Cellular iron governs the host response to malaria.

Malaria and iron deficiency are major global health problems with extensive epidemiological overlap. Iron deficiency-induced anaemia can protect the host from malaria by limiting parasite growth. On the other hand, iron deficiency can significantly disrupt immune cell function. However, the impact of host cell iron scarcity beyond anaemia remains elusive in malaria. To address this, we employed a transgenic mouse model carrying a mutation in the transferrin receptor (TfrcY20H/Y20H), which limits the ability of cells to internalise iron from plasma. At homeostasis TfrcY20H/Y20H mice appear healthy and are not anaemic. However, TfrcY20H/Y20H mice infected with Plasmodium chabaudi chabaudi AS showed significantly higher peak parasitaemia and body weight loss. We found that TfrcY20H/Y20H mice displayed a similar trajectory of malaria-induced anaemia as wild-type mice, and elevated circulating iron did not increase peak parasitaemia. Instead, P. chabaudi infected TfrcY20H/Y20H mice had an impaired innate and adaptive immune response, marked by decreased cell proliferation and cytokine production. Moreover, we demonstrated that these immune cell impairments were cell-intrinsic, as ex vivo iron supplementation fully recovered CD4+ T cell and B cell function. Despite the inhibited immune response and increased parasitaemia, TfrcY20H/Y20H mice displayed mitigated liver damage, characterised by decreased parasite sequestration in the liver and an attenuated hepatic immune response. Together, these results show that host cell iron scarcity inhibits the immune response but prevents excessive hepatic tissue damage during malaria infection. These divergent effects shed light on the role of iron in the complex balance between protection and pathology in malaria.

Uptake of Plasmodium chabaudi hemozoin drives Kupffer cell death and fuels superinfections.

Kupffer cells (KCs) are self-maintained tissue-resident macrophages that line liver sinusoids and play an important role on host defense. It has been demonstrated that upon infection or intense liver inflammation, KCs might be severely depleted and replaced by immature monocytic cells; however, the mechanisms of cell death and the alterations on liver immunity against infections deserves further investigation. We explored the impact of acute Plasmodium infection on KC biology and on the hepatic immune response against secondary infections. Similar to patients, infection with Plasmodium chabaudi induced acute liver damage as determined by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. This was associated with accumulation of hemozoin, increased of proinflammatory response and impaired bacterial and viral clearance, which led to pathogen spread to other organs. In line with this, mice infected with Plasmodium had enhanced mortality during secondary infections, which was associated with increased production of mitochondrial superoxide, lipid peroxidation and increased free iron within KCs-hallmarks of cell death by ferroptosis. Therefore, we revealed that accumulation of iron with KCs, triggered by uptake of circulating hemozoin, is a novel mechanism of macrophage depletion and liver inflammation during malaria, providing novel insights on host susceptibility to secondary infections. Malaria can cause severe liver damage, along with depletion of liver macrophages, which can predispose individuals to secondary infections and enhance the chances of death.

Mistimed malaria parasites re-synchronize with host feeding-fasting rhythms by shortening the duration of intra-erythrocytic development.

AIMS: Malaria parasites exhibit daily rhythms in the intra-erythrocytic development cycle (IDC) that underpins asexual replication in the blood. The IDC schedule is aligned with the timing of host feeding-fasting rhythms. When the IDC schedule is perturbed to become mismatched to host rhythms, it readily reschedules but it is not known how. METHODS: We intensively follow four groups of infections that have different temporal alignments between host rhythms and the IDC schedule for 10 days, before and after the peak in asexual densities. We compare how the duration, synchrony and timing of the IDC differs between parasites in control infections and those forced to reschedule by 12 hours and ask whether the density of parasites affects the rescheduling process. RESULTS AND CONCLUSIONS: Our experiments reveal parasites shorten the IDC duration by 2-3 hours to become realigned to host feeding-fasting rhythms with 5-6 days, in a density-independent manner. Furthermore, parasites are able to reschedule without significant fitness costs for them or their hosts. Understanding the extent of, and limits on, plasticity in the IDC schedule may reveal targets for novel interventions, such as drugs to disrupt IDC regulation and preventing IDC dormancy conferring tolerance to existing drugs.

The plasma membrane calcium ATPase 4 does not influence parasite levels but partially promotes experimental cerebral malaria during murine blood stage malaria.

BACKGROUND: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. METHODS: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. RESULTS: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. CONCLUSIONS: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Differential drivers of intraspecific and interspecific competition during malaria-helminth co-infection.

Various host and parasite factors interact to determine the outcome of infection. We investigated the effects of two factors on the within-host dynamics of malaria in mice: initial infectious dose and co-infection with a helminth that limits the availability of red blood cells (RBCs). Using a statistical, time-series approach to model the within-host 'epidemiology' of malaria, we found that increasing initial dose reduced the time to peak cell-to-cell parasite propagation, but also reduced its magnitude, while helminth co-infection delayed peak cell-to-cell propagation, except at the highest malaria doses. Using a mechanistic model of within-host infection dynamics, we identified dose-dependence in parameters describing host responses to malaria infection and uncovered a plausible explanation of the observed differences in single vs co-infections. Specifically, in co-infections, our model predicted a higher background death rate of RBCs. However, at the highest dose, when intraspecific competition between malaria parasites would be highest, these effects of co-infection were not observed. Such interactions between initial dose and co-infection, although difficult to predict a priori, are key to understanding variation in the severity of disease experienced by hosts and could inform studies of malaria transmission dynamics in nature, where co-infection and low doses are the norm.

Inducible mechanisms of disease tolerance provide an alternative strategy of acquired immunity to malaria.

Immunity to malaria is often considered slow to develop but this only applies to defense mechanisms that function to eliminate parasites (resistance). In contrast, immunity to severe disease can be acquired quickly and without the need for improved pathogen control (tolerance). Using Plasmodium chabaudi, we show that a single malaria episode is sufficient to induce host adaptations that can minimise inflammation, prevent tissue damage and avert endothelium activation, a hallmark of severe disease. Importantly, monocytes are functionally reprogrammed to prevent their differentiation into inflammatory macrophages and instead promote mechanisms of stress tolerance to protect their niche. This alternative fate is not underpinned by epigenetic reprogramming of bone marrow progenitors but appears to be imprinted within the remodelled spleen. Crucially, all of these adaptations operate independently of pathogen load and limit the damage caused by malaria parasites in subsequent infections. Acquired immunity to malaria therefore prioritises host fitness over pathogen clearance.

Malaria is a parasitic infection spread by mosquitoes that causes hundreds of millions of cases each year. People are most likely to die from malaria the first time they are infected ­ usually when they are young children. Among those who survive, however, few will develop severe symptoms again, even though they are often reinfected with as many (or even more) parasites. This indicates that people do not get better at eliminating the parasite. Instead, protection from severe malaria is a form of tolerance - the body learns to limit the damage the infection causes. But exactly which mechanisms have to be engaged to tolerate malaria is unclear. One way to achieve tolerance may be to switch off damaging inflammation. Nahrendorf et al. explored this possibility by comparing the immune response of mice to their first and second infection with malaria parasites. During the first infection of life, immune cells release harmful inflammatory molecules that activate the lining of blood vessels, causing tissue damage and severe symptoms. During the second infection, these immune cells shut down inflammation and instead actively promote tissue health to reduce damage and improve outcome. This change in the immune response occurs despite the fact that the number of parasites is the same in both infections. Nahrendorf et al. also found that the mouse's immune cells 'remembered' to tolerate subsequent infections, even after treatment with a drug that kills all malaria parasites. This was possible because malaria permanently altered the spleen, which reprogrammed the response of the immune cells. A single infection is therefore enough to induce long-lived mechanisms of tolerance that can prevent life-threatening disease. These findings have the potential to change the understanding of immunity to malaria, which currently emphasises the importance of killing parasites. New ways to treat and vaccinate people - and to protect young children from severe malaria - may arise by treating tolerance as an equally important form of host defense.

Transcriptional Memory-Like Imprints and Enhanced Functional Activity in γδ T Cells Following Resolution of Malaria Infection.

γδ T cells play an essential role in the immune response to many pathogens, including Plasmodium. However, long-lasting effects of infection on the γδ T cell population still remain inadequately understood. This study focused on assessing molecular and functional changes that persist in the γδ T cell population following resolution of malaria infection. We investigated transcriptional changes and memory-like functional capacity of malaria pre-exposed γδ T cells using a Plasmodiumchabaudi infection model. We show that multiple genes associated with effector function (chemokines, cytokines and cytotoxicity) and antigen-presentation were upregulated in P. chabaudi-exposed γδ T cells compared to γδ T cells from naïve mice. This transcriptional profile was positively correlated with profiles observed in conventional memory CD8+ T cells and was accompanied by enhanced reactivation upon secondary encounter with Plasmodium-infected red blood cells in vitro. Collectively our data demonstrate that Plasmodium exposure result in "memory-like imprints" in the γδ T cell population and also promotes γδ T cells that can support antigen-presentation during subsequent infections.

Uncovering drivers of dose-dependence and individual variation in malaria infection outcomes.

To understand why some hosts get sicker than others from the same type of infection, it is essential to explain how key processes, such as host responses to infection and parasite growth, are influenced by various biotic and abiotic factors. In many disease systems, the initial infection dose impacts host morbidity and mortality. To explore drivers of dose-dependence and individual variation in infection outcomes, we devised a mathematical model of malaria infection that allowed host and parasite traits to be linear functions (reaction norms) of the initial dose. We fitted the model, using a hierarchical Bayesian approach, to experimental time-series data of acute Plasmodium chabaudi infection across doses spanning seven orders of magnitude. We found evidence for both dose-dependent facilitation and debilitation of host responses. Most importantly, increasing dose reduced the strength of activation of indiscriminate host clearance of red blood cells while increasing the half-life of that response, leading to the maximal response at an intermediate dose. We also explored the causes of diverse infection outcomes across replicate mice receiving the same dose. Besides random noise in the injected dose, we found variation in peak parasite load was due to unobserved individual variation in host responses to clear infected cells. Individual variation in anaemia was likely driven by random variation in parasite burst size, which is linked to the rate of host cells lost to malaria infection. General host vigour in the absence of infection was also correlated with host health during malaria infection. Our work demonstrates that the reaction norm approach provides a useful quantitative framework for examining the impact of a continuous external factor on within-host infection processes.

Caspase-8 mediates inflammation and disease in rodent malaria.

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1ß and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

Host circadian clocks do not set the schedule for the within-host replication of malaria parasites.

Circadian clocks coordinate organisms' activities with daily cycles in their environment. Parasites are subject to daily rhythms in the within-host environment, resulting from clock-control of host activities, including immune responses. Parasites also exhibit rhythms in their activities: the timing of within-host replication by malaria parasites is coordinated to host feeding rhythms. Precisely which host feeding-related rhythm(s) parasites align with and how this is achieved are unknown. Understanding rhythmic replication in malaria parasites matters because it underpins disease symptoms and fuels transmission investment. We test if rhythmicity in parasite replication is coordinated with the host's feeding-related rhythms and/or rhythms driven by the host's canonical circadian clock. We find that parasite rhythms coordinate with the time of day that hosts feed in both wild-type and clock-mutant hosts, whereas parasite rhythms become dampened in clock-mutant hosts that eat continuously. Our results hold whether infections are initiated with synchronous or with desynchronized parasites. We conclude that malaria parasite replication is coordinated to rhythmic host processes that are independent of the core-clock proteins PERIOD 1 and 2; most likely, a periodic nutrient made available when the host digests food. Thus, novel interventions could disrupt parasite rhythms to reduce their fitness, without interference by host clock-controlled homeostasis.

The skeletal muscles of mice infected with Plasmodium berghei and Plasmodium chabaudi reveal a crosstalk between lipid mediators and gene expression.

BACKGROUND: Malaria is one of the most prevalent infectious disease in the world with 3.2 billion humans at risk. Malaria causes splenomegaly and damage in other organs including skeletal muscles. Skeletal muscles comprise nearly 50% of the human body and are largely responsible for the regulation and modulation of overall metabolism. It is essential to understand how malaria damages muscles in order to develop effective preventive measures and/or treatments. Using a pre-clinical animal model, the potential molecular mechanisms of Plasmodium infection affecting skeletal muscles of mice were investigated. METHODS: Mouse Signal Transduction Pathway Finder PCR Array was used to monitor gene expression changes of 10 essential signalling pathways in skeletal muscles from mice infected with Plasmodium berghei and Plasmodium chabaudi. Then, a new targeted-lipidomic approach using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to profile 158 lipid signalling mediators (LMs), mostly eicosanoids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was applied. Finally, 16 key LMs directly associated with inflammation, oxidative stress, and tissue healing in skeletal muscles, were quantified. RESULTS: The results showed that the expression of key genes altered by Plasmodium infection is associated with inflammation, oxidative stress, and atrophy. In support to gene profiling results, lipidomics revealed higher concentrations of LMs in skeletal muscles directly related to inflammatory responses, while on the levels of LMs crucial in resolving inflammation and tissue repair reduced significantly. CONCLUSION: The results provide new insights into the molecular mechanisms of malaria-induced muscle damage and revealed a potential mechanism modulating inflammation in malarial muscles. These pre-clinical studies should help with future clinical studies in humans aimed at monitoring of disease progression and development of specific interventions for the prevention and mitigation of long-term chronic effects on skeletal muscle function.

The malaria parasite has an intrinsic clock.

Malarial rhythmic fevers are the consequence of the synchronous bursting of red blood cells (RBCs) on completion of the malaria parasite asexual cell cycle. Here, we hypothesized that an intrinsic clock in the parasite Plasmodium chabaudi underlies the 24-hour-based rhythms of RBC bursting in mice. We show that parasite rhythms are flexible and lengthen to match the rhythms of hosts with long circadian periods. We also show that malaria rhythms persist even when host food intake is evenly spread across 24 hours, suggesting that host feeding cues are not required for synchrony. Moreover, we find that the parasite population remains synchronous and rhythmic even in an arrhythmic clock mutant host. Thus, we propose that parasite rhythms are generated by the parasite, possibly to anticipate its circadian environment.

Testing possible causes of gametocyte reduction in temporally out-of-synch malaria infections.

BACKGROUND: The intraerythrocytic development cycle (IDC) of the rodent malaria Plasmodium chabaudi is coordinated with host circadian rhythms. When this coordination is disrupted, parasites suffer a 50% reduction in both asexual stages and sexual stage gametocytes over the acute phase of infection. Reduced gametocyte density may not simply follow from a loss of asexuals because investment into gametocytes ("conversion rate") is a plastic trait; furthermore, the densities of both asexuals and gametocytes are highly dynamic during infection. Hence, the reasons for the reduction of gametocytes in infections that are out-of-synch with host circadian rhythms remain unclear. Here, two explanations are tested: first, whether out-of-synch parasites reduce their conversion rate to prioritize asexual replication via reproductive restraint; second, whether out-of-synch gametocytes experience elevated clearance by the host's circadian immune responses. METHODS: First, conversion rate data were analysed from a previous experiment comparing infections of P. chabaudi that were in-synch or 12 h out-of-synch with host circadian rhythms. Second, three new experiments examined whether the inflammatory cytokine TNF varies in its gametocytocidal efficacy according to host time-of-day and gametocyte age. RESULTS: There was no evidence that parasites reduce conversion or that their gametocytes become more vulnerable to TNF when out-of-synch with host circadian rhythms. CONCLUSIONS: The factors causing the reduction of gametocytes in out-of-synch infections remain mysterious. Candidates for future investigation include alternative rhythmic factors involved in innate immune responses and the rhythmicity in essential resources required for gametocyte development. Explaining why it matters for gametocytes to be synchronized to host circadian rhythms might suggest novel approaches to blocking transmission.

A non-lethal malarial infection results in reduced drug metabolizing enzyme expression and drug clearance in mice.

BACKGROUND: Given the central importance of anti-malarial drugs in the treatment of malaria, there is a need to understand the effect of Plasmodium infection on the broad spectrum of drug metabolizing enzymes. Previous studies have shown reduced clearance of quinine, a treatment for Plasmodium infection, in individuals with malaria. METHODS: The hepatic expression of a large panel of drug metabolizing enzymes was studied in the livers of mice infected with the AS strain of Plasmodium chabaudi chabaudi, a nonlethal parasite in most strains of mice with several features that model human Plasmodium infections. C57BL/6J mice were infected with P. chabaudi by intraperitoneal injection of infected erythrocytes and sacrificed at different times after infection. Relative hepatic mRNA levels of various drug metabolizing enzymes, cytokines and acute phase proteins were measured by reverse transcriptase-real time PCR. Relative levels of cytochrome P450 proteins were measured by Western blotting with IR-dye labelled antibodies. Pharmacokinetics of 5 prototypic cytochrome P450 substrate drugs were measured by cassette dosing and high-resolution liquid chromatography-mass spectrometry. The results were analysed by MANOVA and post hoc univariate analysis of variance. RESULTS: The great majority of enzyme mRNAs were down-regulated, with the greatest effects occurring at the peak of parasitaemia 8 days post infection. Protein levels of cytochrome P450 enzymes in the Cyp 2b, 2c, 2d, 2e, 3a and 4a subfamilies were also down-regulated. Several distinct groups differing in their temporal patterns of regulation were identified. The cassette dosing study revealed that at the peak of parasitaemia, the clearances of caffeine, bupropion, tolbutamide and midazolam were markedly reduced by 60-70%. CONCLUSIONS: These findings in a model of uncomplicated human malaria suggest that changes in drug clearance in this condition may be of sufficient magnitude to cause significant alterations in exposure and response of anti-malarial drugs and co-medications.

Plasticity and genetic variation in traits underpinning asexual replication of the rodent malaria parasite, Plasmodium chabaudi.

BACKGROUND: The ability of malaria (Plasmodium) parasites to adjust investment into sexual transmission stages versus asexually replicating stages is well known, but plasticity in other traits underpinning the replication rate of asexual stages in the blood has received less attention. Such traits include burst size (the number of merozoites produced per schizont), the duration of the asexual cycle, and invasion preference for different ages of red blood cell (RBC). METHODS: Here, plasticity [environment (E) effects] and genetic variation [genotype (G) effects] in traits relating to asexual replication rate are examined for 4 genotypes of the rodent malaria parasite Plasmodium chabaudi. An experiment tested whether asexual dynamics differ between parasites infecting control versus anaemic hosts, and whether variation in replication rate can be explained by differences in burst size, asexual cycle, and invasion rates. RESULTS: The within-host environment affected each trait to different extents but generally had similar impacts across genotypes. The dynamics of asexual densities exhibited a genotype by environment effect (G×E), in which one of the genotypes increased replication rate more than the others in anaemic hosts. Burst size and cycle duration varied between the genotypes (G), while burst size increased and cycle duration became longer in anaemic hosts (E). Variation in invasion rates of differently aged RBCs was not explained by environmental or genetic effects. Plasticity in burst size and genotype are the only traits making significant contributions to the increase in asexual densities observed in anaemic hosts, together explaining 46.4% of the variation in replication rate. CONCLUSIONS: That host anaemia induces several species of malaria parasites to alter conversion rate is well documented. Here, previously unknown plasticity in other traits underpinning asexual replication is revealed. These findings contribute to mounting evidence that malaria parasites deploy a suite of sophisticated strategies to maximize fitness by coping with, or exploiting the opportunities provided by, the variable within-host conditions experienced during infections. That genetic variation and genotype by environment interactions also shape these traits highlights their evolutionary potential. Asexual replication rate is a major determinant of virulence and so, understanding the evolution of virulence requires knowledge of the ecological (within-host environment) and genetic drivers of variation among parasites.

Insight into the Effects of Plasmodium chabaudi on Platelets Using Carbon-Fiber Microelectrode Amperometry.

Platelets are anuclear circulating cell bodies within the bloodstream commonly known for their roles in clot formation during vascular injury to prevent blood loss. They also have significant impact in a range of diseases, including malaria. However, the role of platelets in malaria is controversial, with contradicting evidence suggesting either that they assist in destruction of malarial parasites or facilitate a severe form of malaria. Precedent work suggests that the timing of infection is critical in determining whether platelets switch roles from being protective to deleterious. As such, the work herein makes use of the unique mechanistic perspective offered by carbon-fiber microelectrode amperometry (CFMA) to understand how platelet secretion is impacted in malarial infection stages (ascending parasite count versus descending parasite count). Malarial platelet behavior was compared to platelets from noninfected control mice by probing their exocytotic function. Results suggest that mouse malaria caused by the parasite Plasmodium chabaudi, during both ascending and descending infection stages, reduces platelet exocytotic events and delays platelet granule fusion; in addition, platelets are more impacted by the disease early in the infection stages. In all, understanding platelet behavior in the malarial context may present new therapeutic routes to treat or cure malaria.

Adaptive plasticity in the gametocyte conversion rate of malaria parasites.

Sexually reproducing parasites, such as malaria parasites, experience a trade-off between the allocation of resources to asexual replication and the production of sexual forms. Allocation by malaria parasites to sexual forms (the conversion rate) is variable but the evolutionary drivers of this plasticity are poorly understood. We use evolutionary theory for life histories to combine a mathematical model and experiments to reveal that parasites adjust conversion rate according to the dynamics of asexual densities in the blood of the host. Our model predicts the direction of change in conversion rates that returns the greatest fitness after perturbation of asexual densities by different doses of antimalarial drugs. The loss of a high proportion of asexuals is predicted to elicit increased conversion (terminal investment), while smaller losses are managed by reducing conversion (reproductive restraint) to facilitate within-host survival and future transmission. This non-linear pattern of allocation is consistent with adaptive reproductive strategies observed in multicellular organisms. We then empirically estimate conversion rates of the rodent malaria parasite Plasmodium chabaudi in response to the killing of asexual stages by different doses of antimalarial drugs and forecast the short-term fitness consequences of these responses. Our data reveal the predicted non-linear pattern, and this is further supported by analyses of previous experiments that perturb asexual stage densities using drugs or within-host competition, across multiple parasite genotypes. Whilst conversion rates, across all datasets, are most strongly influenced by changes in asexual density, parasites also modulate conversion according to the availability of red blood cell resources. In summary, increasing conversion maximises short-term transmission and reducing conversion facilitates in-host survival and thus, future transmission. Understanding patterns of parasite allocation to reproduction matters because within-host replication is responsible for disease symptoms and between-host transmission determines disease spread.

The Role of BACH2 in T Cells in Experimental Malaria Caused by Plasmodium chabaudi chabaudi AS.

BTB and CNC Homology 1, Basic Leucine Zipper Transcription Factor 2 (BACH2) is a transcription factor best known for its role in B cell development. More recently, it has been associated with T cell functions in inflammatory diseases, and has been proposed as a master transcriptional regulator within the T cell compartment. In this study, we employed T cell-specific Bach2-deficient (B6.Bach2ΔT ) mice to examine the role of this transcription factor in CD4+ T cell functions in vitro and in mice infected with Plasmodium chabaudi AS. We found that under CD4+ T cell polarizing conditions in vitro, Th2, and Th17 helper cell subsets were more active in the absence of Bach2 expression. In mice infected with P. chabaudi AS, although the absence of Bach2 expression by T cells had no effect on blood parasitemia or disease pathology, we found reduced expansion of CD4+ T cells in B6.Bach2ΔT mice, compared with littermate controls. Despite this reduction, we observed increased frequencies of Tbet+ IFNγ+ CD4+ (Th1) cells and IL-10-producing Th1 (Tr1) cells in mice lacking Bach2 expression by T cells. Studies in mixed bone marrow chimeric mice revealed T cell intrinsic effects of BACH2 on hematopoietic cell development, and in particular, the generation of CD4+ and CD8+ T cell subsets. Furthermore, T cell intrinsic BACH2 was needed for efficient expansion of CD4+ T cells during experimental malaria in this immunological setting. We also examined the response of B6.Bach2ΔT mice to a second protozoan parasitic challenge with Leishmania donovani and found similar effects on disease outcome and T cell responses. Together, our findings provide new insights into the role of BACH2 in CD4+ T cell activation during experimental malaria, and highlight an important role for this transcription factor in the development and expansion of T cells under homeostatic conditions, as well as establishing the composition of the effector CD4+ T cell compartment during infection.

Adaptive periodicity in the infectivity of malaria gametocytes to mosquitoes.

Daily rhythms in behaviour, physiology and molecular processes are expected to enable organisms to appropriately schedule activities according to consequences of the daily rotation of the Earth. For parasites, this includes capitalizing on periodicity in transmission opportunities and for hosts/vectors, this may select for rhythms in immune defence. We examine rhythms in the density and infectivity of transmission forms (gametocytes) of rodent malaria parasites in the host's blood, parasite development inside mosquito vectors and potential for onwards transmission. Furthermore, we simultaneously test whether mosquitoes exhibit rhythms in susceptibility. We reveal that at night, gametocytes are twice as infective, despite being less numerous in the blood. Enhanced infectiousness at night interacts with mosquito rhythms to increase sporozoite burdens fourfold when mosquitoes feed during their rest phase. Thus, changes in mosquito biting time (owing to bed nets) may render gametocytes less infective, but this is compensated for by the greater mosquito susceptibility.

Elimination of intravascular thrombi prevents early mortality and reduces gliosis in hyper-inflammatory experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is the most lethal outcome of Plasmodium infection. There are clear correlations between expression of inflammatory cytokines, severe coagulopathies, and mortality in human CM. However, the mechanisms intertwining the coagulation and inflammation pathways, and their roles in CM, are only beginning to be understood. In mice with T cells deficient in the regulatory cytokine IL-10 (IL-10 KO), infection with Plasmodium chabaudi leads to a hyper-inflammatory response and lethal outcome that can be prevented by anti-TNF treatment. However, inflammatory T cells are adherent within the vasculature and not present in the brain parenchyma, suggesting a novel form of cerebral inflammation. We have previously documented behavioral dysfunction and microglial activation in infected IL-10 KO animals suggestive of neurological involvement driven by inflammation. In order to understand the relationship of intravascular inflammation to parenchymal dysfunction, we studied the congestion of vessels with leukocytes and fibrin(ogen) and the relationship of glial cell activation to congested vessels in the brains of P. chabaudi-infected IL-10 KO mice. METHODS: Using immunofluorescence microscopy, we describe severe thrombotic congestion in these animals. We stained for immune cell surface markers (CD45, CD11b, CD4), fibrin(ogen), microglia (Iba-1), and astrocytes (GFAP) in the brain at the peak of behavioral symptoms. Finally, we investigated the roles of inflammatory cytokine tumor necrosis factor (TNF) and coagulation on the pathology observed using neutralizing antibodies and low-molecular weight heparin to inhibit both inflammation and coagulation, respectively. RESULTS: Many blood vessels in the brain were congested with thrombi containing adherent leukocytes, including CD4 T cells and monocytes. Despite containment of the pathogen and leukocytes within the vasculature, activated microglia and astrocytes were prevalent in the parenchyma, particularly clustered near vessels with thrombi. Neutralization of TNF, or the coagulation cascade, significantly reduced both thrombus formation and gliosis in P. chabaudi-infected IL-10 KO mice. CONCLUSIONS: These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature to neuropathology in malaria infection. Strikingly, localization of inflammatory leukocytes within intravascular clots suggests a mechanism for interaction between the two cascades by which cytokines drive local inflammation without considerable cellular infiltration into the brain parenchyma.