Activation of human NK cells by Plasmodium-infected red blood cells.

This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-γ. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinized blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown

NK cells as effectors of acquired immune responses: effector CD4+ T cell-dependent activation of NK cells following vaccination.

We characterized vaccine-induced cellular responses to rabies virus in naive adult volunteers. Contrary to current paradigms, we observed potent and prolonged in vitro NK cell cytokine production and degranulation responses after restimulation of PBMCs with inactivated rabies virus in vaccinated, but not in unvaccinated, individuals. This "recall" NK cell response was absolutely dependent on Ag-specific IL-2 from CD45RO(+) CD4(+) T cells as well as IL-12 and IL-18 from accessory cells. Importantly, NK cells represented over 70% of all IFN-gamma-secreting and degranulating cells in the first 12-18 h after virus rechallenge indicating they may be required for rapid control of infection after vaccination. Activation of NK cells may be a critical function of IL-2-secreting effector memory T cells. Although IL-2-dependent postvaccination NK cell activation has been reported previously, this is the first time the magnitude of this effect and its contribution to the overall vaccine-induced response has been appreciated and the mechanisms of NK activation postvaccination have been elucidated. Our data will allow standard protocols for evaluating vaccine-induced immunity to be adapted to assess NK cell effector responses

The genetics of immune and infection phenotypes in wild mice, Mus musculus domesticus

Wild animals are under constant threat from a wide range of micro- and macroparasites in their environment. Animals make immune responses against parasites, and these are important in affecting the dynamics of parasite populations. Individual animals vary in their anti-parasite immune responses. Genetic polymorphism of immune-related loci contributes to inter-individual differences in immune responses, but most of what we know in this regard comes from studies of humans or laboratory animals; there are very few such studies of wild animals naturally infected with parasites. Here we have investigated the effect of single nucleotide polymorphisms (SNPs) in immune-related loci (the major histocompatibility complex [MHC], and loci coding for cytokines and Toll-like receptors) on a wide range of immune and infection phenotypes in UK wild house mice, Mus musculus domesticus. We found strong associations between SNPs in various MHC and cytokine-coding loci on both immune measures (antibody concentration and cytokine production) and on infection phenotypes (infection with mites, worms and viruses). Our study provides a comprehensive view of how polymorphism of immune-related loci affects immune and infection phenotypes in naturally infected wild rodent populations

Differentiation and adaptation of natural killer cells for anti-malarial immunity

Natural killer cells employ a diverse arsenal of effector mechanisms to target intracellular pathogens. Differentiation of natural killer (NK) cell activation pathways occurs along a continuum from reliance on innate pro-inflammatory cytokines and stress-induced host ligands through to interaction with signals derived from acquired immune responses. Importantly, the degree of functional differentiation of the NK cell lineage influences the magnitude and specificity of interactions with host cells infected with viruses, bacteria, fungi, and parasites. Individual humans possess a vast diversity of distinct NK cell clones, each with the capacity to vary along this functional differentiation pathway, which - when combined - results in unique individual responses to different infections. Here we summarize these NK cell differentiation events, review evidence for direct interaction of malaria-infected host cells with NK cells and assess how innate inflammatory signals induced by malaria parasite-associated molecular patterns influence the indirect activation and function of NK cells. Finally, we discuss evidence that anti-malarial immunity develops in parallel with advancing NK differentiation, coincident with a loss of reliance on inflammatory signals, and a refined capacity of NK cells to target malaria parasites more precisely, particularly through antibody-dependent mechanisms

IL-18-induced expression of high-affinity IL-2R on murine NK cells is essential for NK-cell IFN-γ production during murine Plasmodium yoelii infection.

Early production of pro-inflammatory cytokines, including IFN-γ, is essential for control of blood-stage malaria infections. We have shown that IFN-γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK-cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood-stage infection, expressing CD25 and producing IFN-γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN-γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL-18 signaling is essential for induction of CD25 and synergizes with IL-12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL-infected erythrocytes induced NK-cell CD25 expression and IFN-γ production in a manner that is completely IL-18- and partially IL-12-dependent, and IFN-γ production is enhanced by IL-2. These data suggest that IL-2 signaling via CD25 amplifies IL-18- and IL-12-mediated NK-cell activation during malaria infection

Functional Significance of CD57 Expression on Human NK Cells and Relevance to Disease.

Historically, human NK cells have been identified as CD3(-)CD56(+)CD16(±) lymphocytes. More recently it has been established that CD57 expression defines functionally discrete sub-populations of NK cells. On T cells, CD57 expression has been regarded as a marker of terminal differentiation and (perhaps wrongly) of anergy and senescence. Similarly, CD57 expression seems to identify the final stages of peripheral NK cell maturation; its expression increases with age and is associated with chronic infections, particularly human cytomegalovirus infection. However, CD57(+) NK cells are highly cytotoxic and their presence seems to be beneficial in a number of non-communicable diseases. The purpose of this article is to review our current understanding of CD57 expression as a marker of NK cell function and disease prognosis, as well as to outline areas for further research

Developmental allometry and paediatric malaria

WHO estimates that 80% of mortality due to malaria occurs among infants and young children. Though it has long been established that malaria disproportionately affects children under age five, our understanding of the underlying biological mechanisms for this distribution remains incomplete. Many studies use age as an indicator of exposure, but age may affect malaria burden independently of previous exposure. Not only does the severity of malaria infection change with age, but the clinical manifestation of disease does as well: younger children are more likely to suffer severe anaemia, while older children are more likely to develop cerebral malaria. Intensity of transmission and acquired immunity are important determinants of this age variation, but age differences remain consistent over varying transmission levels. Thus, age differences in clinical presentation may involve inherent age-related factors as well as still-undiscovered facets of acquired immunity, perhaps including the rates at which relevant aspects of immunity are acquired. The concept of "allometry" - the relative growth of a part in relation to that of an entire organism or to a standard - has not previously been applied in the context of malaria infection. However, because malaria affects a number of organs and cells, including the liver, red blood cells, white blood cells, and spleen, which may intrinsically develop at rates partly independent of each other and of a child's overall size, developmental allometry may influence the course and consequences of malaria infection. Here, scattered items of evidence have been collected from a variety of disciplines, aiming to suggest possible research paths for investigating exposure-independent age differences affecting clinical outcomes of malaria infection

Cross-Talk with Myeloid Accessory Cells Regulates Human Natural Killer Cell Interferon-γ Responses to Malaria

Data from a variety of experimental models suggest that natural killer (NK) cells require signals from accessory cells in order to respond optimally to pathogens, but the precise identity of the cells able to provide such signals depends upon the nature of the infectious organism. Here we show that the ability of human NK cells to produce interferon-γ in response to stimulation by Plasmodium falciparum–infected red blood cells (iRBCs) is strictly dependent upon multiple, contact-dependent and cytokine-mediated signals derived from both monocytes and myeloid dendritic cells (mDCs). Contrary to some previous reports, we find that both monocytes and mDCs express an activated phenotype following short-term incubation with iRBCs and secrete pro-inflammatory cytokines. The magnitude of the NK cell response (and of the KIR(−) CD56(bright) NK cell population in particular) is tightly correlated with resting levels of accessory cell maturation, indicating that heterogeneity of the NK response to malaria is a reflection of deep-rooted heterogeneity in the human innate immune system. Moreover, we show that NK cells are required to maintain the maturation status of resting mDCs and monocytes, providing additional evidence for reciprocal regulation of NK cells and accessory cells. However, NK cell–derived signals are not required for activation of accessory cells by either iRBCs or bacterial lipolysaccharide. Together, these data suggest that there may be differences in the sequence of events required for activation of NK cells by non-viral pathogens compared to the classical model of NK activation by virus-infected or major histocompatibility complex–deficient cells. These findings have far-reaching implications for the study of immunity to infection in human populations

Does malaria cause diarrhoea? A systematic review

Malaria is a systemic febrile disease that may progress to prostration, respiratory distress, encephalopathy, anemia, and death. Malaria is also an established risk factor for invasive bacterial disease caused, in the majority of cases, by invasive enteropathogens and in particular by non-Typhoidal Salmonella (NTS). Whilst various malaria-related pathologies have been implicated in the risk of NTS bacteraemia in animal models, including intestinal dysbiosis and loss of gut homeostasis, clinical evidence is lacking. As a first step in gathering such evidence, we conducted a systematic review of clinical and epidemiological studies reporting the prevalence of diarrhoea among malaria cases and vice versa. Database searches for "plasmodium" and "diarrhoea" identified 1,771 articles; a search for "plasmodium" and "gastroenteritis" identified a further 215 articles. After review, 66 articles specified an association between the search terms and referred primarily, but not exclusively, to Plasmodium falciparum infections. Overall, between 1.6 and 44% of patients with acute malaria infection reported symptoms of diarrhoea (812 of 7,267 individuals, 11%) whereas 5-42% of patients presenting to hospital with diarrhoea had an underlying malaria parasite infection (totaling 749 of 2,937 individuals, 26%). However, given the broad range of estimates, a paucity of purposeful case control or longitudinal studies, and varied or poorly specified definitions of diarrhoea, the literature provides limited evidence to draw any firm conclusions. The relationship between malaria and gastrointestinal disturbance thus remains unclear. Carefully designed case-control studies and prospective longitudinal studies are required to confidently assess the prevalence and significance of intestinal manifestations of malaria parasite infection