Innate immune responses to malaria-infected erythrocytes in pregnant women: Effects of gravidity, malaria infection, and geographic location.

BACKGROUND: Malaria in pregnancy causes maternal, fetal and neonatal morbidity and mortality, and maternal innate immune responses are implicated in pathogenesis of these complications. The effects of malaria exposure and obstetric and demographic factors on the early maternal immune response are poorly understood. METHODS: Peripheral blood mononuclear cell responses to Plasmodium falciparum-infected erythrocytes and phytohemagglutinin were compared between pregnant women from Papua New Guinea (malaria-exposed) with and without current malaria infection and from Australia (unexposed). Elicited levels of inflammatory cytokines at 48 h and 24 h (interferon γ, IFN-γ only) and the cellular sources of IFN-γ were analysed. RESULTS: Among Papua New Guinean women, microscopic malaria at enrolment did not alter peripheral blood mononuclear cell responses. Compared to samples from Australia, cells from Papua New Guinean women secreted more inflammatory cytokines tumor necrosis factor-α, interleukin 1ß, interleukin 6 and IFN-γ; p<0.001 for all assays, and more natural killer cells produced IFN-γ in response to infected erythrocytes and phytohemagglutinin. In both populations, cytokine responses were not affected by gravidity, except that in the Papua New Guinean cohort multigravid women had higher IFN-γ secretion at 24 h (p = 0.029) and an increased proportion of IFN-γ+ Vδ2 γδ T cells (p = 0.003). Cytokine levels elicited by a pregnancy malaria-specific CSA binding parasite line, CS2, were broadly similar to those elicited by CD36-binding line P6A1. CONCLUSIONS: Geographic location and, to some extent, gravidity influence maternal innate immunity to malaria.

Intermittent screening and treatment with dihydroartemisinin-piperaquine and intermittent preventive therapy with sulfadoxine-pyrimethamine have similar effects on malaria antibody in pregnant Malawian women.

In a randomised trial comparing intermittent screening and treatment (IST) with dihydroartemisinin-piperaquine (DP) and intermittent preventive therapy against malaria in pregnancy (IPT) with sulfadoxine-pyrimethamine (SP) in Malawi, the impacts of IST-DP and IPT-SP on the development and maintenance of malaria antibody immunity were compared. Pregnant Malawian women were randomised to receive IST-DP or IPT-SP. In a nested study, paired enrolment and delivery plasma samples from 681 women were assayed for antibodies against recombinant antigens and for IgG and opsonising antibodies to antigens found on infected erythrocytes (IEs). At delivery, antibody responses did not differ between study arms. Between enrolment and delivery, antibodies to recombinant antigens decreased, whereas antibodies to IEs including opsonising antibodies remained stable. Overall, changes in antibody responses over pregnancy did not differ by treatment arm. Stratifying by gravidity, antibody to schizont extract decreased more in multigravidae receiving IST-DP than IPT-SP. There was minimal impact of treatment arm on the development and maintenance of malaria immunity. While antibodies to recombinant antigens declined between enrolment and delivery, antibodies directed against IEs tended to be more stable, suggesting longer-lasting protection.Clinical trial registration: Pa n African Clinical Trials Registry (PACTR201103000280319) 14/03/2011. URL: http://www.isrctn.com/ISRCTN69800930 .

STAT1 Signaling in Astrocytes Is Essential for Control of Infection in the Central Nervous System.

The local production of gamma interferon (IFN-γ) is important to control Toxoplasma gondii in the brain, but the basis for these protective effects is not fully understood. The studies presented here reveal that the ability of IFN-γ to inhibit parasite replication in astrocytes in vitro is dependent on signal transducer and activator of transcription 1 (STAT1) and that mice that specifically lack STAT1 in astrocytes are unable to limit parasite replication in the central nervous system (CNS). This susceptibility is associated with a loss of antimicrobial pathways and increased cyst formation in astrocytes. These results identify a critical role for astrocytes in limiting the replication of an important opportunistic pathogen. IMPORTANCE: Astrocytes are the most numerous cell type in the brain, and they are activated in response to many types of neuroinflammation, but their function in the control of CNS-specific infection is unclear. The parasite Toxoplasma gondii is one of the few clinically relevant microorganisms that naturally infects astrocytes, and the studies presented here establish that the ability of astrocytes to inhibit parasite replication is essential for the local control of this opportunistic pathogen. Together, these studies establish a key role for astrocytes as effector cells and in the coordination of many aspects of the protective immune response that operates in the brain.

Malaria preventive therapy in pregnancy and its potential impact on immunity to malaria in an area of declining transmission.

BACKGROUND: Regular anti-malarial therapy in pregnancy, a pillar of malaria control, may affect malaria immunity, with therapeutic implications in regions of reducing transmission. METHODS: Plasma antibodies to leading vaccine candidate merozoite antigens and opsonizing antibodies to endothelial-binding and placental-binding infected erythrocytes were quantified in pregnant Melanesian women receiving sulfadoxine-pyrimethamine (SP) with chloroquine taken once, or three courses of SP with azithromycin. RESULTS: Malaria prevalence was low. Between enrolment and delivery, antibodies to recombinant antigens declined in both groups (p<0.0001). In contrast, median levels of opsonizing antibodies did not change, although levels for some individuals changed significantly. In multivariate analysis, the malaria prevention regimen did not influence antibody levels. CONCLUSION: Different preventive anti-malarial chemotherapy regimens used during pregnancy had limited impact on malarial-immunity in a low-transmission region of Papua New Guinea. TRIAL REGISTRATIONS: NCT01136850.

Decreasing malaria prevalence and its potential consequences for immunity in pregnant women.

BACKGROUND: As malaria control is intensified, pregnant women may be less exposed to malaria, thus affecting the acquisition of protective antibody. METHODS: Plasma samples were collected from Malawian and Papua New Guinean (PNG) pregnant women enrolled over 7-year periods, during which malaria prevalence fell by over two thirds. Immunoglobulin G (IgG) levels to schizont extract, merozoite antigens, and VAR2CSA-DBL5ε were measured by enzyme-linked immunosorbent assay (ELISA). Levels of IgG to variant surface antigens of infected erythrocytes (IEs) and merozoites and levels of opsonizing IgG to IEs were measured by flow cytometry. RESULTS: In both settings, levels of antibodies in pregnant women to recombinant antigens and to intact IEs but not of opsonizing antibodies decreased over time. After adjustment for coverage with insecticide-treated bed nets (ITNs), these differences disappeared in the Malawian cohort, whereas in the PNG cohort, time was independently associated with a decrease in several antibody responses measured by ELISA. CONCLUSIONS: The impact of falling parasite prevalence on anti-Plasmodium falciparum serological indicators in pregnant women varies by setting. Increased ITN coverage may affect development of antibodies to recombinant antigens, but levels of opsonizing IgG remained stable over time. Opsonizing IgG against placental-binding IEs may persist, thus offering longer-lasting protection against malaria during pregnancy.

The aryl hydrocarbon receptor promotes IL-10 production by NK cells.

The cytokine IL-10 has an important role in limiting inflammation in many settings, including toxoplasmosis. In the present studies, an IL-10 reporter mouse was used to identify the sources of this cytokine following challenge with Toxoplasma gondii. During infection, multiple cell types expressed the IL-10 reporter but NK cells were a major early source of this cytokine. These IL-10 reporter(+) NK cells expressed high levels of the IL-12 target genes T-bet, KLRG1, and IFN-γ, and IL-12 depletion abrogated reporter expression. However, IL-12 signaling alone was not sufficient to promote NK cell IL-10, and activation of the aryl hydrocarbon receptor (AHR) was also required for maximal IL-10 production. NK cells basally expressed the AHR, relevant chaperone proteins, and the AHR nuclear translocator, which heterodimerizes with the AHR to form a competent transcription factor. In vitro studies revealed that IL-12 stimulation increased NK cell AHR levels, and the AHR and AHR nuclear translocator were required for optimal production of IL-10. Additionally, NK cells isolated from T. gondii-infected Ahr(-/-) mice had impaired expression of IL-10, which was associated with increased resistance to this infection. Taken together, these data identify the AHR as a critical cofactor involved in NK cell production of IL-10.

Low antibody levels to pregnancy-specific malaria antigens and heightened cytokine responses associated with severe malaria in pregnancy.

BACKGROUND: Pregnant women living in unstable malaria transmission settings may develop severe malaria (SM). The pathogenesis of SM in pregnancy is poorly understood. METHODS: To determine whether SM in pregnancy is associated with lower malarial antibody responses and higher cytokine responses, plasma samples were collected from 121 Sudanese pregnant women of whom 39 were diagnosed with SM. Antibodies to pregnancy-specific and non-pregnancy-specific Plasmodium falciparum variant surface antigens (VSA) and concentrations of cytokines TNF, IFNγ, IL-1ß, IL-6, IL-8 and IL-10 were measured. RESULTS: Pregnant women with SM demonstrated significantly lower antibody levels to pregnancy-specific VSA (P = .020) and higher plasma IFNγ (P = .020), IL-10 (P = .0002) and IL-6 levels (P < .0001) than uninfected pregnant women. Concentrations of inflammatory cytokines IL-1ß (P = .001), IL-6 (P = .004) and IL-8 (P = .020) were inversely correlated with antibodies to VAR2CSA-DBL5 in pregnant women with SM. Lower haemoglobin levels and higher parasite densities were associated with lack of pregnancy-specific antibodies (P = .028) and higher levels of inflammatory cytokines, in particular IL-6 and IL-8. CONCLUSIONS: Pregnant women with SM lack pregnancy-specific malaria immunity, and this correlates with heightened inflammatory cytokine concentrations, low haemoglobin levels and high parasite density, suggesting that failure of antibody to control parasitaemia may contribute to SM pathogenesis.

Pivotal advance: peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells.

Breaking the long-held paradigm that primary B cells are not phagocytic, several studies have demonstrated recently that B cells from fish, amphibians, and reptilians have a significant phagocytic capacity. Whether such capacity has remained conserved in certain mammalian B cell subsets is presently an enigma. Here, we report a previously unrecognized ability of PerC B-1a and B-1b lymphocytes to phagocytose latex beads and bacteria. In contrast, B-2 lymphocytes had an almost negligible ability to internalize these particles. Upon phagocytosis, B-1a and B-1b cells were able to mature their phagosomes into phagolysosomes and displayed the ability to kill internalized bacteria. Importantly, B-1a and B-1b cells effectively present antigen recovered from phagocytosed particles to CD4(+) T cells. However, these cells showed a much lower competence to present soluble antigen or antigen from large, noninternalized particles. B-1 B cells presented particulate and soluble antigen to CD4(+) T cells more efficiently than macrophages, whereas DCs were the most potent APCs. The novel phagocytic and microbicidal abilities identified in B-1 B lymphocytes strengthen the innate nature that has long been attributed to these cells. In the context of adaptive immunity, we show that these innate immune processes are relevant, as they enable B-1 B cells to present phagocytosable particulate antigen. These capacities position these cells at the crossroads that link innate with adaptive immune processes. In a broader context, these newly identified capacities of B-1 B cells further support the previously recognized functional, developmental, and evolutionary relationships between these cells and macrophages.

Critical roles for LIGHT and its receptors in generating T cell-mediated immunity during Leishmania donovani infection.

LIGHT (TNFSF14) is a member of the TNF superfamily involved in inflammation and defence against infection. LIGHT signals via two cell-bound receptors; herpes virus entry mediator (HVEM) and lymphotoxin-beta receptor (LTßR). We found that LIGHT is critical for control of hepatic parasite growth in mice with visceral leishmaniasis (VL) caused by infection with the protozoan parasite Leishmania donovani. LIGHT-HVEM signalling is essential for early dendritic cell IL-12/IL-23p40 production, and the generation of IFNγ- and TNF-producing T cells that control hepatic infection. However, we also discovered that LIGHT-LTßR interactions suppress anti-parasitic immunity in the liver in the first 7 days of infection by mechanisms that restrict both CD4(+) T cell function and TNF-dependent microbicidal mechanisms. Thus, we have identified distinct roles for LIGHT in infection, and show that manipulation of interactions between LIGHT and its receptors may be used for therapeutic advantage.

Analysis of behavior and trafficking of dendritic cells within the brain during toxoplasmic encephalitis.

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site.

A study of the TNF/LTA/LTB locus and susceptibility to severe malaria in highland papuan children and adults.

BACKGROUND: Severe malaria (SM) syndromes caused by Plasmodium falciparum infection result in major morbidity and mortality each year. However, only a fraction of P. falciparum infections develop into SM, implicating host genetic factors as important determinants of disease outcome. Previous studies indicate that tumour necrosis factor (TNF) and lymphotoxin alpha (LTα) may be important for the development of cerebral malaria (CM) and other SM syndromes. METHODS: An extensive analysis was conducted of single nucleotide polymorphisms (SNPs) in the TNF, LTA and LTB genes in highland Papuan children and adults, a population historically unexposed to malaria that has migrated to a malaria endemic region. Generated P-values for SNPs spanning the LTA/TNF/LTB locus were corrected for multiple testing of all the SNPs and haplotype blocks within the region tested through 10,000 permutations. A global P-value of < 0.05 was considered statistically significant. RESULTS: No associations between SNPs in the TNF/LTA/LTB locus and susceptibility to SM in highland Papuan children and adults were found. CONCLUSIONS: These results support the notion that unique selective pressure on the TNF/LTA/LTB locus in different populations has influenced the contribution of the gene products from this region to SM susceptibility.

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria.

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-gamma and lymphotoxin alpha, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4(+) and CD8(+) T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4(+) T cell responses by helminth coinfection amplified CD4(+) T cell-mediated parasite sequestration, whereas vaccination could generate CD4(+) T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Age-related susceptibility to severe malaria associated with galectin-2 in highland Papuans.

BACKGROUND: Age and host genetics are important determinants of malaria severity. Lymphotoxin-alpha (LTalpha) has been associated with the development of cerebral malaria (CM) and other severe malaria (SM) syndromes. Mutations in genes regulating LTalpha production contribute to other acute vascular diseases and may contribute to malaria pathogenesis. METHODS: We tested the association between rs7291467, a single-nucleotide polymorphism (SNP) in the LTalpha-related gene encoding galectin-2 (LGALS2), disease severity, and function in a case-control study of ethnic Highland Papuan adults and children with SM (n = 380) and asymptomatic malaria-exposed controls (n = 356) originating from a non-malaria-endemic region but residing in a lowland malaria-endemic area of Papua, Indonesia. RESULTS: The LGALS2 SNP showed a significant association with susceptibility to SM (including CM), in children (odds ratio, 2.02 [95% confidence interval, 1.14-3.57]) but not in adults. In SM, the C allele at rs7291467 was associated with enhanced galectin-2 transcript levels. In a separate group of Tanzanian children originating from a malaria-endemic region, we found preservation of the major ancestral LGALS2 allele and no association with susceptibility to CM. CONCLUSIONS: Results suggest differences in the inflammatory contribution to the development of SM between children and adults in the same population and potential differences between individuals originating from malaria-endemic and non-malaria-endemic areas.

TNF family members and malaria: old observations, new insights and future directions.

Tumor necrosis factor (TNF) has long been recognized to promote malaria parasite killing, but also to contribute to the development of severe malaria disease. The precise molecular mechanisms that influence these different outcomes in malaria patients are not well understood, but the virulence and drug-resistance phenotype of malaria parasites and the genetic background and age of patients are likely to be important determinants. In the past few years, important roles for other TNF family members in host immune responses to malaria parasites and the induction of disease pathology have been discovered. In this review, we will summarize these more recent findings and highlight major gaps in our current knowledge. We will also discuss future research strategies that may allow us to better understand the sometimes subtle and intricate effects of TNF family molecules during malaria infection.

Soluble lymphotoxin is an important effector molecule in GVHD and GVL.

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTalpha3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTalpha have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTalpha3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTalpha is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTalpha- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta(-/-) donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTalpha were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTalpha3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

Cutting edge: selective blockade of LIGHT-lymphotoxin beta receptor signaling protects mice from experimental cerebral malaria caused by Plasmodium berghei ANKA.

Studies in experimental cerebral malaria (ECM) in mice have identified T cells and TNF family members as critical mediators of pathology. In this study we report a role for LIGHT-lymphotoxin beta Receptor (LTbetaR) signaling in the development of ECM and control of parasite growth. Specific blockade of LIGHT-LTbetaR, but not LIGHT-herpesvirus entry mediator interactions, abrogated the accumulation of parasites and the recruitment of pathogenic CD8(+) T cells and monocytes to the brain during infection without affecting early activation of CD4(+) T cells, CD8(+) T cells, or NK cells. Importantly, blockade of LIGHT-LTbetaR signaling caused the expansion of splenic monocytes and an overall enhanced capacity to remove and process Ag during infection, as well as reduced systemic cytokine levels when control mice displayed severe ECM symptoms. In summary, we have discovered a novel pathogenic role for LIGHT and LTbetaR in ECM, identifying this TNF family receptor-ligand interaction as an important immune regulator during experimental malaria.

Common strategies to prevent and modulate experimental cerebral malaria in mouse strains with different susceptibilities.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, predominantly experienced by children and nonimmune adults, which results in significant mortality and long-term sequelae. Previous studies have reported distinct susceptibility gene loci in CBA/CaH (CBA) and C57BL/6 (B6) mice with experimental CM (ECM) caused by infection with Plasmodium berghei ANKA. Here we present an analysis of genome-wide expression profiles in brain tissue taken from B6 and CBA mice with ECM and report significant heterogeneity between the two mouse strains. Upon comparison of the leukocyte composition of ECM brain tissue, microglia were expanded in B6 mice but not CBA mice. Furthermore, circulating levels of gamma interferon, interleukin-10, and interleukin-6 were significantly higher in the serum of B6 mice than in that of CBA mice with ECM. Two therapeutic strategies were applied to B6 and CBA mice, i.e., (i) depletion of regulatory T (Treg) cells prior to infection and (ii) depletion of CD8(+) T cells after the establishment of ECM. Despite the described differences between susceptible mouse strains, depletion of Treg cells before infection attenuated ECM in both B6 and CBA mice. In addition, the depletion of CD8(+) T cells when ECM symptoms are apparent leads to abrogation of ECM in B6 mice and a lack of progression of ECM in CBA mice. These results may have important implications for the development of effective treatments for human CM.

Activation of invariant NKT cells exacerbates experimental visceral leishmaniasis.

We report that natural killer T (NKT) cells play only a minor physiological role in protection from Leishmania donovani infection in C57BL/6 mice. Furthermore, attempts at therapeutic activation of invariant NKT (iNKT) cells with alpha-galactosylceramide (alpha-GalCer) during L. donovani infection exacerbated, rather than ameliorated, experimental visceral leishmaniasis. The inability of alpha-GalCer to promote anti-parasitic immunity did not result from inefficient antigen presentation caused by infection because alpha-GalCer-loaded bone marrow-derived dendritic cells were also unable to improve disease resolution. The immune-dampening affect of alpha-GalCer correlated with a bias towards increased IL-4 production by iNKT cells following alpha-GalCer stimulation in infected mice compared to naïve controls. However, studies in IL-4-deficient mice, and IL-4 neutralisation in cytokine-sufficient mice revealed that alpha-GalCer-induced IL-4 production during infection had only a minor role in impaired parasite control. Analysis of liver cell composition following alpha-GalCer stimulation during an established L. donovani infection revealed important differences, predominantly a decrease in IFNgamma+ CD8+ T cells, compared with control-treated mice. Our data clearly illustrate the double-edged sword of NKT cell-based therapy, showing that in some circumstances, such as when sub-clinical or chronic infections exist, iNKT cell activation can have adverse outcomes.

A role for natural regulatory T cells in the pathogenesis of experimental cerebral malaria.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection that is responsible for a significant number of deaths in children and nonimmune adults. A failure to control blood parasitemia and subsequent sequestration of parasites to brain microvasculature are thought to be key events in many CM cases. Here, we show for the first time, to our knowledge, that CD4(+)CD25(+)Foxp3(+) natural regulatory T (Treg) cells contribute to pathogenesis by modulating immune responses in P. berghei ANKA (PbA)-infected mice. Depletion of Treg cells with anti-CD25 monoclonal antibody protected mice from experimental CM. The accumulation of parasites in the vasculature and brain was reduced in these animals, resulting in significantly lower parasite burdens compared with control animals. Mice lacking Treg cells had increased numbers of activated CD4(+) and CD8(+) T cells in the spleen and lymph nodes, but CD8(+) T-cell recruitment to the brain was selectively reduced in these mice. Importantly, a non-Treg-cell source of interleukin-10 was critical in preventing experimental CM. Finally, we show that therapeutic administration of anti-CD25 monoclonal antibody, even when blood parasitemia is established, can prevent disease, confirming a critical and paradoxical role for Treg cells in experimental CM pathogenesis.

Cutting edge: conventional dendritic cells are the critical APC required for the induction of experimental cerebral malaria.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum infection, causing significant morbidity and mortality among young children and nonimmune adults in the developing world. Although previous work on experimental CM has identified T cells as key mediators of pathology, the APCs and subsets therein required to initiate immunopathology remain unknown. In this study, we show that conventional dendritic cells but not plasmacytoid dendritic cells are required for the induction of malaria parasite-specific CD4+ T cell responses and subsequent experimental CM. These data have important implications for the development of malaria vaccines and the therapeutic management of CM.