Download 2016 department of medicine research day

2016 DEPARTMENT OF MEDICINE RESEARCH DAY
Title of Poster: Hepcidin protects against extracellular infections by eliminating nontransferrin-bound iron
Presenter: Debora Stefanova
Division: Pulmonary and Critical Care Medicine
☐ Faculty ☐ Fellow ☐ Resident ☐ Post-doc Research Fellow ☒ Graduate Student ☐ Medical Student ☐Other
Principal Investigator/Mentor: Elizabeta Nemeth Co-Investigators: Antoan Raychev, Joao Arezes, Kathryn Michels,
Barbara Dillon, Marcus Horwitz, Borna Mehrad, Tomas Ganz, Yonca Bulut
Thematic Poster Category:
Atherosclerosis
Infections, Injury and Repair, Inflammation, Host Defense, Immunology, Hemostasis and
Abstract
The production of the iron-regulatory hormone hepcidin is strongly induced by infection and
inflammation, causing hypoferremia. This is proposed to be a host defense response to limit microbial
replication. Patients with hereditary hemochromatosis or β-thalassemia, who are hepcidin-deficient
and iron-overloaded, are susceptible to infections with gram-negative bacteria. The specific
mechanism by which hepcidin and hypoferremia mediate resistance to infection, and the spectrum of
microbes affected, are poorly understood.
We tested the role of hepcidin in infections with several groups of bacteria: 1) siderophilic gramnegative Yersinia enterocolitica and Vibrio vulnificus, known to have increased virulence in patients
with iron overload; 2) clinically common gram-negative Klebsiella pneumoniae and gram-positive
Staphylococcus aureus; and 3) primarily intracellular Mycobacterium tuberculosis.
In vivo role of hepcidin and iron was assessed by comparing survival and tissue bacterial burden
between wild type (WT) and hepcidin knockout (KO) mice. Hepcidin KO mice were tested both in ironloaded and iron-depleted condition to assess for any iron-independent roles of hepcidin in infection.
Hepcidin played the most prominent host defense role in infections with siderophilic pathogens Y.
enterocolitica and V. vulnificus. At inocula where WT mice were resistant and had 0% mortality, ironloaded hepcidin KO mice were highly susceptible to these infections and displayed 100% mortality
(Figure 1). Compared to WT mice, iron-loaded hepcidin KO were also more susceptible to infection
with K. pneumoniae, and displayed more rapid and higher mortality (Figure 1). In all of these models,
treatment of infected iron-loaded hepcidin KO mice with hepcidin analogs prevented or decreased
mortality. Short-term administration of hepcidin analogs altered serum iron concentrations but not
iron stores, indicating that only extracellular iron availability plays a role in infection with these
extracellular, gram-negative pathogens. Additional experiments indicated that the protective effect of
hepcidin and minihepcidin was not caused by any direct antimicrobial activity but by their effect on
extracellular iron.
In contrast, in mouse models of infection with gram-positive S. aureus or with M. tuberculosis, there
was no difference in susceptibility between hepcidin KO and WT mice. We further tested the effect of
parenteral iron administration in WT mice infected with S. aureus and M. tuberculosis but iron loading
of these mice had no effect on bacterial burden. These results indicated that neither hepcidin nor iron
availability affect the virulence of these bacteria.
We next examined the mechanism by which hepcidin deficiency promotes the growth of gramnegative pathogens. Hepcidin causes a decrease in iron-transferrin concentrations, however iron-
transferrin is accessible only to a few highly host-adapted pathogens and most bacteria cannot utilize
this form of iron. On the other hand, non-transferrin-bound iron (NTBI), a form of iron that appears in
the circulation when the iron-binding capacity of transferrin is exceeded, may be readily utilizable by
microbes. Indeed, mice that had increased susceptibility to infection in our models also had detectable
NTBI in plasma. We developed an in vitro model to test the dependence of bacterial growth on irontransferrin or NTBI concentrations. Human plasma was supplemented with graded amounts of ferric
ammonium citrate and agar plates generated with a range of increasing transferrin saturation and
eventually increasing NTBI concentrations. V. vulnificus, Y. enterocolitica and K. pneumoniae only
grew when sufficient iron was added to saturate transferrin and generate NTBI. Similarly, bacterial
CFU counts in vivo correlated with the presence of NTBI in mouse models but not with transferrin
saturation when NTBI was absent.
Using mouse models of different bacterial infections, we demonstrated that hepcidin is essential for
resistance to infection with certain gram-negative pathogens, but plays no role in the susceptibility to
intracellular M. tuberculosis or gram-positive S. aureus. We further provide evidence that the primary
host defense function of hepcidin is the elimination of NTBI, and this can be potentiated by the use of
synthetic hepcidin analogues to prevent progression of infection and associated mortality.
Figure 1. Survival of WT and iron-overloaded hepcidin KO mice after infection with V. vulnificus, Y.
enterocolitica or K. pneumoniae.