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

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.

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.

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.