Geoffrey T. Hart

Malaria Immunity in Man and Mosquito: Insights into Unsolved Mysteries of a Deadly Infectious Disease*

Malaria is a mosquito-borne disease caused by parasites of the obligate intracellular Apicomplexa phylum the most deadly of which, Plasmodium falciparum, prevails in Africa. Malaria imposes a huge health burden on the worlds most vulnerable populations, claiming the lives of nearly one million children and pregnant women each year. Although there is keen interest in eradicating malaria, we do not yet have the necessary tools to meet this challenge, including an effective malaria vaccine and adequate vector control strategies. Here we review what is known about the mechanisms at play in immune resistance to malaria in both the human and mosquito hosts at each step in the parasites complex life cycle with a view toward developing the tools that will contribute to the prevention of disease and death and, ultimately, to the goal of malaria eradication. In so doing, we hope to inspire immunologists to participate in defeating this devastating disease.

Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.

Kruppel-Like Factor 2 Is Required for Trafficking but Not Quiescence in Postactivated T Cells

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8+ T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P1) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle–related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activatedCD8+ T cell differentiation and argue against a physiological role in cell cycle regulation.

Quantitative Gene Expression Profiling Implicates Genes for Susceptibility and Resistance to Alveolar Bone Loss

ABSTRACT Periodontal disease is one of the most prevalent chronic inflammatory diseases. There is a genetic component to susceptibility and resistance to this disease. Using a mouse model, we investigated the progression of alveolar bone loss by gene expression profiling of susceptible and resistant mouse strains (BALB/cByJ and A/J, respectively). We employed a novel and sensitive quantitative real-time PCR method to compare basal RNA transcription of a 48-gene set in the gingiva and the spleen and the subsequent changes in gene expression due to Porphyromonas gingivalis oral infection. Basal expression of interleukin-1 beta (Il1b) and tumor necrosis factor alpha (Tnf) mRNA was higher in the gingiva of the susceptible BALB/cByJ mice than in the gingiva of resistant A/J mice. Gingival Il1b gene expression increased further and Stat6 gene expression was turned on after P. gingivalis infection in BALB/cByJ mice but not in A/J mice. The basal expression of interleukin-15 (Il15) in the gingiva and the basal expression of p-selectin (Selp) in the spleen were higher in the resistant A/J mice than in the susceptible BALB/cByJ mice. In the resistant A/J mice the expression of no genes detectably changed in the gingiva after infection. These results suggest a molecular phenotype in which discrete sets of differentially expressed genes are associated with genetically determined susceptibility (Il1b, Tnf, and Stat6) or resistance (Il15 and Selp) to alveolar bone loss, providing insight into the genetic etiology of this complex disease.

Targeting glutamine metabolism rescues mice from late-stage cerebral malaria

Significance Cerebral malaria (CM) is a deadly complication of Plasmodium falciparum infection in African children despite effective antimalarial treatment. Once signs of neurologic disease have commenced, there is no adjunctive treatment for CM, and overall mortality remains high. Thus, a treatment that arrests disease and promotes healing in the late stages is urgently needed. Here we report, in an animal model of CM, that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) is an effective therapy even when treatment is initiated after infected animals show neurological signs of disease. Within hours of DON treatment blood–brain barrier integrity was restored, and brain swelling was reduced. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children. The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15–25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-l-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood–brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8+ effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood–brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8+ T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.

Krüppel-like Factors in Lymphocyte Biology

The Krüppel-like factor family of transcription factors plays an important role in differentiation, function, and homeostasis of many cell types. While their role in lymphocytes is still being determined, it is clear that these factors influence processes as varied as lymphocyte quiescence, trafficking, differentiation, and function. This review will present an overview of how these factors operate and coordinate with each other in lymphocyte regulation.

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature.

Inhibiting the Mammalian Target of Rapamycin Blocks the Development of Experimental Cerebral Malaria

ABSTRACT Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human hosts immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. IMPORTANCE Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection is cerebral malaria with a fatality rate of 15 to 20%, despite treatment with antimalarial drugs. Cerebral malaria takes a second toll on African children, leaving survivors at high risk of debilitating neurological defects. At present, we have no effective adjunctive therapies for cerebral malaria, and developing such therapies would have a large impact on saving young lives in Africa. Here we report results that open a new avenue for the development of highly selective adjunctive therapies for cerebral malaria by targeting pathways that regulate host and parasite metabolism. Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection is cerebral malaria with a fatality rate of 15 to 20%, despite treatment with antimalarial drugs. Cerebral malaria takes a second toll on African children, leaving survivors at high risk of debilitating neurological defects. At present, we have no effective adjunctive therapies for cerebral malaria, and developing such therapies would have a large impact on saving young lives in Africa. Here we report results that open a new avenue for the development of highly selective adjunctive therapies for cerebral malaria by targeting pathways that regulate host and parasite metabolism.

Cutting edge: Krüppel-like factor 2 is required for phenotypic maintenance but not development of B1 B cells

Several recent studies reported that Krüppel-like factor (KLF)2 controls trafficking, development, and function of B cells. Conditional B cell KLF2 knockout mice have increased numbers of marginal zone B cells and decreased numbers of B1 phenoytpe cells. However, it was unclear whether KLF2 is required for B1 B cell development, survival, or phenotypic maintenance. We show that B1 phenotype B cells are present in neonatal mice with B cell-specific KLF2 deficiency, suggesting that B1 differentiation can occur even in the absence of KLF2. Furthermore, by use of an inducible knockout strategy, we show that deletion of KLF2 in mature B1 cells causes loss of phenotypic markers associated with B1 cell identity, but it has a minimal effect on short-term cell survival. Taken together, our findings suggest that KLF2 is necessary for the maintenance of B1 cell identity rather than differentiation or survival of the population.

Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells

SUMMARY Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA‐infected red blood cells develop a fatal CM‐like disease caused by CD8+ T cell‐mediated pathology. We found that treatment with interleukin‐15 complex (IL‐15C) prevented ECM, whereas IL‐2C treatment had no effect. IL‐15C‐expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL‐15C treatment also decreased CD8+ T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL‐15C induced NK cells to express IL‐10, which was required for IL‐15C‐mediated protection against ECM. Finally, we show that ALT‐803, a modified human IL‐15C, mediates similar induction of IL‐10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine‐stimulated NK cells in the prevention of a pathogenic immune response. Graphical Abstract Figure. No caption available. HighlightsIL‐15 complex (IL‐5C) treatment protects mice from experimental cerebral malaria (ECM)NK cell‐derived IL‐10 is required for IL‐15C‐mediated survival from ECMIL‐15C inhibits CD8+ T cell activation and cytokine production in the brainHuman NK cells also produce IL‐10 after cytokine stimulation In Brief NK cells can display both pro‐inflammatory and regulatory function, but their role in the pathogenesis of malaria is not fully understood. Burrack et al. demonstrate that IL‐15 complex (IL‐15C) therapy prevents mice from succumbing to experimental cerebral malaria (ECM). IL‐15C treatment stimulates NK cells to produce IL‐10, suppressing the pathogenic CD8+ T cell response during ECM.