Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Single-cell RNA sequencing and analysis of rodent blood stage Plasmodium.

Bulk RNA sequencing of Plasmodium spp., the causative parasite of malaria, fails to discriminate developmental-stage-specific gene regulation. Here, we provide a protocol that uses single-cell RNA sequencing of FACS-sorted Plasmodium-chabaudi-chabaudi-AS-infected red blood cells (iRBCs) to characterize developmental-stage-specific modulation of gene expression during malaria blood stage. We describe steps for infecting mice, monitoring disease progression, preparing iRBCs, and single-cell sequencing iRBCs. We then detail procedures for analyzing scRNA-seq data. For complete details on the use and execution of this protocol, please refer to Ramos et al.1.

Renal control of life-threatening malarial anemia.

Iron recycling prevents the development of anemia under homeostatic conditions. Whether iron recycling was co-opted as a defense strategy to prevent the development of anemia in response to infection is unclear. We find that in severe Plasmodium falciparum malaria, the onset of life-threatening anemia is associated with acute kidney injury (AKI), irrespective of parasite load. Using a well-established experimental rodent model of malaria anemia, we identify a transcriptional response that endows renal proximal tubule epithelial cells (RPTECs) with the capacity to store and recycle iron during P. chabaudi chabaudi (Pcc) infection. This response encompasses the induction of ferroportin 1/SLC40A1, which exports iron from RPTECs and counteracts AKI while supporting compensatory erythropoiesis and preventing the onset of life-threatening malarial anemia. Iron recycling by myeloid cells is dispensable to this protective response, suggesting that RPTECs provide an iron-recycling salvage pathway that prevents the pathogenesis of life-threatening malarial anemia.

A hypometabolic defense strategy against malaria.

Hypoglycemia is a clinical hallmark of severe malaria, the often-lethal outcome of Plasmodium falciparum infection. Here, we report that malaria-associated hypoglycemia emerges from a non-canonical resistance mechanism, whereby the infected host reduces glycemia to starve Plasmodium. This hypometabolic response is elicited by labile heme, a byproduct of hemolysis that induces illness-induced anorexia and represses hepatic glucose production. While transient repression of hepatic glucose production prevents unfettered immune-mediated inflammation, organ damage, and anemia, when sustained over time it leads to hypoglycemia, compromising host energy expenditure and adaptive thermoregulation. The latter arrests the development of asexual stages of Plasmodium via a mechanism associated with parasite mitochondrial dysfunction. In response, Plasmodium activates a transcriptional program associated with the reduction of virulence and sexual differentiation toward the generation of transmissible gametocytes. In conclusion, malaria-associated hypoglycemia represents a trade-off of a hypometabolic-based defense strategy that balances parasite virulence versus transmission.

Evaluating antidisease immunity to malaria and implications for vaccine design.

Immunity to malaria could be categorized broadly as antiparasite or antidisease immunity. While most vaccine research efforts have focused on antiparasite immunity, the evidence from endemic populations suggest that antidisease immunity is an important component of natural immunity to malaria. The processes that mediate antidisease immunity have, however, attracted little to no attention, and most interests have been directed towards the antibody responses. This review evaluates the evidence for antidisease immunity in endemic areas and discusses the possible mechanisms responsible for it. Given the key role that inflammation plays in the pathogenesis of malaria, regulation of the inflammatory response appears to be a major mechanism for antidisease immunity in naturally exposed individuals.

Patterns of inflammatory responses and parasite tolerance vary with malaria transmission intensity.

BACKGROUND: In individuals living in malaria-endemic regions, parasitaemia thresholds for the onset of clinical symptoms vary with transmission intensity. The mechanisms that mediate this relationship are however, unclear. Since inflammatory responses to parasite infection contribute to the clinical manifestation of malaria, this study investigated inflammatory cytokine responses in children with malaria from areas of different transmission intensities (ranging from low to high). METHODS: Blood samples were obtained from children confirmed with malaria at community hospitals in three areas with differing transmission intensities. Cytokine levels were assessed using the Luminex®-based magnetic bead array system, and levels were compared across sites using appropriate statistical tests. The relative contributions of age, gender, parasitaemia and transmission intensity on cytokine levels were investigated using multivariate regression analysis. RESULTS: Parasite density increased with increasing transmission intensity in children presenting to hospital with symptomatic malaria, indicating that the parasitaemia threshold for clinical malaria increases with increasing transmission intensity. Furthermore, levels of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-6, IL-8, and IL-12, decreased with increasing transmission intensity, and correlated significantly with parasitaemia levels in the low transmission area but not in high transmission areas. Similarly, levels of anti-inflammatory cytokines, including IL-4, IL-7, IL-10 and IL-13, decreased with increasing transmission intensity, with IL-10 showing strong correlation with parasitaemia levels in the low transmission area. Multiple linear regression analyses revealed that transmission intensity was a stronger predictor of cytokine levels than age, gender and parasitaemia. CONCLUSION: Taken together, the data demonstrate a strong relationship between the prevailing transmission intensity, parasitaemia levels and the magnitude of inflammatory responses induced during clinical malaria.