Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia

Abstract

Malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth. We investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged <5 years between August 1998 and October 2019 (n=75,034). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malarial anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA (n=33); (3) NTS (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. SMA and severe anemia without malaria were associated with a 2-fold or more increased risk of NTS bacteremia, while other malaria phenotypes were not associated with increased NTS risk. Children with SMA had lower hepcidin/ferritin ratios (0.10; interquartile range [IQR]: 0.03-0.19) than those with CM (0.24; IQR: 0.14-0.69; P=0.006) or asymptomatic malaria (0.19; IQR: 0.09-0.46; P=0.01) indicating suppressed hepcidin levels. Children with SMA+NTS had lower hepcidin levels (9.3 ng/mL; IQR: 4.7-49.8) and hepcidin/ferritin ratios (0.03; IQR: 0.01-0.22) than those with NTS alone (105.8 ng/mL; IQR: 17.3-233.3; P=0.02 and 0.31; IQR: 0.06-0.66; P=0.007, respectively). Since hepcidin degrades ferroportin on the Salmonella-containing vacuole, we hypothesize that reduced hepcidin in children with SMA might contribute to NTS growth by modulating iron availability for bacterial growth. Further studies are needed to understand how the hepcidin-ferroportin axis might mediate susceptibility to NTS in severely anemic children.

Figure 1.

The hepcidin-link between severe malarial anemia and non-typhoidal Salmonella bacteremia. Low hepcidin levels in children with severe malarial anemia (SMA) may contribute to the risk of non-typhoidal Salmonella (NTS) bacteremia. (A) During malaria infection, proinflammatory responses and parasitemia induce the expression of hepcidin, the main iron regulatory hormone. Hepcidin degrades ferroportin (FPN) on the macrophage membrane and the Salmonella-containing vacuole (SCV), resulting in decreased iron availability for NTS bacteria. The bacteria may also utilize other iron acquisition strategies such as transferrin through transferrin receptors (TfR) in early endosomes. Proinflammatory responses, including production of interleukin (IL)-6 and interferon-gamma (IFN-γ), mediate killing of NTS through formation of reactive oxygen species (ROS) and other pathways. (B) In SMA, increased hemolysis and erythropoietic drive induce production of erythroferrone (ERFE), a hormone that downregulates hepcidin synthesis. This results in increased expression of FPN on the surface of the macrophage and the SCV. Heme from hemolyzed parasitized red blood cells (pRBC) and the haptoglobin-hemoglobin (Hp-Hb) complex is broken down by heme oxygenase-1 (HO-1) into equimolar amounts of iron, biliverdin and carbon monoxide. HO-1 and hemebreakdown products downregulate immune responses and promote an anti-inflammatory environment. The net effect of low hepcidin, increased HO-1, SMA-induced anti-inflammatory cytokines such as IL-10 and increased intra-SCV iron levels is increased proliferation of NTS bacteria. DMT-1: divalent metal transporter 1; Cp: ceruloplasmin.

Figure 2.

Selection of study participants. All children aged ≤60 months with complete age and hemoglobin data admitted between August 1998 and October 2019 were included in the retrospective epidemiological analysis. Children with concomitant severe malaria and non-typhoidal Salmonella (NTS), and whose specimens were available in the Kilifi biobank, were enrolled into the iron and hepcidin sub-study. Each child was then matched with two hospitalized children with NTS alone, severe malaria anemia (SMA) alone, and cerebral malaria (CM) alone based on age and sex.

Figure 3.

Iron and inflammatory biomarkers in children with malaria. Circulating levels of (A) hepcidin, (B) ferritin, (C) hepcidin/ferritin ratio, (D) soluble transferrin receptors (sTfR), (E) C-reactive protein (CRP), and (F) parasite density. P values from pairwise comparisons were determined by the Wilcoxon rank-sum test. NS’ (not significant) indicates P>0.05. AM: asymptomatic malaria; CM: cerebral malaria; SMA: severe malaria anemia.

Figure 4.

Iron and inflammatory biomarkers in children with severe malaria anemia and/or non-typhoidal Salmonella bacteremia. Circulating levels of (A) hepcidin, (B) ferritin, (C) hepcidin/ferritin ratio, (D) soluble transferrin receptors (sTfR), (E) Creactive protein (CRP), and (F) parasite density in children with severe malarial anemia (SMA) and/or non-typhoidal Salmonella (NTS) bacteremia. P values from pairwise comparisons were determined by the Wilcoxon rank-sum test. NS’ (not significant) indicates P>0.05.