Polymorphisms of iron regulation in susceptibility and manifestation of malaria

FRANK MOCKENHAUPT (Charité) in partnership with Melanie Rug (ANU) and Gaétan Burgio (ANU)

Iron is essential for Plasmodium: iron deficiency (ID) inhibits parasite growth in vitro, increases phagocytosis of parasitized murine erythrocytes, and protects against malaria in rodents and humans. Upregulation due to blood stage malaria of the iron master-regulator hepcidin reduces parasite survival at the hepatic bottleneck and thereby curtails secondary Plasmodium infections. Various polymorphisms influence iron status whereas hepcidin levels are regulated by pro- and anti-inflammatory cytokines [1, 2]. The Mockenhaupt group has shown that in pregnant women, ID [3] as well as a ferroportin polymorphism associates with reduced placental malaria. The group hypothesizes that common polymorphisms of iron and hepcidin regulation influence, directly or via ID, risk and manifestation of P. falciparum and P. vivax malaria. Moreover, they hypothesize that enhanced phagocytosis of iron-deficient parasitized RBCs [4] occurs and that this goes along with an enhanced activation of innate immune responses.

In this project, polymorphisms in genes involved in iron/hepcidin regulation (e.g. SLCIIA2, SLC40A1, TF, TNFa, IL-10) will be genotyped and compared between malaria cases and controls using available samples and prospectively recruited groups. The latter will involve MD doctoral students contributing to on-site recruitment in Africa and Asia. Polymorphisms will be related to biomarkers of iron status and to the odds of various entities and syndromes of malaria. For growth and phagocytosis assays, red blood cells with and without ID, iron chelator treatment, and/or polymorphisms of interest (obtained from patients and volunteers) will be used to cultivate P. falciparum (each, groups of 10-20). Parasite multiplication will be monitored by microscopy, flow cytometry, and RT-PCR. P. falciparum-infected RBCs (>90% parasitaemia, MACS separated) and monocytes from non-autologous, malaria naïve donors will be co-cultivated for 24-48h; phagocytosis rates and activity will be assessed microscopically and by ELISA and surface marker expression. For the assessment of activation of innate immune responses, cytokine concentrations in supernatants will be measured by ELISA, cytokine mRNA levels by RT-PCR, and surface marker expression by flow cytometry. For the identification of pathways involved in potentially modified recognition and activation, monoclonal antibodies and siRNA techniques will be used.

Surface variation of infected RBCs, e.g. in ID, will be identified by high resolution scanning microscopy in cooperation with the Rug lab, and analysis of surface membrane components and adhesion properties will be done in collaboration with the Maier lab. Identified relevant polymorphisms will be tested for significance by using CRISPR/Cas9 mediated genome-editing technology of erythroblasts and subsequent monitoring of parasite growth in cooperation with the Burgio lab. Screening of polymorphisms among available clinical samples and prospectively recruited groups will be extended to include platelet-related variants (Brendan McMorran), riboflavin deficiency (Kevin Saliba), and other host polymorphisms (Simon Foote).

Interlinkages: Alexander Maier (ANU), Brendan McMorran (ANU), Kevin Saliba (ANU), Simon Foote (ANU).

References:

(1) Spottiswood, N. et al. (2014) Front. Pharmacol. 5:124

(2) McLaren, C.E. et al. (2011) PLoS One 6:e17390

(3) Danquah, I. et al.  (2008) J. Infect. Dis. 198:1573-1574

(4) Matsuzaki-Moriya, C. et al. (2011) Eur. J. Immunol. 41:1365-1375