The relationship between malarial anaemia, neutrophil function and susceptibility to invasive bacterial disease.

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

It is well documented that children with a recent history of acute malaria infection are at increased risk of developing severe, life threatening bacterial infections and we have recently described a biological mechanism that explains this association. Malaria causes destruction of red blood cells (haemolysis), releasing haemoglobin into the blood stream. Haemoglobin is degraded to heme and the heme is detoxified by an enzyme, heme-ozygenase-1 (HO-1). We found that HO-1 causes abnormal differentiation and loss of function of a white blood cell population (neutrophils) that are essential for killing bacteria. Thus, the anaemia caused by malaria infection causes the neutrophil dysfunction which predisposes to severe bacterial infections.

Severe bacterial infections are much more common in areas where malaria is endemic than in countries where malaria is absent, and as malaria has begun to be controlled, the incidence of severe bacterial disease has also fallen. However, most children who develop severe bacterial infections do not have the history of a very recent episode of acute malaria infection. This has led us to hypothesise that chronic, low grade malaria infections (which are often, erroneously, described as "asymptomatic" infections and which are known to contribute to chronic malarial anaemia) may also increase the risk of severe bacterial infection. In most malaria endemic settings, the major burden of malaria infection is due to these "asymptomatic" cases, which greatly outnumber those with classical malaria symptoms. "Asymptomatically" infected individuals may thus represent a large, hitherto unrecognized, population that is at high risk of developing severe bacterial infection due to persistent neutrophil dysfunction.

Thus, the central hypothesis underpinning this study is that chronic "asymptomatic" malaria leads to persistent haemolysis and neutrophil dysfunction, resulting in a decreased ability to control secondary bacterial infections. In addition, we hypothesise that treatment of chronic "asymptomatic" malaria will restore neutrophil function. To test this hypothesis we will determine the extent to which chronic, low grade "asymptomatic" malaria infection affects neutrophil function and the ability to control bacterial infections. We will begin by exploring the association between severity and duration of malarial anaemia, neutrophil function and susceptibility to bacterial disease in a mouse model system and then determine the extent to which these findings are relevant in humans by carrying out a cross-sectional study of Gambian children with chronic, subclinical malaria infection and anaemia. Finally, we will carry out a small proof-of-principle study to determine whether treating children with anti-malarial drugs will restore neutrophil function. If so, this may provide the justification for future clinical trials to determine whether public health programmes to treat "asymptomatic" malaria would reduce the incidence of severe bacterial infections in malaria endemic populations.

Technical Summary

A recent episode of clinical malaria is a known risk for subsequent life-threatening invasive bacterial disease, due primarily (but not exclusively) to non-Typhoidal Salmonella (NTS) infection. We have shown that malaria-induced haemolysis increases plasma concentrations of heme and heme-oxygenase-1 (HO-1) which in turn leads to profound neutrophil dysfunction and failure to control NTS. Observations in a mouse model (Cunnington et al, 2012, Nature Medicine) were subsequently verified in children with acute malaria infection (Cunnington et al, 2012, J. Immunol). We have also shown that neutrophil dysfunction is compounded by reduced resistance to bacterial colonisation (Mooney et al, 2015, Sci Rep.) and impaired mucosal immunity (Mooney et al, 2014, Mucosal Immunol.) leading to dissemination of bacteria from the gut to the periphery (Lokken, Mooney et al, 2014, PLoS Pathog.). However, in most malaria endemic settings, the major burden of malarial anaemia is due to chronic, low grade (often, erroneously, described as "asymptomatic") infections. "Asymptomatically" infected individuals thus represent a large, hitherto unrecognized, population that may be at high risk of invasive bacterial disease due to persistent neutrophil dysfunction. If so, treatment of "asymptomatic" infections might restore neutrophil function and reduce the burden of bacteraemia in malaria-endemic communities.

The aim of this research is to determine the extent to which persistent, low density, "asymptomatic" malaria infection impairs immunity to secondary bacterial infections; to further characterise neutrophil dysfunction; and to test whether anti-malarial treatment reverses these defects and restores functional immunity to NTS. Preliminary experiments in mouse models of anaemia will be followed by cross-sectional studies of Gambian children with persistent "asymptomatic" malaria and a proof-of-principle study of the impact of antimalarial therapy on neutrophil function.

Planned Impact

This work may be useful to public health professionals and health policy makers, especially those working within malaria control programmes. If our study confirms a role for chronic, subclinical malaria infections in neutrophil dysfunction and increased susceptibility to invasive bacterial disease, it will impact upon the ongoing debate about the public health relevance of these infections. The risks and benefits of treating subclinical malaria infections need to take account of the individual's wellbeing within the context of sustainable malaria control strategies that may lead to elimination and eradication. Concerns about widespread use of antimalarial drugs leading to more rapid selection and spread of resistant parasites, combined with the costs and logistic challenges of mass drug administration, have tended to swing the balance of opinion against treatment. Our research will inform this debate by revealing some of the consequences of these subclinical infections and the scale of benefits afforded by treatment, and by beginning to identify individuals or epidemiological settings where treatment might be most beneficial. This may persuade public health researchers/policy advisors to commission and/or fund a series of public health trials to directly measure the benefits of treating subclinical malaria infections and to identify the settings where such an intervention might be most effective. If such trials were successful, there would then need to be a debate about the pros and cons of treatment and how to integrate this into routine malaria control. In all likelihood, this process will take at least five years.

Our work may also benefit clinicians by alerting them to the wider consequences of recurrent or persistent haemolysis, informing decisions about whether certain patients might benefit from prophylactic antibiotic treatment, and extending their understanding of the risk factors for invasive bacterial disease. Although the notion that haemolysis may increase the risk of bacterial disease is fairly well-established, it has often been assumed that this is simply due to the increased bioavailability of iron, on which many bacteria thrive. The notion that these patients may also have a global - and long lasting - neutrophil dysfunction would add a new dimension to the understanding of infection risk. This may lead to changes in clinical practice, ideally preceded by clinical trials of the benefits of prophylactic antimicrobial therapy in children recovering from malaria (especially malarial anaemia) or with other forms of haemolytic disease. These impacts could happen very quickly (months to years).

Our work may also be of interest to the pharmaceutical industry in that novel insights into neutrophil biology, and the elucidation of novel pathways of neutrophil dysfunction, may reveal new targets for therapeutic intervention and drug development. This might include drugs to antagonise the pathways associated with heme and HO-1, in order to restore normal neutrophil function, or agonists to reduce neutrophil function in inflammatory diseases associated with excessive neutrophil activation. The time scale for these impacts is likely to be years for the identification of candidate drugs and decades for their eventual licensing and prescription.

Finally, the PDRA employed here, Dr Jason Mooney, will extend his experience of working in animal models of infection to carrying out field based, immune-epidemiological research. Very few infectious disease researchers have the breadth of skills and experience to rapidly translate their fundamental research to relevant clinical settings; even fewer have experience of working in low and middle income countries. This project will allow Dr Mooney to develop a rare combination of skills to apply to infectious disease research in academia, industry or global health policy.

Publications

10 25 50
 
Description Anaemia is common among sepsis patients, but the consequences of sepsis-associated anaemia are poorly understood. Here, our objective was to determine the prevalence and extent of anaemia, haemolysis and inflammation after early hospital admission. We hypothesised that inflammation- or infection-induced haemolysis contributes to sepsis-associated anaemia and that this will lead to expression of HO-1. In this study, plasma obtained from seventy adult patients within 12 hours of admission to intensive care due to sepsis were analysed for anaemia, haemolysis and inflammatory markers by ELISA and microbead array. The majority of patients were anaemic with evidence of haemolysis. Interestingly, concentrations of both haemoglobin and IL-10 were moderately positively correlated with HO-1 concentration whereas HO-1 concentration was weakly negatively correlated with haemopexin . Anaemia, while common, was not associated with HO-1 concentration. After adjusting for confounding, HO-1 induction appears to be associated primarily with IL-10 concentration rather than haemolysis. Disease severity at diagnosis was correlated with early plasma IL-10 (r = 0.35, p = 0.003) and HO-1 (r = 0.24, p = 0.048) concentrations. Notably, admission levels of haem, HO-1, and IL-10 were indicators of survival.

Also, the haemolysis associated with clinical episodes of malaria results in the liberation of haem, which activates the enzyme haem oxygenase-1 (HO-1). HO-1 has been shown to reduce neutrophil function and increase susceptibility to invasive bacterial disease. However, the majority of community-associated malaria infections are subclinical, often termed "asymptomatic" and the consequences of low-grade haemolysis during subclinical malaria infection are unknown. As part of an ongoing study of subclinical malaria in Burkina Faso, 23 children with subclinical Plasmodium falciparum infections (determined by qPCR) were compared with 21 village-matched uninfected control children. Infected children showed evidence of persistent haemolysis over 35 days, with raised plasma haem and HO-1 concentrations. Concentrations of IL-10, which can also directly activate HO-1, were also higher in infected children compared to uninfected children. Regression analysis revealed that HO-1 was associated with haemolysis, but not with parasite density, anaemia or IL-10 concentration. This study reveals that subclinical P. falciparum malaria infection is associated with sustained haemolysis and the induction of HO-1. Given the association between HO-1, neutrophil dysfunction and increased risk of Salmonella bacteraemia, prolonged HO-1 induction may explain epidemiological associations and geographic overlap between malaria and invasive bacterial disease. Further studies are needed to understand the consequences of persistent subclinical malaria infection, low-grade haemolysis and raised HO-1 on immune cell function and risk of comorbidities.
Exploitation Route Data analysis is still ongoing so final conclusions are not yet known, but a potential outcome is a clinical trial to clear subclinical malarial infections with the aim of reducing the incidence of invasive bacterial disease.
Sectors Healthcare

 
Description COSMIC study Nijmegen Burkina 
Organisation Radboud University Nijmegen
Country Netherlands 
Sector Academic/University 
PI Contribution We analysed samples from malaria infected children in Burkina Faso
Collaborator Contribution They provided samples for us to analyse and metadata
Impact Paper in final stages of preparation; submission imminent
Start Year 2017
 
Description KCL Manu Sepsis project 
Organisation King's College London
Department School of Medicine KCL
Country United Kingdom 
Sector Academic/University 
PI Contribution We analysed samples from a large study of sepsis patients looking for evidence of hemolysis and HO-1 induction
Collaborator Contribution They provided samples for analysis and metadata
Impact Paper currently sunder review for publication
Start Year 2016
 
Description MRC Gambia 
Organisation Medical Research Council (MRC)
Department MRC Unit, The Gambia
Country Gambia 
Sector Public 
PI Contribution Joint research activities - field work in the Gambia
Collaborator Contribution Supporting field research activities in The Gambia
Impact First field surveys completed; follow up studies ongoing
Start Year 2017
 
Description Research seminars - various 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research seminars given at numerous universities in the UK, Europe and North America.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017
 
Description Women and Girls in Science Day St Andrews 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact UNESCO Women and Girls in Science Day lecture at the University of St Andrews
Year(s) Of Engagement Activity 2018
URL https://byretheatre.com/events/combatting-malaria-prof-eleanor-riley/