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M. Nairz et al.
ed serum-spiked RPMI treated with 100 mM desferasirox (DFX) to deplete the medium of chelatable iron. Alternatively, we added 100 mM FeSO4 to saturate any iron-binding factors (e.g. transferrin, lactoferrin and Lcn2). S. Tm. WT growth was only inhibited by serum from Hfe-/- mice, possibly due to the presence of high Lcn2.13 In serum from AlfpCre+ Hfefl/fl mice, bacterial growth was strongly enhanced, while it was not affected in serum from LysMCre+ Hfefl/fl (Figure 4A). In addition, growth of S. Tm. WT was strongly restricted by the presence of the iron chelator DFX and enhanced by the addition of FeSO4, independent of the Hfe status of the mice the sera were derived from (Figure 4A). In liquid cultures of iron uptake mutant S. Tm. strains, growth was most pronouncedly
inhibited in the case of the triple mutant (entC, feo and sitABCD deletion). Importantly, we saw that the iron-rich serum of AlfpCre+ Hfefl/fl mice facilitated extracellular growth of S. Tm., an effect that was reduced by the lack of all three iron uptake systems (Figure 4B) or abolished by iron chelation (Figure 4A). Notably, the addition of recom- binant murine Lcn2 reduced the growth of S. Tm. in a dose-dependent fashion, even though it did not abolish the differences between AlfpCre– Hfefl/fl and AlfpCre+ Hfefl/fl mice (Figure 4C). This suggests that in the presence of high Lcn2 concentrations, iron uptake pathways of Salmonella not targeted by Lcn2, such as the feo and sitABCD systems, are able to compensate. Apparently, feo and sitABCD can maintain a sufficient supply of iron for
A BCD
E FGH
I JKL
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+ Hfefl/fl in E-H) or macrophages (LysMCre+ Hfefl/fl in I-L), respec- tively, compared to matched controls. Mice were infected with 500 colony forming units (CFU) of S. enterica serovar Typhimurim by intraperitoneal injection and mon- itored 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- Hfefl/fl, n=9 for AlfpCre+ Hfefl/fl, n=16 for LysMCre- Hfefl/fl, n=15 for LysMCre+ Hfefl/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- Hfefl/fl, n=14 for AlfpCre+ Hfefl/fl, n=10 for LysMCre- Hfefl/fl, n=10 for LysMCre+ Hfefl/fl.
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haematologica | 2021; 106(12)