Hepcidin-mediated iron sequestration protects against bacterial dissemination during pneumonia

The analogue lowered median bacterial loads in lungs and blood of infected hepcidin-deficient mice by more than 99%.

“The treatment also resulted in > 99% reduction in median lung and blood bacterial burden in infected hepcidin-deficient mice and 40% reduction in mortality compared with vehicle alone (Figure 7, C and D).”

Iron-treated mice showed a large rise in plasma hepcidin after acute iron loading compared with PBS-treated mice.

“Similar to prior reports (31), mice treated with iron also had marked elevation of plasma hepcidin as compared with mice treated with PBS in response to acute iron loading (Figure 5D), indicating that hepcidin induction alone is not sufficient to protect mice from fatal infection if plasma iron levels are high and supporting the hypothesis that the beneficial effects of hepcidin are mediated via”

In mice with Klebsiella pneumoniae infection, total iron levels in BAL change significantly over time (P < 0.001 by linear trend).

“(B and C) Calculated total iron levels in whole lung homogenate per mouse, and bronchoalveolar lavage fluid (BAL) recovered in a volume of 1 ml per mouse.”

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  “Data shown as mean ± SE; n = 4-6 animals per time point, representative of 2 independent experiments; ***P < 0.001 (1-way ANOVA, multiple comparison for linear trend).”

These iron-depleted knockout mice had less stored body iron than wild-type mice.

“We found that iron-depleted hepcidin -/-animals did not develop hyperferremia during infection, had lower body iron stores than WT mice, and were no more susceptible to infection compared with WT mice (Supplemental Figure 3, A and B).”

Wild-type mice showed a rapid drop in plasma iron after Klebsiella pneumonia infection.

“As expected, plasma iron concentration decreased quickly in WT mice following infection (Figure 1A), similar to prior reports with acute inflammatory stimuli (9).”

Blocking IL-6 impaired the host's defense against K. pneumoniae.

“In addition, we found that IL-6 neutralization disrupted host defense against K. pneumoniae (Figure 3A), in corroboration with previous reports (22).”

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In uninfected and K. pneumoniae-infected mice, treatment with IL-6 neutralizing antibody alters plasma hepcidin protein levels compared to isotype control (P < 0.01, one-way ANOVA Tukey).

“(B and C) Plasma hepcidin protein and iron levels in uninfected and infected animals treated with isotype or IL-6 neutralizing antibody.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars) combined from 2 experiments; n = 8-9 animals per group; **P < 0.01, one way ANOVA Tukey post-test.”

With plasma from three separate donors, ferric iron allowed K. pneumoniae to grow when labile iron appeared, not simply according to how much iron citrate was added.

“Interestingly, we found that, in plasma preparations from 3 independent donors, addition of ferric iron resulted in K. pneumoniae out-growth coincided with the appearance of labile iron in the plasma, rather than with the total amount of iron citrate added.”

Infection caused robust induction of hepcidin mRNA in liver but not in peripheral blood leukocytes or lungs.

“We thus measured hepcidin transcription in the liver, blood, and lungs during infection and found a robust induction of hepcidin mRNA in the liver (Figure 2B) but not in peripheral blood leukocytes or lungs (Figure 2, C and D), suggesting that liver is the main source of hepcidin during the infection and elevations in lung hepcidin protein likely represent extravasation from the blood.”

Unlike wild-type mice, hepcidin-deficient mice showed rising plasma iron during infection.

“In contrast to reduction in plasma iron during infection in WT mice, plasma iron in hepcidin-deficient mice became more elevated during the infection (Figure 4A), indicating that hepcidin is essential for controlling plasma iron levels following infection.”

Hepcidin-deficient mice were far more vulnerable to bacterial pneumonia, dying at 100% versus 50% in wild-type controls.

“We next assessed the contribution of hepcidin-mediated iron sequestration to host defense against K. pneumoniae and found that hepcidin-deficient animals were markedly more susceptible to bacterial pneumonia, demonstrating 100% mortality compared with 50% mortality in WT controls (Figure 4C), comparable to mice treated with exogenous iron during infection (Figure 1, D and E).”

Most iron in whole lung homogenates was intracellular.

“We found that most lung iron was intracellular, and extracellular iron levels were low and did not change appreciably during infection.”

Iron-depleted knockout mice were not more vulnerable to infection than wild-type mice.

“We found that iron-depleted hepcidin -/-animals did not develop hyperferremia during infection, had lower body iron stores than WT mice, and were no more susceptible to infection compared with WT mice (Supplemental Figure 3, A and B).”

IL-6 neutralization also removed the accompanying low plasma iron.

“In addition, IL-6 neutralization abrogated the increase in plasma hepcidin during the infection and eliminated the associated hypoferremia (Figure 3, B and C), indicating that hepcidin induction during pneumonia is dependent on IL-6.”

Hepcidin-knockout mice made iron deficient did not become hyperferremic during infection.

“We found that iron-depleted hepcidin -/-animals did not develop hyperferremia during infection, had lower body iron stores than WT mice, and were no more susceptible to infection compared with WT mice (Supplemental Figure 3, A and B).”

Circulating neutrophils and both Ly6C-high and Ly6C-low monocyte populations were comparable in wild-type and hepcidin-deficient mice before and two days after infection.

“We found similar concentrations of circulating neutrophils, and Ly6C hi and Ly6C lo monocytes in WT and hepcidin-deficient mice at baseline and 2 days following infection.”

In mice with Klebsiella pneumoniae infection, plasma iron concentration changes significantly over time (P < 0.001 by linear trend).

“Data shown as mean ± SE; n = 4-6 animals per time point, representative of 2 independent experiments; ***P < 0.001 (1-way ANOVA, multiple comparison for linear trend).”

When made iron deficient, hepcidin-knockout mice were protected from infection.

“Our data suggest that the hepcidin-dependent protection was mediated by reducing plasma iron, as indicated by our observation that WT mice treated with iron citrate were susceptible to K. pneumoniae infection despite robust hepcidin induction (Figure 1, E and F) and that hepcidin-deficient mice were protected from infection if starved of iron (Supplemental Figure 3B).”

In mice with Klebsiella pneumoniae infection, total iron levels in whole lung homogenate change significantly over time (P < 0.001 by linear trend).

“(B and C) Calculated total iron levels in whole lung homogenate per mouse, and bronchoalveolar lavage fluid (BAL) recovered in a volume of 1 ml per mouse.”

• Also stated in: § R E S E A R C H A R T I C L E · plain

  “Data shown as mean ± SE; n = 4-6 animals per time point, representative of 2 independent experiments; ***P < 0.001 (1-way ANOVA, multiple comparison for linear trend).”

IL-6 neutralization prevented the infection-induced rise in plasma hepcidin.

“In addition, IL-6 neutralization abrogated the increase in plasma hepcidin during the infection and eliminated the associated hypoferremia (Figure 3, B and C), indicating that hepcidin induction during pneumonia is dependent on IL-6.”

Wild-type mice given iron citrate remained vulnerable to K. pneumoniae even though hepcidin was strongly induced.

“Our data suggest that the hepcidin-dependent protection was mediated by reducing plasma iron, as indicated by our observation that WT mice treated with iron citrate were susceptible to K. pneumoniae infection despite robust hepcidin induction (Figure 1, E and F) and that hepcidin-deficient mice were protected from infection if starved of iron (Supplemental Figure 3B).”

Infected wild-type and hepcidin-deficient mice produced similar amounts of reactive oxygen species in lung and blood leukocytes.

“Since oxidative burst is an iron-dependent process, we compared the generation of reactive oxygen species in lung and blood leukocyte populations and found no difference between infected WT and hepcidin-deficient mice (Supplemental Figure 4).”

Survival during K. pneumoniae pneumonia differs between WT and hepcidin -/- mice (P < 0.001, Mantel-Cox).

“(C) Survival of WT and hepcidin -/-mice during pneumonia.n = 26-30 mice per group, combined from 3 independent experiments; ***P < 0.001 Mantel-Cox test.”

After intravenous K. pneumoniae challenge, wild-type mice pretreated with ferric ammonium citrate had more bacteria in blood at 24 hours than PBS controls.

“After 24 hours, mice treated with ferric ammonium citrate had higher blood bacterial content than mice treated with PBS, indicating that elevated plasma iron directly mediates increased growth of K. pneumoniae in the blood stream (Figure 5C).”

Blocking IL-6 led to greater bacterial loads in blood and lungs two days after infection.

“We found that immunoneutralization of IL-6 resulted in higher blood and lung bacterial burden 2 days following infection (Figure 3A).”

Ferric ammonium citrate-treated mice also carried more bacteria in their lungs.

“Interestingly, mice treated with ferric ammonium citrate also had higher bacterial burdens in the lungs.”

The same iron treatment accompanied higher bacterial loads in the lungs and blood.

“This transient increase in iron availability during the first 24 hours after infection was associated with a markedly increased mortality from pneumonia associated with increased bacterial burden in the lung and the blood (Figure 1, E and F).”

In uninfected and K. pneumoniae-infected mice, treatment with IL-6 neutralizing antibody alters plasma iron levels compared to isotype control (P < 0.01, one-way ANOVA Tukey).

“(B and C) Plasma hepcidin protein and iron levels in uninfected and infected animals treated with isotype or IL-6 neutralizing antibody.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars) combined from 2 experiments; n = 8-9 animals per group; **P < 0.01, one way ANOVA Tukey post-test.”

Lung and liver bacterial loads were about ten times higher in hepcidin-deficient than wild-type mice.

“Hepcidin-deficient mice exhibited 23% greater incidence of bacteremia and > 100-fold higher median blood bacterial concentration, as well as ~10-fold higher bacterial burden in the lungs and liver as compared with WT mice (Figure 4D).”

In mice challenged intrapulmonary with K. pneumoniae, ferric ammonium citrate treatment prior to and 24 h after challenge reduces survival compared to PBS (P < 0.001, Mantel-Cox).

“(E) Mice were treated with either PBS or ferric ammonium citrate (iron) 30 minutes prior to intrapulmonary K. pneumoniae challenge and again 24 hours thereafter.n = 6-12 per group, combined result of 2 experiments; ***P < 0.001 Mantel-Cox test.”

In WT mice challenged intravenously with K. pneumoniae, iron citrate treatment prior to challenge alters bacterial burden in lungs and blood after 24 h (P < 0.05, Mann-Whitney U).

“(C and D) Bacterial burden and plasma hepcidin protein in WT mice treated with either PBS or iron citrate 30 minutes prior to i.v.challenge with K. pneumoniae.Lungs and blood were harvested after 24 hours.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars).n = 11 mice per group, combined form 2 experiments; *P < 0.”

In WT and hepcidin -/- mice, lung nonheme iron levels differ before and 2 days after K. pneumoniae infection (P < 0.01 and P < 0.0001, one-way ANOVA Tukey).

“(A and B) Comparison of plasma or lung nonheme iron levels in WT and hepcidin -/-mice before and 2 days after infection.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars) of data from 2 experiments; n = 4-9 per group; ** and **** denote P values of <0.01 and <0.0001 by one-way ANOVA with Tukey post test,”

In mice challenged intrapulmonary with K. pneumoniae, intraperitoneal ferric ammonium citrate increases plasma iron compared to PBS (P < 0.0001, two-way ANOVA).

“(D) Plasma iron following i.p. injection with PBS or ferric ammonium citrate (iron) 30 minutes prior to intrapulmonary K. pneumoniae challenge.n = 6-8 per group per time point, combined result of 2 experiments; ****P < 0.0001, two-way ANOVA.”

In human plasma, K. pneumoniae growth was inhibited unless iron was added.

“K. pneumoniae growth was suppressed in plasma but was permitted by iron supplementation, consistent with reports that transferrin inhibits bacterial growth (30) (Figure 5B).”

Mortality fell by 40% relative to vehicle alone.

“The treatment also resulted in > 99% reduction in median lung and blood bacterial burden in infected hepcidin-deficient mice and 40% reduction in mortality compared with vehicle alone (Figure 7, C and D).”

Hepcidin-deficient mice had >100-fold higher median blood bacterial concentration compared with WT mice.

“Hepcidin-deficient mice exhibited 23% greater incidence of bacteremia and > 100-fold higher median blood bacterial concentration, as well as ~10-fold higher bacterial burden in the lungs and liver as compared with WT mice (Figure 4D).”

Neutrophils from wild-type and hepcidin-deficient mice killed bacteria equally well ex vivo.

“Similarly, neutrophils from WT and hepcidin-deficient mice exhibited comparable ex vivo bacterial killing (Figure 6G).”

A hepcidin analogue lacking the β-defensin motif needed for antimicrobial action still rescued hepcidin-deficient mice.

“Similarly, administration of a hepcidin analogue, which lacks the β-defensin motif required for its antimicrobial activity (37), rescued the phenotype of hepcidin-deficient mice (Figure 7, C and D).”

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The hepcidin concentrations in plasma are far below those needed to kill bacteria in vitro.

“Arguing against such a direct microbicidal role, we report plasma hepcidin levels at concentrations much lower than those required to mediate killing in vitro (39).”

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Hepcidin-deficient mice exhibited 23% greater incidence of bacteremia compared with WT mice.

“Hepcidin-deficient mice exhibited 23% greater incidence of bacteremia and > 100-fold higher median blood bacterial concentration, as well as ~10-fold higher bacterial burden in the lungs and liver as compared with WT mice (Figure 4D).”

In mice with K. pneumoniae infection, bacterial burden on day 2 differs between WT and hepcidin -/- mice in the indicated organs (P < 0.01 and P < 0.0001, Mann-Whitney U).

“(D) Bacterial burden on day 2 of infection in WT and hepcidin -/-mice in indicated organs.Pooled data from 3 independent experiments; n = 23-26.Horizontal lines represent median and each circle represents one animal; animals with no detectable bacteria are reported to have a bacterial burden of 1 CFU on the logarithmic scale; ** and **** denote P values of <0.01 and <0.0001, respectively, using”

In mice infected with K. pneumoniae, treatment with IL-6 neutralizing antibody alters bacterial burden on day 2 compared to isotype control (P < 0.05 and P < 0.0001, Mann-Whitney U).

“(A) Animals were treated daily with an isotype control or IL-6 neutralizing monoclonal antibody and assessed for bacterial burden on day 2 of infection.Horizontal lines represent median, and each circle represents 1 animal; animals with no detectable bacteria are reported to have a bacterial burden of 1 CFU on the logarithmic scale; * and **** denote P values of <0.05 and <0.0001 by Mann-Whitney U”

In bronchoalveolar lavage, alveolar macrophages, neutrophils, and monocyte-derived macrophages did not differ between wild-type and hepcidin-deficient mice before or after infection.

“The number of BAL alveolar macrophages, neutrophils, and monocyte-derived macrophages were also similar between WT and hepcidin-deficient hosts, both at baseline and following infection, arguing against a defect in leukocyte quantity or recruitment in the absence of hepcidin.”

In WT and hepcidin -/- mice, plasma nonheme iron levels differ before and 2 days after K. pneumoniae infection (P < 0.01 and P < 0.0001, one-way ANOVA Tukey).

“(A and B) Comparison of plasma or lung nonheme iron levels in WT and hepcidin -/-mice before and 2 days after infection.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars) of data from 2 experiments; n = 4-9 per group; ** and **** denote P values of <0.01 and <0.0001 by one-way ANOVA with Tukey post test,”

In hepcidin-deficient mice, minihepcidin treatment alters plasma nonheme iron levels compared to vehicle (P < 0.001, t test).

“(A and B) Nonheme iron levels in the plasma and lung of vehicle-treated and minihepcidin-treated hepcidin-deficient mice.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars).n = 17-19 per group combined from 2 independent experiments; ****P < 0.001, t test.”

In WT mice challenged intravenously with K. pneumoniae, iron citrate treatment prior to challenge alters plasma hepcidin protein after 24 h (P < 0.01, t test).

“(C and D) Bacterial burden and plasma hepcidin protein in WT mice treated with either PBS or iron citrate 30 minutes prior to i.v.challenge with K. pneumoniae.Lungs and blood were harvested after 24 hours.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars).n = 11 mice per group, combined form 2 experiments; *P < 0.”

There are no significant differences in leukocyte responses in BAL and blood between WT and hepcidin -/- mice before and 2 days after K. pneumoniae infection.

“(A-F) BAL and blood were harvested from WT and hepcidin -/-mice before and 2 days following infection.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars).n = 4-5 per group, one way ANOVA Tukey post-test.No significant differences were found for any WT to hepcidin -/-comparison.”

In hepcidin-deficient mice with K. pneumoniae infection, minihepcidin treatment alters bacterial burden in blood and lungs compared to vehicle (P < 0.05 and P < 0.01, Mann-Whitney U).

“(C) Bacterial burden in blood and lungs.Horizontal lines represent median and each circle represents 1 animal; animals with no detectable bacteria are reported to have a bacterial burden of 1 CFU on the logarithmic scale; n = 17-19 per group, combined data from 2 experiments; * and ** denote P values <0.05 and <0.01 by Mann-Whitney U test, respectively.”

In hepcidin-deficient mice, minihepcidin treatment alters lung nonheme iron levels compared to vehicle (P < 0.001, t test).

“(A and B) Nonheme iron levels in the plasma and lung of vehicle-treated and minihepcidin-treated hepcidin-deficient mice.Box and whisker plots show median (line within box), upper and lower quartiles (upper and lower box boundaries), and total range (bars).n = 17-19 per group combined from 2 independent experiments; ****P < 0.001, t test.”

There is no significant difference in killing of K. pneumoniae by freshly isolated neutrophils from WT versus hepcidin -/- mice after 1 hour coincubation.

“(G) Freshly isolated neutrophils from WT and hepcidin -/-mice were coincubated with 2 × 10 5 CFU of K. pneumoniae for 1 hour.Total surviving bacteria were enumerated from each well.n = 3 independent experiments per group; data were analyzed using a 2-way ANOVA, and no differences were found between groups”

In hepcidin-deficient mice with K. pneumoniae infection, treatment with hepcidin analogue on days 0-5 alters survival compared to vehicle (P < 0.05, Mantel-Cox).

“(D) Survival of hepcidin-deficient mice treated with either vehicle or hepcidin analogue days 0-5; n = 16 per group, combined result of 2 experiments; *P < 0.05 by Mantel-Cox test.”

Spread of Gram-negative bacteria from the initial infection site into the blood is particularly linked to death.

“Dissemination of these organisms from the primary site of infection to the bloodstream is especially associated with mortality (2,3).”

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People with iron overload are unusually vulnerable to Vibrio vulnificus.

“Hepcidin-mediated hypoferremia is essential for protection of the host against Vibrio vulnificus in a murine model (11), consistent with the clinical observation that individuals with iron-overload conditions are uniquely susceptible to this organism (12).”

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In mice, hepcidin-driven low plasma iron is required to defend against Vibrio vulnificus.

“Hepcidin-mediated hypoferremia is essential for protection of the host against Vibrio vulnificus in a murine model (11), consistent with the clinical observation that individuals with iron-overload conditions are uniquely susceptible to this organism (12).”

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Aerobic Gram-negative bacteria require their iron uptake systems for virulence.

“While the iron acquisition mechanisms of aerobic Gram-negative bacteria are known to be essential to their virulence (5), the host mechanisms of iron restriction in these infections are incompletely defined.”

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Removing iron restriction might make some people more prone to Gram-negative sepsis.

“In this context, we posit that relieving the iron-restricted state could predispose some individuals to Gram-negative sepsis.”

• AssumptionRests on an assumption: The mouse Klebsiella pneumonia model is sufficiently representative of human Gram-negative pneumonia for clinical and therapeutic conclusions.

Acutely lowering plasma iron during infection in hepcidin-deficient hosts could be a reasonable treatment approach.

“These data provide evidence that the acute induction of hypoferremia during infection in hepcidin-deficient hosts may represent a plausible therapeutic strategy.”

• AssumptionRests on an assumption: The mouse Klebsiella pneumonia model is sufficiently representative of human Gram-negative pneumonia for clinical and therapeutic conclusions.

Spread of microbes from the initial infection site into the blood is a major complication of Gram-negative bacterial infections.

“We reasoned that the bloodstream dissemination of microorganisms from the primary site of infection is a critical complication in Gram-negative bacterial infections.”

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For pneumonia and other localized infections from aerobic Gram-negative rods, bacteria in the blood correlate strongly with sepsis and mortality.

“In the context of pneumonia, as well as other focal infections caused by aerobic Gram-negative bacilli, bacteremia is strongly associated with sepsis and death (13,14).”

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Hepcidin's benefit appears to work through lowering plasma iron, not through an iron-independent mechanism.

“Similar to prior reports (31), mice treated with iron also had marked elevation of plasma hepcidin as compared with mice treated with PBS in response to acute iron loading (Figure 5D), indicating that hepcidin induction alone is not sufficient to protect mice from fatal infection if plasma iron levels are high and supporting the hypothesis that the beneficial effects of hepcidin are mediated via”

Hepcidin-mediated iron sequestration protects against Gram-negative pneumonia.

“Taken together, these data suggest that hepcidin-mediated iron sequestration protects against Gram-negative pneumonia.”

• AssumptionRests on an assumption: The mouse Klebsiella pneumonia model is sufficiently representative of human Gram-negative pneumonia for clinical and therapeutic conclusions.

The growth support from ferric ammonium citrate in plasma comes from its iron, not from it serving as a carbon or nitrogen source.

“These data suggest that ferric ammonium citrate supports K. pneumoniae growth in plasma by providing iron, rather than acting as a carbon or nitrogen source.”

Giving iron citrate was enough to allow bacterial overgrowth in wild-type mice.

“We showed that hepcidin was necessary for protection against bacterial dissemination in Gram-negative pneumonia and that treatment with iron citrate was sufficient to permit bacterial outgrowth in WT mice.”

Raising hepcidin by itself does not protect mice from lethal infection when plasma iron remains high.

“Similar to prior reports (31), mice treated with iron also had marked elevation of plasma hepcidin as compared with mice treated with PBS in response to acute iron loading (Figure 5D), indicating that hepcidin induction alone is not sufficient to protect mice from fatal infection if plasma iron levels are high and supporting the hypothesis that the beneficial effects of hepcidin are mediated via”

The results suggest that even bacteria with high-affinity iron uptake systems can be thwarted by host iron restriction.

“Our findings indicate that even bacteria equipped with these high-affinity acquisition systems are susceptible to a host mechanism that limits iron availability in the host environment.”

When bacteremia is higher, bacteria spreading from blood into the lung likely add to the lung bacterial load.

“We interpret this as evidence that, in mice with increased bacteremia, bacterial seeding of the lung from the bloodstream contributes to higher lung bacterial content.”

During pneumonia, hepcidin induction requires IL-6.

“In addition, IL-6 neutralization abrogated the increase in plasma hepcidin during the infection and eliminated the associated hypoferremia (Figure 3, B and C), indicating that hepcidin induction during pneumonia is dependent on IL-6.”

Reduced hepcidin production could be an overlooked factor that makes hosts more susceptible to infection.

“Our data suggest that impaired hepcidin production may represent an unappreciated mechanism predisposing these hosts to infection.”

• AssumptionRests on an assumption: The mouse Klebsiella pneumonia model is sufficiently representative of human Gram-negative pneumonia for clinical and therapeutic conclusions.

The ability of hepcidin to restrain bacterial growth during infection depends on iron.

“Taken together, these data suggest that the hepcidin-mediated control of bacterial growth during infection is iron dependent.”

After infection, hepcidin is required to keep plasma iron in check.

“In contrast to reduction in plasma iron during infection in WT mice, plasma iron in hepcidin-deficient mice became more elevated during the infection (Figure 4A), indicating that hepcidin is essential for controlling plasma iron levels following infection.”

In Gram-negative pneumonia, hepcidin was required to prevent bacteria from spreading.

“We showed that hepcidin was necessary for protection against bacterial dissemination in Gram-negative pneumonia and that treatment with iron citrate was sufficient to permit bacterial outgrowth in WT mice.”

• AssumptionRests on an assumption: The mouse Klebsiella pneumonia model is sufficiently representative of human Gram-negative pneumonia for clinical and therapeutic conclusions.

Limiting iron in plasma is enough to provide protection.

“Furthermore, we found that hepcidin-deficient mice treated with minihepcidin were robustly protected from infection despite having tissue iron overload, suggesting that restriction of iron in the plasma is sufficient for protection (Figure 7, C and D).”

Hepcidin's protective effect depended on lowering plasma iron.

“Our data suggest that the hepcidin-dependent protection was mediated by reducing plasma iron, as indicated by our observation that WT mice treated with iron citrate were susceptible to K. pneumoniae infection despite robust hepcidin induction (Figure 1, E and F) and that hepcidin-deficient mice were protected from infection if starved of iron (Supplemental Figure 3B).”