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Cerebral Malaria (CM), the deadliest complication ofPlasmodiuminfection, is a complex and unpredictable disease. However, our understanding of the host and parasite factors that cause CM is limited. Using a mouse model of CM, experimental CM (ECM), we performed a three-way comparison between: ECM susceptible C57BL/6 mice infected with ECM-causingPlasmodiumANKA (PbANKA) parasites (ANKA(C57BL/6)); ECM resistant Balb/c mice infected withPbANKA (ANKA(Balb/c)); and C57BL/6 mice infected withPbNK65 that does not cause ECM (NK65(C57BL/6)). All ANKA(C57BL/6)mice developed CM. In contrast in ANKA(Balb/c)and NK65(C57BL/6)infections do not result in CM and proceed similarly in terms of parasite growth, disease course and host immune response.. However, parasite gene expression in (ANKA(C57BL/6)) was remarkably different than in ANKA(Balb/c)but similar to the gene expression in NK65(C57BL/6). Thus,PbANKA has a ECM-specific gene expression profile that is only activated in susceptible hosts providing evidence that the host has a critical influence on the outcome of infection.

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host’s immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

ApiAP2 transcription factor PBANKA_011210 is a novel Plasmodium virulence factor that blocks induction of host’s immune response., The acquisition of malaria immunity is both remarkably slow and unpredictable. At present, we know little about the malaria parasite genes that influence the host’s ability to mount a protective immune response. Here, we show that a single-nucleotide polymorphism (SNP) resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in the rodent malaria parasite Plasmodium berghei (Pb) NK65 allowed infected mice to mount a T helper cell 1 (TH1)–type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F infections resulted in an early interferon-γ response and a later expansion of germinal centers, resulting in high levels of infected red blood cell–specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections.

Key events in T cell–dependent antibody responses, including affinity maturation, are dependent on the B cell’s presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells’ capture and presentation of antigen. The presentation of antigen by germinal-center B cells to follicular T cells engenders the process of antibody affinity maturation and humoral memory. Pierce and colleagues show that TLR9 signaling in B cells antagonizes B cell–mediated antigen presentation, which leads to the enhanced generation of short-lived plasma cells and the production of lower-affinity antibodies.

B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce Abs and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. In this study we show that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete Abs and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs, with implications for the use of CpG-A and CpG-B as immunomodulators.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.

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., Author Summary Cerebral malaria (CM) is a severe and potentially fatal complication of malaria in humans that results in swelling and bleeding within the brain. The mechanisms that cause this fatal disease in humans are not completely understood. We studied an animal model known as experimental cerebral malaria to learn more about the factors that drive this disease process. Using a technique referred to as intravital microscopy, we captured movies of immune cells operating in the living brain as the disease developed. At the peak of disease, we observed evidence of immune cells interacting with and aggregating along blood vessels throughout the brain. These interactions were directly associated vascular leakage. This caused the brain to swell, which gave rise to an unsustainable pressure that ultimately killed neurons responsible for heart and lung function. The fatal swelling was induced by immune cells (referred to as T cells) interacting with bits of parasite presented by blood vessels in the brain. Removal of this parasite presentation protected the mice from fatal disease. We also evaluated a straightforward therapy that involved intravenous administration of antibodies that interfered with T cell sticking to blood vessels. Our movies revealed that this therapeutic approach rapidly displaced T cells from the blood vessels in the brain and prevented fatal disease.

The signaling cascades initiated by binding of the antigen to B cell receptor (BCR) leads to rapid increases in cellular respiration, nutrient uptake and expression of various activation markers. However, unless a second, independent stimulus is received soon after antigen binding, BCR signaling does not lead to B cell proliferation and differentiation. Instead, BCR signaling alone leads to a dysregulation of cellular calcium homeostasis which in turn causes mitochondrial dysfunction and a phenomenon called activation induced cell death. The second signal can be provided by Toll like receptor (TLR) signaling, indicating the presence of an acute infection or through B cell-T cell interactions that rules out that the initial antigen was a prohibited self-antigen. Additionally, there is an interplay between TLR versus T cell mediated second signals, namely that TLR signaling trumped the need for the antigen-stimulated B cell to interact with T cells to receive a ‘go’ signal. TLR stimulation decreased B cells’ ability to capture, process and present antigens to solicit help from T cells. Rather TLRs directly boosted B cell antibody and cytokine secretion and proliferation. Thus, TLR signals drive B cells toward rapid T cell-independent differentiation to plasma cells providing rapid neutralizing antibody production to fight off infections quickly. In contrast B cells that receive their survival signals from T cells are induced to undergo a time-consuming process that is necessary for antibody affinity maturation and long-lasting immune memory. Therefore, the TLR driven shortcut to quick antibody comes with a price which is the absence of “crème de la crème” antibodies that would protect the body from subsequent infections for years to come.

Activation of B cells to proliferate and differentiate into antibody secreting cells is regulated to avoid unwanted antibody responses by two temporally distinct signals, the first provided by antigen binding to the B cell receptor (BCR) and the second by T helper cells. Despite the central role of these two signals in driving antibody responses, our understanding of the consequences of each signal is incomplete. Here using in vitro and in vivo models, we investigated the metabolic changes that accompany B cell activation and show that antigen binding to the BCR signals for rapid increases in both oxidative phosphorylation and glycolysis, however early events following antigen engagement including spreading and contraction of antigen bound BCR and internalization of the bound antigen depend solely on the energy produced by oxidative phosphorylation. Changes in gene expression, mitochondrial performance and glucose uptake follow the initial activation, preparing the cells to respond to a second signal. However, in the absence of a second signal B cells undergo progressive loss of mitochondria function and glycolytic capacity ultimately leading to apoptosis. Mitochondria dysfunction is a result of the gradual accumulation of intracellular calcium which leads to inefficient oxidative phosphorylation and increased ROS production and eventually swollen patches of mitochondria. This process can be avoided for approximately nine hours after B cell antigen binding by either T helper cells or by signaling through Toll-like receptor 9. Thus, antigen binding to B cells appears to activate a metabolic program that imposes a short time window in which B cells either receive a second signal and survive or alternatively face elimination.

Key events in T cell-dependent antibody responses, including the formation of germinal centers and affinity selection, are dependent on the ability of B cells to capture antigen and present it to follicular helper T cells. Despite its importance, little is known about the regulation of this critical B cell function. Here we show that although TLR9 signaling induced B cell proliferation, secretion of cytokines and IgM, it blocked the ability of antigen-specific B cells to capture, process and present antigen. In the presence of the TLR9 agonist, CpG, B cells were less able to form synapses with antigen-containing membranes and to pull antigen from them. CpG also diminished the ability of the BCR to deliver internalized antigen to antigen processing compartments resulting in fewer MHC-peptide complexes on the B cell surface. Additionally CpG blocked the BCR-induced increases in the expression of both MHC class II and important co-receptors, including CD86, resulting in the inability of B cells to activate antigen-specific CD4+ T cells. A principle component analysis of RNA seq data showed little overlap between B cells stimulated through the BCR or TLR9 alone or together indicating that TLR9 antagonism of BCR-dependent antigen presentation is through the activation of a novel transcriptional program. In a chimeric mouse model and in a human clinical trial the TLR9 agonist, CpG, enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling may enhance the magnitude of an antibody response at the expense of the ability of B cells to engage in germinal center events that are highly dependent on antigen capture and presentation.

B cells are key components of the humoral immune response. Two forms of B cell activation come from engagement of the B cell receptor (BCR) with foreign antigens and from pathogen-associated molecular patters (PAMPs) binding to toll-like receptors (TLRs). For TLRs, MyD88 and TRIF are the major adaptors that lead to NFkB and IRF3 activation respectively. Among various TLRs that are present in B cells, TLR9 is only able to use MyD88 and TLR3 only signals through TRIF, while TLR4 uses both pathways. Therefore, these TLRs provide excellent tools to investigate the roles of TRIF and MyD88 pathways in B cell function and the interplay between them. Here, using stimulation through these three different TLRs either individually or in combination with each other and with/or without anti-IgM, we observed that there is synergy and antagonism between these pathways in terms of activation induced metabolic boost, cytokine production, IgM secretion, expression of various cell surface markers, and antigen presentation. We identified the TRIF pathway as an inducer of Type-I interferon secretion by B cells, which eventually favors the expression of costimulatory molecules such as CD86. On the other hand, MyD88 stimulation antagonized the expression of costimulatory molecules and dramatically induced rapid proliferation and production of IgM as well as proinflammatory cytokines such as IL-6 and TNF. Using B cells obtained from MyD88 or TRIF KO mice, we showed that the interplay between pathways is of limited scale; however, for TLR4 signaling MyD88 has a largely permissive effect while TRIF determines the magnitude of response. These results further our understanding of the complex TLR signaling machinery in B lymphocytes.

Malaria is a global health concern which affects over 200 million individuals worldwide. Although the immune system rapidly responds to Plasmodium infection with specific antibodies, natural antibodies fail to establish long term protection, often leading to repetitive infections and chronicity. Recent studies showed that in a chronic malaria setting both B cells and T cells induce expression of T-bet transcription factor. It is not completely understood whether this is a part of the specific host defense or a result of disease pathogenesis. Here using transgenic mouse models, we show that knockout of T-bet or IFN-g similarly reduces survival of mice compared to WT upon infection with Plasmodium chabaudi, therefore suggesting a protective role for T-bet. To determine the specific role T-bet plays in B cell responses to Plasmodium, we first identified that T-bet expression in mouse B cells are triggered by dual stimulation of IFN-g and B cell receptor. Similarly using Tbet-zsgreen transgenic mice we showed that conditions specific to Plasmodium chabaudi infection drive T-bet expression in multiple B cell subsets in a time dependent fashion. However, no such induction is shown when mice are immunized with T-dependent protein antigens adjuvanted with Alum. On the other hand, bone marrow chimeric animals reconstituted with equal amounts of congenically labeled WT and T-bet KO bone marrow cells showed that for most B cell subsets the lack of T-bet does not change the ratio of WT and KO cells upon Plasmodium infection indicating that T-bet expression may not be equally critical for all B cell responses to infection. These findings suggest a complex role of T-bet in the development of humoral responses.

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.

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 host’s 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.

B cells express the innate immune system receptor, Toll like receptor 9 (TLR9), a potent regulator of B cell function, that signals in response to unmethylated CpG sequences in microbial DNA. A dichotomy in the function of the two major classes of CpG-containing oligonucleotides, namely CpG-A and CpG-B, appears to exist with CpG-A restricted to inducing type 1 interferon (type 1 IFN) in innate immune cells and CpG-B to activating B cells to proliferate and produce antibody and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost the innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. We provide evidence that CpG-A activated B cells in vitro in a TLR9 dependent fashion resulting in the production of IL-6, proliferation, and alterations in the expression of a variety of B cell surface markers, albeit in a less robust fashion as compared to CpG-B. Neither CpGB nor CpG-A alone induced B cells to express type 1 IFN. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B induced a robust type 1 IFN response in B cells both in vitro and in vivo. Conversely, CpG-B-DOTAP but not CpGA-DOTAP promoted B cell germinal center responses in vivo and the production of high affinity antibodies. We also provide evidence that difference in the function of CpGs versus DOTAP-associated-CpGs may be related to their intracellular trafficking in B cells since DOTAP enhances the trafficking of CpG-B but not CpG-A to late endosomal compartments. These findings have important implications for the use of CpG-A and CpG-B to augment type 1 IFN production versus B cell responses in vivo.

Key events in T cell-dependent antibody responses are dependent on the ability of B cells to capture antigen and process and present it to follicular helper T cells. Despite its importance, little is known about the regulation of this critical B cell function. Here we focus on the impact of signaling through the innate immune receptor, Toll like receptor 9 (TLR9), on the outcome of a B cell’s encounter with antigen. We show that although TLR9 signaling induced B cell proliferation, and secretion of cytokines and IgM, it blocked the ability of antigen-specific B cells to capture, process and present antigen. In the presence of the TLR9 agonist, CpG, B cells were less able to form synapses with antigen-containing membranes and to pull antigen from them. CpG also diminished the ability of the BCR to traffick antigen within the cell to antigen processing compartments resulting in fewer MHC-peptide complexes on the B cell surface. TLR signaling also antagonized the BCR-induced increases in the expression of both MHC class II and important co-receptors, including CD86, resulting in a decrease in the duration of antigen-specific B cell-T helper cell interaction and the inability of B cells to activate antigen-specific T cells. Chimeric mice that contained either wild type B cells or B cells deficient in TLR signaling when immunized with antigen adjuvanted with CpG showed that the loss of the B cells’ ability to respond to CpG resulted in lower antigen-specific IgM responses but enhanced high affinity antigen-specific IgG responses. Thus, B cell intrinsic TLR9 signaling may ensure rapid B cell expansion and antibody secretion at the expense of the ability of B cells to engage in germinal center events that are highly dependent antigen capture and presentation.

The Apetela2 (AP2) family of transcription factors is a major regulator of Plasmodium parasite gene expression. Twenty seven members of this family have been identified since their discovery in 2005. However, our understanding which AP2 members regulate key features of Plasmodium biology is incomplete. Here we describe the impact of a single nucleotide polymorphism (SNP) in the active site of the AP2 protein, PBANKA_011210, on the outcome of infection in mice. Plasmodium berghei Anka (PbA), the only rodent parasite that causes experimental cerebral malaria (ECM), codes for a phenylalanine at position 1823 whereas all other Plasmodium parasite strains, none of which cause ECM in mice, including PbNK65, have a serine in this position. Using CRISPR, we modified the PbNK65 parasite (PbNK65-WT) to encode the PbA SNP (PbNK65-PbA). The course of infection with PbNK65-WT or PbNK65-PbA were similar and showed no ECM pathology indicating that the PbA AP2 SNP is not sufficient to cause ECM. However, mice infected with PbNK65-PbA generated dramatically higher germinal center responses and produced higher titers of parasite-specific antibodies. When infected mice were treated with sub-therapeutic doses of the anti-malarial, chloroquine, to lower the parasitemias PbNK65-PbA-infected mice cleared the infection whereas mice infected with PbNK65-WT were unable to control the infection resulting severe anemia and death. These results reveal a novel role of AP2 proteins in controlling host immunity to Plasmodium infections.

Severe malaria, including cerebral malaria (CM) and placental malaria (PM), have been recognized to have many of the features of uncontrolled inflammation. We recently showed that in mice genetic susceptibility to the lethal inflammatory autoimmune disease, systemic lupus erythematosus (SLE), conferred resistance to CM. Protection appeared to be mediated by immune mechanisms that allowed SLE-prone mice, prior to the onset of overt SLE symptoms, to better control their inflammatory response to Plasmodium infection. Here we extend these findings to ask does SLE susceptibility have 1) a cost to reproductive fitness and/or 2) an effect on PM in mice? The rates of conception for WT and SLE susceptible (SLE(s)) mice were similar as were the number and viability of fetuses in pregnant WT and SLE(s) mice indicating that SLE susceptibility does not have a reproductive cost. We found that Plasmodium chabaudi AS (Pc) infection disrupted early stages of pregnancy before the placenta was completely formed resulting in massive decidual necrosis 8 days after conception. Pc-infected pregnant SLE(s) mice had significantly more fetuses (∼1.8 fold) but SLE did not significantly affect fetal viability in infected animals. This was despite the fact that Pc-infected pregnant SLE(s) mice had more severe symptoms of malaria as compared to Pc-infected pregnant WT mice. Thus, although SLE susceptibility was not protective in PM in mice it also did not have a negative impact on reproductive fitness.

B cells express the endosomal, DNA sensing, Toll-like receptor 9 (TLR9) that, in response to CpG oligonucleotides, signals through the adaptor protein MyD88 and activates the NF-kB pathway leading to the transcriptional activation of proinflammatory cytokines. In dendritic cells TLR9-MyD88 signaling is bifurcated into two distinct pathways, the NF-kB pathway and an IRF7-dependent pathway that results in the activation of type 1 interferon (IFN) genes. In these cells, monomeric CpG-B activates the NF-kB-dependent pathway and multimeric CpG-A or CpG-A coupled to the liposomal transfection reagent, Dotap (CpG-A-Dotap), activates the IRF-7-dependent pathway. Here we provide evidence that the B cell TLR9 signaling pathway is similarly bifurcated. Both CpG-B and CpG-B-Dotap potently activate the NF-kB-dependent pathway leading to IL-6 synthesis but neither trigger an IFN response. In contrast, CpG-A-Dotap but not CpG-A alone stimulates an IFN response as shown by increases in the levels of mRNA encoding IFN and in the amount of IFN protein produced and also by the activation of B cells from IFN-reporter mice both in vitro and in vivo. By confocal microscopy CpG-A and CpG-B appear to traffic to different intracellular vesicles. However, the cellular machinery that mediates the IL-6 and IFN pathways seems to be linked in that pretreatment of B cells with CpG-B blocks the subsequent activation of the IFN pathway by CpG-A-Dotap. It will be of interest to determine if natural DNA containing ligands might trigger the two pathways in B cells.

B cells express both an adaptive antigen-specific B cell receptor (BCR) and innate Toll-like receptors (TLRs) allowing the functional outcome of the B cell’s engagement with antigen to be modulated in response to pathogen-derived TLR ligands. In T cell-dependent responses, the BCR both signals for B cell proliferation and differentiation and also internalizes bound antigen for processing and presentation to helper T cells. Here we show that although the presence of the TLR9 ligand, CpG, does not affect early BCR mediated events following antigen binding including phosphorylation of key components of the signaling cascade and the formation of BCR clusters on cell surface, CpG alters the outcome of BCR signaling, resulting in unique transcriptional profiles, enhanced proliferation and differentiation to antibody secreting cells. In contrast, CpG dramatically limits the ability of B cells to process and present antigen to helper T cells. In the presence of CpG, BCR-induced upregulation of CD86 and MHC class II expressions are antagonized and antigen internalized by the BCR is not properly trafficked to antigen processing compartments resulting in reduced numbers of peptide-MHC class II complexes on the B cell surface. Indeed, CpG treated antigen-specific B cells show a reduced ability to maintain contact with antigen-specific T cells and to activate antigen-specific helper T cell proliferation in vitro. These results suggest that TLR9 activation in T cell dependent responses would drive B cell toward proliferation and antibody secretion and away from events that are highly dependent on the ability of B cells to present antigen to helper T cells, such as entry into germinal centers and affinity selection.

Malaria is an infectious disease caused by parasites of the Plasmodium spp. In endemic areas, in approximately 1% of cases, almost exclusively in young children, malaria becomes severe resulting in nearly seven hundred thousand deaths each year in Africa alone. Cerebral malaria (CM) is a common form of severe malaria and one for which we have no effective adjunctive therapy. The kinase, mammalian target of rapamycin (mTOR), through its regulation of cellular metabolism, is a central regulator of immune responses and drugs that inhibit the mTOR pathway have been shown to be antiparasitic, raising the possibility that mTOR inhibitors could be effective adjunctive therapies for CM. Here we show in a mouse model of CM, experimental CM (ECM), that the mTOR inhibitor rapamycin protects against ECM when administered within the first four days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood brain barrier and brain hemorrhaging, decreased influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells 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 and elevated the levels of peripheral parasitemia. 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.