments of Laboratory Medicine and Microbiology, University of Washington, for providing Salmonella mutants and intellectualinput.
Funding
This work was supported by grants from the Austrian Research Fund (FWF sponsored doctoral programme W-1253 HOROS to GW and stand-alone project P 33062, to MN), the Christian Doppler Society (to GW), the Tyrolean Research Fund (TWF, to MN) and by the ‘Verein zur Förderung von Forschung und Weiterbildung in Infektiologie und Immunologie an der Medizinischen Universität Innsbruck’. CM was funded through a postdoctoral scholarship from the Medical Faculty of Heidelberg University, Germany and the Virtual Liver Network funding initiative (BMBF). MUM was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 1036).
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