Hepcidin Protects against Lethal Escherichia coli Sepsis in Mice Inoculated with Isolates from Septic Patients

Abstract

Iron is an essential micronutrient for most microbes and their hosts. Mammalian hosts respond to infection by inducing the iron-regulatory hormone hepcidin, which causes iron sequestration and a rapid decrease in the plasma and extracellular iron concentration (hypoferremia). Previous studies showed that hepcidin regulation of iron is essential for protection from infection-associated mortality with the siderophilic pathogens Yersinia enterocolitica and Vibrio vulnificus However, the evolutionary conservation of the hypoferremic response to infection suggests that not only rare siderophilic bacteria but also common pathogens may be targeted by this mechanism. We tested 10 clinical isolates of Escherichia coli from children with sepsis and found that both genetic iron overload (by hepcidin-1 knockout [HKO]) and iatrogenic iron overload (by intravenous iron) potentiated infection with 8 out of the 10 studied isolates: after peritoneal injection of E. coli, iron-loaded mice developed sepsis with 60% to 100% mortality within 24 h, while control wild-type mice suffered 0% mortality. Using one strain for more detailed study, we show that iron overload allows rapid bacterial multiplication and dissemination. We further found that the presence of non-transferrin-bound iron (NTBI) in the circulation is more important than total plasma or tissue iron in rendering mice susceptible to infection and mortality. Postinfection treatment of HKO mice with just two doses of the hepcidin agonist PR73 abolished NTBI and completely prevented sepsis-associated mortality. We demonstrate that the siderophilic phenotype extends to clinically common pathogens. The use of hepcidin agonists promises to be an effective early intervention in patients with infections and dysregulated iron metabolism.

Keywords: Escherichia coli; NTBI; hepcidin; infection; iron; sepsis.

FIG 1

Genetic as well as iatrogenic iron overload enhances sepsis-associated mortality in a mouse model of E. coli infection (A and B) Survival (A) and the number of bacterial CFU in blood and liver (B) after infection of wild-type (WT) or naturally iron-loaded hepcidin-1 knockout (IL-HKO) mice with 10⁴ CFU/mouse E. coli (isolate 1). Dashed line, limit of detection. (C and D) Survival (C) and number of bacterial CFU in blood and liver (D) after infection of IL-HKO or dietary iron-depleted HKO (ID-HKO) mice with 10⁴ CFU of E. coli (isolate 1) per mouse. Dashed line, limit of detection. (E and F) Survival of WT mice that were injected i.v. with saline, 2 to 4 mg iron sucrose (E), or 2 mg iron dextran (F) and 4 h later infected i.p. with 10⁴ CFU of E. coli (isolate 1) per mouse. The results of two separate experiments were combined for panels E and F. In the experiment whose results are shown in panel E, two different batches of iron sucrose were used and pretested prior to these experiments. In pilot experiments, the first batch promoted susceptibility to infection at 2 mg/mouse. In pilot studies with the second batch, 2 mg/mouse did not promote infection, while 4 mg/mouse did, consistent with the reported variability of rapid labile iron release from such preparations (44).

FIG 2

E. coli infection rapidly induces systemic inflammation, increases hepcidin levels, and causes hypoferremia. WT mice were injected i.p. with either saline or 10⁴ CFU of E. coli (isolate 1) per mouse. Tissues were collected at 4 and 16 h postinfection for analysis. We measured liver Saa1 levels as a marker of systemic inflammation (A), hepcidin protein (B), hepcidin mRNA (C), and the serum iron concentration (D). Inflammation induces the initial rise in serum hepcidin protein and mRNA levels, followed by hypoferremia and a reactive decrease in hepcidin protein and mRNA levels.

FIG 3

Minihepcidin prevents E. coli sepsis-associated mortality in a model of hereditary hemochromatosis. Naturally iron-loaded HKO mice were infected i.p. with 10⁴ CFU of E. coli (isolate 1) per mouse and treated with solvent (SO) or 100 nmol minihepcidin (MH) at +3 and +24 h p.i. Survival was monitored (A), and in two repeat experiments, tissues were collected at 16 h p.i. to assess the bacterial burden in the blood and liver (B). Dashed line, limit of detection.

FIG 4

Non-transferrin-bound iron (NTBI) rather than total iron promotes susceptibility to E. coli sepsis. (A to C) Liver iron, serum iron, and non-transferrin-bound iron measurements for WT (white boxes) and IL-HKO (red boxes) mice (A), IL-HKO mice treated i.p. either with a single dose of solvent (red boxes) or 100 nmol minihepcidin (white boxes) at 14 h prior to tissue collection (B), or IL-HKO (red boxes) and ID-HKO (white boxes) mice (C). All mice were infected i.p. with 10⁴ CFU of E. coli (isolate 1) per mouse, and tissues were collected at 14 to 16 h postinfection (p.i.). IL, iron loaded; ID, iron depleted. (D) Human plasma was supplemented with 0 to 60 μM ferric ammonium citrate (FAC) and used to make agar plates. E. coli isolate 1 was plated and incubated for 38 to 41 h at 37°C, and the number of bacterial CFU was documented by photography. The results of two separate experiments using plasma from different donors are shown. Conditions below the red line had detectable NTBI.