Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus - PubMed (original) (raw)
Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus
João Arezes et al. Cell Host Microbe. 2015.
Abstract
Hereditary hemochromatosis, an iron overload disease caused by a deficiency in the iron-regulatory hormone hepcidin, is associated with lethal infections by siderophilic bacteria. To elucidate the mechanisms of this susceptibility, we infected wild-type and hepcidin-deficient mice with the siderophilic bacterium Vibrio vulnificus and found that hepcidin deficiency results in increased bacteremia and decreased survival of infected mice, which can be partially ameliorated by dietary iron depletion. Additionally, timely administration of hepcidin agonists to hepcidin-deficient mice induces hypoferremia that decreases bacterial loads and rescues these mice from death, regardless of initial iron levels. Studies of Vibrio vulnificus growth ex vivo show that high iron sera from hepcidin-deficient mice support extraordinarily rapid bacterial growth and that this is inhibited in hypoferremic sera. Our findings demonstrate that hepcidin-mediated hypoferremia is a host defense mechanism against siderophilic pathogens and suggest that hepcidin agonists may improve infection outcomes in patients with hereditary hemochromatosis or thalassemia.
Copyright © 2015 Elsevier Inc. All rights reserved.
Figures
Figure 1. Vibrio vulnificus infection is highly lethal in hepcidin-deficient mice
Kaplan-Meier survival curves for iron depleted (A) and iron loaded (B) mice, after infection with 1x103 (dashed lines) or 1x105 (solid lines) CFU of V. vulnificus (n=4–11 in each group). WT and Hamp1−/− mice were iron-depleted or iron-loaded by dietary modification (WT: 4 ppm or 10,000 ppm Fe diet for 2 weeks; Hamp1−/−: 4 ppm or standard diet for 4–6 weeks, see Methods). By multifactorial Kaplan-Meier Log-Rank analysis, differences in survival between WT and Hamp1 −/− mice were significant in both iron depleted (combined CFU, p<0.05), and iron-loaded conditions (combined CFU, p<0.001). (C) Liver iron stores were measured in a parallel set of mice and confirmed the effective modulation of iron stores by dietary iron manipulation (n=5–10 per group). For liver iron measurements, statistical significance was assessed using student’s t test if data were normally distributed (**p<0.01) or Mann-Whitney U test if they were not normally distributed (++p<0.01). See also Figure S1.
Figure 2. Local V. vulnificus proliferation induces vasodilation, leukostasis and erythrocyte sludging
Skin sections at the site of injection with saline (A, C, E and G) or 1 x 103 CFU of V. vulnificus (B, D, F, H) in Hamp1−/− mice. Sections were stained with hematoxylin and eosin (A–D) or Gram stain and tartrazine (E–H). Panels A–B and magnified in C–D: Dilated venules (v) and arterioles (a), leukostasis and erythrocyte sludging are seen in the skin of _V. vulnificus-_infected mice compared with saline-injected controls**. Panels E–F** and magnified in G–H: Numerous bacteria (purple) are seen in perivascular spaces of infected mice (arrow). See also figure S2.
Figure 3. Iron-dependent dissemination of V. vulnificus in blood and liver
Wild-type and Hamp1−/− mice were iron-depleted or iron-loaded by dietary modification (WT: 4 ppm or 10,000 ppm Fe diet for 2 weeks; Hamp1−/−: 4 ppm or standard diet for 4–6 weeks, see Methods), and infected with V. vulnificus (n=5–10 per group). (A) and (B) Bacterial counts in blood and liver 16 h after infection with 300 CFU (A) and 1x105CFU (B). Each symbol represents one mouse (iron depleted in blue and iron loaded in red); black solid lines represent the mean of CFU counts; black dotted line represents the lower limit of detection of CFU counts (calculated as half of the minimum detectable CFU counts). (C) Serum iron levels of WT (1x105CFU) and Hamp1−/−(300 CFU) mice (white fill = saline groups, grey fill = V. vulnificus groups). Unlike WT mice which decreased their serum iron to the mean of ~30 μM, Hamp1−/− mice did not develop marked hypoferremia after infection. (D) Measurement of non-transferrin bound iron in serum of iron-depleted and iron-loaded WT and Hamp1−/− mice prior to infection (n=4–6 per group). Statistical significance was assessed using student’s t test if data were normally distributed (*p<0.05; **p<0.01; ***p<0.001) or Mann-Whitney U test if they were not (+p<0.05; ++p<0.01). See also Figure S3.
Figure 4. WT mice respond to V. vulnificus infection by rapidly increasing plasma hepcidin concentration
WT mice, either iron-depleted (blue) or iron-loaded (red), were injected with 105 CFU V. vulnificus (solid lines) or saline (dashed lines) and euthanized after 3, 6 or 10 hours. (A) Hepatic Hamp1 mRNA expression. (B) Serum hepcidin concentration. (C) Serum iron concentration. (D) The inflammatory response to infection was confirmed by serum IL-6 assay. Each point represents the mean ± standard deviation (n=5). Statistical significance was assessed using student’s t test if data were normally distributed (*p<0.05; **p<0.01; ***p<0.001) or Mann-Whitney U test if they were not (+p<0.05; ++p<0.01). See also Figure S4.
Figure 5. Minihepcidin PR73 decreases infection by V. vulnificus in Hamp1−/− mice
Hamp1−/− mice (either iron-depleted or iron-loaded) were treated with 100 nmol of PR73 (red symbols) or solvent (white symbols) 24h and 3h before infection with 300 CFU V. vulnificus. Mice were euthanized 16h after infection. N=10–11 per group. (A) Serum iron was markedly decreased in PR73-injected mice, with no difference in serum iron between solvent-injected iron-depleted and iron-loaded mice. (B) Liver iron was higher in iron-loaded than iron-depleted mice as expected. PR73 administration did not result in significant changes in liver iron stores. (C) and (D) V. vulnificus was undetectable in blood and liver in PR73-treated mice, in contrast to mice treated with saline. Each dot represents one mouse; black solid lines represent the mean of CFU counts. The black dotted line represents the lower limit of detection of CFU counts (calculated as half of the minimum detectable CFU counts). Statistical significance was assessed using student’s t test if data were normally distributed (**p<0.01) or Mann-Whitney U test if they were not (+p<0.05; ++p<0.01). See also Figure S5.
Figure 6. Minihepcidin PR73 prevents death due to V. vulnificus infection in Hamp1−/− mice
Kaplan-Meier survival curves, n=4–5 per group. Both iron-depleted (A) and iron-loaded (B) Hamp1−/− mice survived the infection with 103 and 105 CFU V. vulnificus when treated with 100 nmol PR73 (red) before the infection. (C) Hamp1−/− mice were resistant to infection even if PR73 was administered 3h after infection. Statistically significant differences in survival between solvent- and PR73-treated mice were assessed using the Log-Rank survival analyzes: p<0.05 for iron-depleted, 103 CFU; p<0.01 for the other groups. See also Figure S6.
Figure 7. Minihepcidin PR73 acts in serum by slowing bacterial growth
Serum was collected from iron-depleted or iron-loaded Hamp1−/− mice that were injected with PR73 or solvent, and from iron-depleted or iron-loaded WT mice (not treated with PR73). Serum iron concentrations are shown in Table S2. V. vulnificus carrying the non-replicating marker plasmid pGTR905 was incubated in these sera in vitro for 2 h, and CFU were determined either on plates without chloramphenicol (total bacteria) or plates with chloramphenicol and arabinose (allows growth of only plasmid-containing bacteria). (A) PR73 greatly reduced total bacterial yield, which reflects the sum of bacteriostatic and bactericidal effects. (B) PR73 only slightly reduced the yield of plasmid-containing bacteria indicating only a small bactericidal effect. (C) The number of total bacteria was much higher in serum from iron-loaded WT mice than in serum from iron-depleted mice, as expected. (D) Different serum iron concentrations did not affect the yield of plasmid-containing bacteria indicating that hypoferremia by itself does not have a bactericidal effect. Each vertical bar represents the mean, and error bars represent standard deviation for 3 independent experiments (with 3 replicates in each experiment). The black dotted line represents the number of plasmid-containing bacteria after growth that diluted plasmid copy number to 1 plasmid per bacterium (thus bacteria in the original inoculum carried 5 plasmids per bacterium). Statistical significance was assessed using student’s t test (*p<0.05; **p<0.01; ***p<0.001). See also Figure S7.
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