Download Collin-Jamal Smith The Role of Ferroportin in Macrophage

Collin-Jamal Smith
The Role of Ferroportin in Macrophage-Mediated Immunity
From before birth until death, macrophages exhibit an integral role in tissue homeostasis.
Their phagocytic abilities are used for clearing cell debris, as observed in embryogenic apoptosis
(Böse et al., 2004), and for destroying pathogens, as observed in innate and adaptive immune
responses. One role of macrophages in the reticuloendothelial system (spleen, liver, bone
marrow, and other lymphoid tissues) is eryptosis, or the phagocytosis and elimination of
senescent erythrocytes in circulation; a consequence of this is the elevation of intracellular iron
(Fe2+) levels. The iron is either stored in ferritin proteins or exported out of the cell to the plasma
via the ferroportin protein.
Ferroportin-driven iron export is regulated by the presence hepcidin. Hepcidin, a peptide
of hepatic origin, binds to ferroportin and transports it to a lysosome within which it is degraded.
Consequently, high levels of hepcidin result in low levels of ferroportin and high levels of iron
inside macrophages; the reverse of this also holds true. Among other stimuli, hepcidin synthesis
is induced by excessive iron levels in the blood and the presence of lipopolysaccharide. Since
ferroportin is the only known transporter of iron into the plasma and hepcidin is the primary
regulator of ferroportin, disorders whose symptoms involve detrimentally high iron levels are
commonly caused by the disruption of the ferroportin/hepcidin equilibrium (Abboud et al.,
2000). Aberrations from this equilibrium commonly have genetic origins; mutations in the Hfe
gene, for example, enervate the body’s ability to sense serum iron levels. As a result, hepcidin is
under expressed, allowing the overexpression of ferroportin and the over-export of iron into the
blood. Similarly, autosomal dominant mutations in the ferroportin gene cause acute variation in
iron homeostasis. One group of mutations is characterized by a delocalization from the plasma
membrane to the center of the cell; this dissonance suffers from iron export ebbing, but boasts
hepcidin resistance. Intracellular iron concentrations experience a pronounced augmentation
under these conditions. The other group of mutations produces proteins with typical iron
exporting capabilities, but its interactions with hepcidin are dysfunctional. Evasion of
counterbalancing degradation causes a concurrent rise in serum iron levels.
Various pathogens, such as Mycobacterium tuberculosis, Chlamydia trachomatis, and
Legionella pneumophila, grow inside macrophages; moreover, their growth and virulence are
elevated under conditions of iron abundance in vivo and in vitro. In Lounis, N. et al., 2001,
researchers tested the influence of iron administration on the growth of M. tuberculosis in 8 week
old Balb/C female mice. Thirty mice were treated with 50 mg/kg of polymaltose ferric
hydroxide three times a week for two weeks before infection. The control group contained
thirty mice which did not receive the iron supplement prior to infection. Each of the
sixty mice was then injected intravenously with 7.2×103 colony forming units (CFU) of the
virulent H37Rv strain of M. tuberculosis; 42 days later, they were sacrificed. Amounts of
M. tuberculosis were quantified using cultures of the lungs and the spleen. The cultures were
homogenized, diluted, plated in triplicate on medium, and recorded after 6 weeks of incubation
at 37°C. A significant difference increase in spleen weights and a significant increase in both
lung and spleen M. tuberculosis counts (p<0.001) was observed in the iron-loaded mice. The
results were bolstered by the absence of a significant difference between the body weights of
the iron-fed mice and control mice. Thus, the iron-loading of mice was correlated with an
enhancement in the multiplication of M.tuberculosis in both the lungs and the spleen.
In addition to the aforementioned study, many experiments have demonstrated a
connection between bacterial proliferation and the iron homeostasis of the host cell. Other
evidence suggests that the iron status of a macrophage affects other mechanisms of immune
responses, such as the synthesis of nitric oxide (NO) by the inducible nitric oxide synthase
(iNOS). Infection with virulent M. tuberculosis is known to induce iNOS and, consequently, the
production of microbicidal NO in murine macrophages. In Chan J. et al. (1992), primary
murine peritoneal macrophages were harvested from 8 week old Balb/C female mice. M.
tuberculosis was labeled with [3H]uracil; it is known that 80% of [3H]uracil is assimilated into
the RNA of mycobacteria while the remaining portion ends up in DNA. After 7 days, the M.
tuberculosis cultures were used to infect the murine macrophages. The anti-mycobacterial
activity was measured by metabolic labeling; NO2- levels were quantified by sterilizing the
culture supernatant, applying the Griess reagent, and measuring its absorption at 540nm. As
shown by Figure 1, increases in NO2- were concomitant with increases in [3H]uracil suppression,
illustrating the mycobactericidal effects of NO2-. The results provided support for the view that
the L-arginine-dependent production of reactive nitrogen intermediates was the principal
mechanism by which murine macrophages killed and inhibited virulent M. tuberculosis.
Figure 1: NO2- at acidic pH effectively inhibits M. tuberculosis.
Figure 2: Summary of Ferroportin/Hepcidin/NO control of Intracellular Fe2+ Levels and
Bacterial Growth.
Figure 2 is a summary and revision of the components of the iron homeostasis
mechanism as described previously.
There is a clear connection between iron homeostasis and macrophage anti-microbial
function. This connection warranted the investigation of ferroportin’s effect on NO production
in response to bacterial assaults (Johnson et al. 2010). J774 murine macrophages stably
overexpressing ferroportin and a matched vector control were generated using retroviral
transduction. Cells were cultured in antibiotic-free DMEM (nutrient-rich medium). Total nitrite
concentration was obtained using the Griess reaction. Ferroportin and iNOS levels were detected
using fluorescent antibodies and a Zeiss Axitome Fluorescent microscope. mRNA was isolated
and analyzed using quantitative Real Time PCR. Protein levels were examined using Western
Blotting and the Bradford Assay. Statistical analysis was completed using ANOVA and the
Student’s t test.
Figure 3: Ferroportin Overexpression Limits Early Intracellular M. tuberculosis Growth.
As illustrated by the red staining in graph A, the J774 mutant (FPN.RV2) is expressing
more of the ferroportin protein (the darker stain indicates a larger amount) when compared to the
control (GFP.RV). Moreover, graph B shows that the mutant is expressing less ferritin, which
means less iron is being stored inside the cell. This bifold decrease in intracellular iron has a
detrimental effect on the growth of M. tuberculosis as displayed by graph C.
Figure 4: iNOS mRNA is Induced in Ferroportin Overexpressing Cells.
The relative expression of iNOS mRNA in macrophages exposed to M. tuberculosis (top
two graphs on the right side) and lipopolysaccharide (bottom two graphs on the right side) is
generally and significantly induced relative to the control (graph on the left).
Figure 5: Ferroportin Overexpression Inhibits NO Production and iNOS Protein
Expression following LPS Treatment
Contrary to the increase in iNOS mRNA shown in Figure 4, the level of NO in the mutant
FPN.RV2 macrophages is significantly lower than the control. Graph A shows the highly
significant different in µM of nitrite, graph B shows the lack of iNOS protein in the mutant via a
western blot, and graph C shows the lack of red staining due to iNOS protein after 24 hours of
incubation.
Collectively, these findings suggest a novel role for the iron export protein ferroportin in
modulation of the innate immune response through post-transcriptional control of iNOS
expression.
Works Cited
Abboud, S. and D. J. Haile. 2000. A novel mammalian iron-regulated protein involved
in intracellular iron metabolism. J. Biol. Chem. 275:19906-19912.
Chan, J., Y. Xing, R. S. Magliozzo, and B. R. Bloom. 1992. Killing of virulent Mycobacterium
tuberculosis by reactive nitrogen intermediates produced by activated murine
macrophages. J. Exp. Med. 175:1111-1122.
Johnson, E. E., A. Sandgren, B. J. Cherayil, M. Murray, and M. Wessling-Resnick. "The Role of
Ferroportin in Macrophage-mediated Immunity." Infection and Immunity (2010).
Pubmed. Web. 22 Sept. 2010. <http://www.ncbi.nlm.nih.gov/pubmed/20837712>.
Lounis, N., C. Truffot -Pernot, J. Grosset, V. R. Gordeuk, and J. R. Boelaert . 2001. 9
Iron and Mycobacterium tuberculosis infection. J. Clin. Virol. 20:123-126.