In sub-Saharan Africa, invasive non-Typhoid Salmonella (NTS) is a common and often fatal complication of Plasmodium falciparum infection. Induction of heme oxygenase-1 (HO-1) mediates tolerance to the cytotoxic effects of heme during malarial hemolysis but might impair resistance to NTS by limiting production of bactericidal reactive oxygen species. We show that co-infection of mice with Plasmodium yoelii 17XNL (Py17XNL) and S. typhimurium causes acute, fatal bacteremia with increased bacterial load; features reproduced by phenylhydrazine hemolysis or hemin administration. S. typhimurium localized predominantly in granulocytes. Py17XNL, phenylhydrazine and hemin caused premature mobilization of granulocytes from bone marrow with a quantitative defect in the oxidative burst. Inhibition of HO by tin protoporphyrin abrogated the impairment of resistance to S. typhimurium by hemolysis. Thus a mechanism of tolerance to one infection, malaria, impairs resistance to another, NTS. Furthermore, HO inhibitors may be useful adjunctive therapy for NTS infection in the context of hemolysis.

Neutrophils are essential innate immune cells that extrude chromatin in the form of neutrophil extracellular traps (NETs) when they die. This form of cell death has potent immunostimulatory activity. We show that heme-induced NETs are essential for malaria pathogenesis. Using patient samples and a mouse model, we define two mechanisms of NET-mediated inflammation of the vasculature: activation of emergency granulopoiesis via granulocyte colony-stimulating factor production and induction of the endothelial cytoadhesion receptor intercellular adhesion molecule–1. Soluble NET components facilitate parasite sequestration and mediate tissue destruction. We demonstrate that neutrophils have a key role in malaria immunopathology and propose inhibition of NETs as a treatment strategy in vascular infections.

It is not known why people are more susceptible to bacterial infections such as non-Typhoid Salmonella (NTS) during and after a malaria infection but, in mice, malarial hemolysis impairs resistance to NTS by impairing the neutrophil oxidative burst. This acquired neutrophil dysfunction is a consequence of induction of the cytoprotective, heme degrading enzyme heme oxygenase-1 (HO-1) in neutrophil progenitors in bone marrow. In this study, we assessed whether neutrophil dysfunction occurs in humans with malaria and how this relates to hemolysis. We evaluated neutrophil function in 58 Gambian children with Plasmodium falciparum malaria (55 (95%) with uncomplicated disease), and examined associations with erythrocyte count, haptoglobin, hemopexin, plasma heme, expression of receptors for heme uptake, and HO-1 induction. Malaria caused the appearance of a dominant population of neutrophils with reduced oxidative burst activity, which gradually normalized over 8 weeks of follow-up. The degree of neutrophil impairment correlated significantly with markers of hemolysis and HO-1 induction. HO-1 expression was increased in blood during acute malaria, but at a cellular level HO-1 expression was modulated by changes in surface expression of the haptoglobin receptor (CD163). These findings demonstrate that neutrophil dysfunction occurs in P. falciparum malaria and support the relevance of the mechanistic studies in mice. Furthermore, they suggest the presence of a regulatory pathway to limit HO-1 induction by hemolysis in the context of infection, and indicate new targets for therapeutic intervention to abrogate the susceptibility to bacterial infection in the context of hemolysis in humans.

Heme oxygenase 1 (HO-1) is an essential enzyme induced by heme and multiple stimuli associated with critical illness. In humans, polymorphisms in the HMOX1 gene promoter may influence the magnitude of HO-1 expression. In many diseases including murine malaria, HO-1 induction produces protective anti-inflammatory effects, but observations from patients suggest these may be limited to a narrow range of HO-1 induction, prompting us to investigate the role of HO-1 in malaria infection. In 307 Gambian children with either severe or uncomplicated P. falciparum malaria, we characterized the associations of HMOX1 promoter polymorphisms, HMOX1 mRNA inducibility, HO-1 protein levels in leucocytes (flow cytometry), and plasma (ELISA) with disease severity. The (GT) n repeat polymorphism in the HMOX1 promoter was associated with HMOX1 mRNA expression in white blood cells in vitro , and with severe disease and death, while high HO-1 levels were associated with severe disease. Neutrophils were the main HO-1-expressing cells in peripheral blood, and HMOX1 mRNA expression was upregulated by heme-moieties of lysed erythrocytes. We provide mechanistic evidence that induction of HMOX1 expression in neutrophils potentiates the respiratory burst, and propose this may be part of the causal pathway explaining the association between short (GT) n repeats and increased disease severity in malaria and other critical illnesses. Our findings suggest a genetic predisposition to higher levels of HO-1 is associated with severe illness, and enhances the neutrophil burst leading to oxidative damage of endothelial cells. These add important information to the discussion about possible therapeutic manipulation of HO-1 in critically ill patients.

The pathogenesis of infectious diseases depends on the interaction of host and pathogen. In Plasmodium falciparum malaria, host and parasite processes can be assessed by dual RNA-sequencing of blood from infected patients. Here we performed dual transcriptome analyses on samples from 46 malaria-infected Gambian children to reveal mechanisms driving the systemic pathophysiology of severe malaria. Integrating these transcriptomic data with estimates of parasite load and detailed clinical information allowed consideration of potentially confounding effects due to differing leukocyte proportions in blood, parasite developmental stage, and whole-body pathogen load. We report hundreds of human and parasite genes differentially expressed between severe and uncomplicated malaria, with distinct profiles associated with coma, hyperlactatemia, and thrombocytopenia. High expression of neutrophil granule-related genes was consistently associated with all severe malaria phenotypes. We observed severity-associated variation in the expression of parasite genes which determine cytoadhesion to vascular endothelium, rigidity of infected erythrocytes, and parasite growth rate. Up to 99% of human differential gene expression in severe malaria was driven by differences in parasite load, whereas parasite gene expression showed little association with parasite load. Co-expression analyses revealed interactions between human and P. falciparum, with prominent co-regulation of translation genes in severe malaria between host and parasite. Multivariate analyses suggested that increased expression of granulopoiesis and interferon-γ related genes, together with inadequate suppression of type-1 interferon signalling, best explained severity of infection. These findings provide a framework for understanding the contributions of host and parasite to the pathogenesis of severe malaria and identifying targets for adjunctive therapy.

Summary

Plasmodium falciparum malaria and non-typhoid Salmonella (NTS) bacteraemia are both major causes of morbidity and mortality in children in sub-Saharan Africa. Co-infections are expected to occur because of their overlapping geographical distribution, but accumulating evidence indicates that malaria is a risk factor for NTS bacteraemia. A literature review was undertaken to provide an overview of the evidence available for this association, the epidemiology of malaria-NTS co-infection (including the highest risk groups), the underlying mechanisms, and the clinical consequences of this association, in children in sub-Saharan Africa. The burden of malaria-NTS co-infection is highest in young children (especially those less than three years old). Malaria is one of the risk factors for NTS bacteraemia in children, and the risk is higher with severe malaria, especially severe malarial anaemia. There is insufficient evidence to determine whether asymptomatic parasitaemia is a risk factor for NTS bacteraemia. Many mechanisms have been proposed to explain how malaria causes susceptibility to NTS, ranging from macrophage dysfunction to increased gut permeability, but the most consistent evidence is that malarial haemolysis creates conditions which favour bacterial growth, by increasing iron availability and by impairing neutrophil function. Few discriminatory clinical features have been described for those with malaria and NTS co-infection, except for a higher risk of anaemia compared to those with either infection alone. Children with malaria and NTS bacteraemia co-infection have higher case fatality rates compared to those with malaria alone, and similar to those with bacteraemia alone. Antimicrobial resistance is becoming widespread in invasive NTS serotypes, making empirical treatment problematic, and increasing the need for prevention measures. Observational studies indicate that interventions to reduce malaria transmission might also have a substantial impact on decreasing the incidence of NTS bacteraemia.

Severe malaria defines individuals at increased risk of death from their infection. Proposed pathogenic mechanisms include parasite sequestration, inflammation, and endothelial dysfunction. Severe malaria is not a single entity, manifesting with distinct syndromes such as severe anemia, severe respiratory distress or coma, each characterized by differences in epidemiology, underlying biology, and risk of death. The relative contribution of the various pathogenic mechanisms may differ between syndromes, and this is supported by accumulating evidence, which challenges sequestration as the initiating event. Here we propose that high parasite biomass is the common initiating feature, but subtle variations in the interaction between the host and parasite exist, and understanding these differences may be crucial to improve outcomes in patients with severe malaria. KeywordsSevere malaria; sequestration; inflammation; endothelial activation; biomass Severe malariaSymptomatic malaria occurs during the erythrocytic stage (see Glossary) of infection with protozoal parasites of the genus Plasmodium, when parasites replicate within red blood cells (RBCs). The term severe malaria applies to manifestations associated with an increased risk of death or other adverse outcome (such as brain injury). Of the six Plasmodium species known to infect humans, Plasmodium falciparum causes the greatest burden of disease and accounts for up to 1.24 million deaths annually, mostly in African children [1]. The definition of severe malaria is based on World Health Organization criteria [2] and includes clinical and laboratory features, which are predictive of death in those receiving antimalarial treatment. However, the vast majority of children with severe malaria can be recognized from the presence of just three clinical syndromes: (i) coma (cerebral malaria); (ii) severe © 2013 Elsevier Ltd. All rights reserved. Corresponding Author: Cunnington, A.J. (a.cunnington@imperial.ac.uk). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access What causes severe malaria?The pathogenesis of severe malaria is hotly debated; some authors suggest that sequestration (Box 1) is the overriding pathogenic mechanism [17], whilst others believe that inflammatory processes are more important [18] ( Figure 1A). An accumulating body of evidence indicates that vascular endothelial dysfunction is also important [15] and could be the interface between sequestration and inflammation [19]. Establishing the causal role of any single mechanism in severe malaria in humans is difficult, and no existing animal ...