Tmprss6 Depletion-Mediated Iron Deficiency Attenuates the Clinical Course of Plasmodium Berghei Infection in Mice

Background Anaemia and malaria cause a great burden of disease and commonly co-exist. Field studies have linked iron deficiency with decreased malaria risk but may be confounded. We aimed to define the association between iron status and malaria risk in an in vivo experimental system. Using the P. berghei mouse malaria model, we assessed the impact of both severe iron overload and iron deficiency using inducible hepcidin (Hamp) knockout (iHamp-KO), Tmprss6 knockout (Tmprss6-KO) and Tmprss6 knockdown (Tmprss6-KD) mouse models. Homozygous mutations of HAMP (encoding hepcidin) cause severe iron overload, while homozygous inactivation of TMPRSS6 leads to hepcidin overexpression and iron deficiency.

MethodsP. berghei infection of C57BL/6 mice is an experimental model of cerebral and severe malaria. The majority of mice develop neurological symptoms between day 6-10 post infection, surviving mice usually develop hyperparasitaemia and symptoms of severe anaemia by day 20. Mice were infected with PbmCherryLuci sporozoites by intravenous injection. Luciferase activity was used to assess liver infection and egress. Mice were analysed by IVIS at 24, 36, 52 and 55/56 hours post infection. From day 3 post infection peripheral parasitaemia was assessed daily. Mice were euthanised if they showed signs of cerebral malaria (such as loss of self-righting reflex and hind-limb paresis), severe anaemia, or if parasitaemia reached >20% (iHamp-KO and Tmprss6-KO) or >30% (Tmprss6-KD) in line with animal ethics requirements.

iHamp-KO mice received tamoxifen intraperitoneally daily for 5 days 3 months prior to infection to induce Hamp deletion. Controls were iHamp-KO littermates that received vehicle alone. Tmprss6-KO mice were infected at about 24 weeks of age and compared to littermate heterozygote controls which are known to have a negligible phenotype. Tmprss6-KD was achieved through treatment of WT mice with siTMP, a GalNAc siRNA conjugate targeting Tmprss6. Mice received siTMP 5mg/kg s.c. 3 weekly starting 8 weeks prior to P. berghei infection, or a single treatment on day 3 (expected onset of blood stage infection) or day 6 (expected onset of high risk period for cerebral malaria), while controls received PBS.

Results iHamp-KO mice had lower peak liver infection (p=0.0097), but egress was not affected, and there was no effect on overall survival or survival from cerebral malaria. Tmprss6-KO mice showed higher liver infection compared with heterozygous littermate controls at all timepoints, but egress was not different. Tmprss6-KO mice had improved overall survival compared with heterozygous controls (p=0.0004) and improved survival from cerebral malaria (p=0.0004). All 7 heterozygous controls succumbed to cerebral malaria by day 10, compared to 1 of 6 Tmprss6-KO animals (Figure 1). We further investigated effects of iron deficiency on malaria severity using Tmprss6 knockdown. Mice treated with siTMP for 8-10 weeks had markedly reduced Tmprss6 and elevated Hamp expression in the liver, and lower haemoglobin, MCV, and plasma iron. Tmprss6-KD mice, treated 8 weeks prior to P. berghei infection exhibited reduced parasitaemia, and took longer to reach 10%, 20% and 30% parasitaemia (Figure 2). There was no change in survival from cerebral malaria. Tmprss6-KD after infection did not change parasitaemia or survival.

Conclusion Experimental iron deficiency via Tmprss6 downregulation attenuated malaria disease phenotype in mice. These data provide experimental evidence to support observational data that iron deficiency provides protection against malaria infection.

Schaeper:Silence Therapeutics PLC: Current Employment, Current holder of stock options in a privately-held company. Pasricha:Vifor Pharma: Consultancy; Keros Therapeutics: Consultancy.