Infection-induced plasmablasts are a nutrient sink that impairs humoral immunity to malaria.

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation.

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

IL-27 signalling regulates glycolysis in Th1 cells to limit immunopathology during infection.

Inflammation is critical for controlling pathogens, but also responsible for symptoms of infectious diseases. IL-27 is an important regulator of inflammation and can limit development of IFNγ-producing Tbet+ CD4+ T (Th1) cells. IL-27 is thought to do this by stimulating IL-10 production by CD4+ T cells, but the underlying mechanisms of these immunoregulatory pathways are not clear. Here we studied the role of IL-27 signalling in experimental visceral leishmaniasis (VL) caused by infection of C57BL/6 mice with the human pathogen Leishmania donovani. We found IL-27 signalling was critical for the development of IL-10-producing Th1 (Tr1) cells during infection. Furthermore, in the absence of IL-27 signalling, there was improved control of parasite growth, but accelerated splenic pathology characterised by the loss of marginal zone macrophages. Critically, we discovered that IL-27 signalling limited glycolysis in Th1 cells during infection that in turn attenuated inflammation. Furthermore, the modulation of glycolysis in the absence of IL-27 signalling restricted tissue pathology without compromising anti-parasitic immunity. Together, these findings identify a novel mechanism by which IL-27 mediates immune regulation during disease by regulating cellular metabolism.

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Distinct Roles for CD4+ Foxp3+ Regulatory T Cells and IL-10-Mediated Immunoregulatory Mechanisms during Experimental Visceral Leishmaniasis Caused by Leishmania donovani.

The outcome of intracellular parasitic infection can be determined by the immunoregulatory activities of natural regulatory CD4+ Foxp3+ T (Treg) cells and the anti-inflammatory cytokine IL-10. These mechanisms protect tissue but can also suppress antiparasitic CD4+ T cell responses. The specific contribution of these regulatory pathways during human parasitic diseases remains unclear. In this study, we investigated the roles of Treg cells and IL-10 during experimental visceral leishmaniasis caused by Leishmania donovani infection of C57BL/6 mice. We report only a limited contribution of Treg cells in suppressing antiparasitic immunity, but important roles in delaying the development of splenic pathology and restricting leukocyte expansion. We next employed a range of cell-specific, IL-10- and IL-10R-deficient mice and found these Treg cell functions were independent of IL-10. Instead, conventional CD4+ T cells and dendritic cells were the most important cellular sources of IL-10, and the absence of IL-10 in either cell population resulted in greater control of parasite growth but also caused accelerated breakdown in splenic microarchitecture. We also found that T cells, dendritic cells, and other myeloid cells were the main IL-10-responding cells because in the absence of IL-10R expression by these cell populations, there was greater expansion of parasite-specific CD4+ T cell responses associated with improved control of parasite growth. Again, however, there was also an accelerated breakdown in splenic microarchitecture in these animals. Together, these findings identify distinct, cell-specific, immunoregulatory networks established during experimental visceral leishmaniasis that could be manipulated for clinical advantage.

Interleukin 2 is an Upstream Regulator of CD4+ T Cells From Visceral Leishmaniasis Patients With Therapeutic Potential.

Control of visceral leishmaniasis (VL) caused by Leishmania donovani requires interferon-γ production by CD4+ T cells. In VL patients, antiparasitic CD4+ T-cell responses are ineffective for unknown reasons. In this study, we measured the expression of genes associated with various immune functions in these cells from VL patients and compared them to CD4+ T cells from the same patients after drug treatment and from endemic controls. We found reduced GATA3, RORC, and FOXP3 gene expression in CD4+ T cells of VL patients, associated with reduced Th2, Th17, and FOXP3+CD4+ T regulatory cell frequencies in VL patient blood. Interleukin 2 (IL-2) was an important upstream regulator of CD4+ T cells from VL patients, and functional studies demonstrated the therapeutic potential of IL-2 for improving antiparasitic immunity. Together, these results provide new insights into the characteristics of CD4+ T cells from VL patients that can be used to improve antiparasitic immune responses.

A molecular signature for CD8+ T cells from visceral leishmaniasis patients.

CD8+ T-cell function is compromised in chronic diseases such as visceral leishmaniasis (VL). However, little is known about the changes in gene expression that cause CD8+ T-cell dysfunction during VL. We used targeted transcriptional profiling of peripheral blood CD8+ T cells from VL patients pre- and post-anti-parasitic drug treatment, and compared them with the same cell population from healthy endemic controls to assess their activation, differentiation and functional status during disease. We found a predominance of downregulated immune genes in CD8+ T cells from VL patients. However, genes encoding several notable immune checkpoint molecules, including LAG-3, TIM-3 and CTLA-4, cytolytic molecules, such as granzymes A, B and H and perforin, as well as SOCS3, STAT1, JAK2 and JAK3 cytokine signalling genes were found to be increasingly expressed by VL patient CD8+ T cells. Additional studies confirmed increased expression of the inhibitory receptors LAG3 and TIM3 on VL patient CD8+ T cells, thereby identifying these molecules as potential targets to improve antigen-specific CD8+ T-cell responses during disease.

Loss of complement regulatory proteins on red blood cells in mild malarial anaemia and in Plasmodium falciparum induced blood-stage infection.

BACKGROUND: Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. METHODS: The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. RESULTS: CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. CONCLUSIONS: Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.

Early Changes in CD4+ T-Cell Activation During Blood-Stage Plasmodium falciparum Infection.

We examined transcriptional changes in CD4+ T cells during blood-stage Plasmodium falciparum infection in individuals without a history of previous parasite exposure. Transcription of CXCL8 (encoding interleukin 8) in CD4+ T cells was identified as an early biomarker of submicroscopic P. falciparum infection, with predictive power for parasite growth. Following antiparasitic drug treatment, a CD4+ T-cell regulatory phenotype developed. PD1 expression on CD49b+CD4+ T (putative type I regulatory T) cells after drug treatment negatively correlated with earlier parasite growth. Blockade of PD1 but no other immune checkpoint molecules tested increased interferon γ and interleukin 10 production in an ex vivo antigen-specific cellular assay at the peak of infection. These results demonstrate the early development of an immunoregulatory CD4+ T-cell phenotype in blood-stage P. falciparum infection and show that a selective immune checkpoint blockade may be used to modulate early developing antiparasitic immunoregulatory pathways as part of malaria vaccine and/or drug treatment protocols.

IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection.

Parasite-specific antibodies protect against blood-stage Plasmodium infection. However, in malaria-endemic regions, it takes many months for naturally-exposed individuals to develop robust humoral immunity. Explanations for this have focused on antigenic variation by Plasmodium, but have considered less whether host production of parasite-specific antibody is sub-optimal. In particular, it is unclear whether host immune factors might limit antibody responses. Here, we explored the effect of Type I Interferon signalling via IFNAR1 on CD4+ T-cell and B-cell responses in two non-lethal murine models of malaria, P. chabaudi chabaudi AS (PcAS) and P. yoelii 17XNL (Py17XNL) infection. Firstly, we demonstrated that CD4+ T-cells and ICOS-signalling were crucial for generating germinal centre (GC) B-cells, plasmablasts and parasite-specific antibodies, and likewise that T follicular helper (Tfh) cell responses relied on B cells. Next, we found that IFNAR1-signalling impeded the resolution of non-lethal blood-stage infection, which was associated with impaired production of parasite-specific IgM and several IgG sub-classes. Consistent with this, GC B-cell formation, Ig-class switching, plasmablast and Tfh differentiation were all impaired by IFNAR1-signalling. IFNAR1-signalling proceeded via conventional dendritic cells, and acted early by limiting activation, proliferation and ICOS expression by CD4+ T-cells, by restricting the localization of activated CD4+ T-cells adjacent to and within B-cell areas of the spleen, and by simultaneously suppressing Th1 and Tfh responses. Finally, IFNAR1-deficiency accelerated humoral immune responses and parasite control by boosting ICOS-signalling. Thus, we provide evidence of a host innate cytokine response that impedes the onset of humoral immunity during experimental malaria.

Blimp-1-Dependent IL-10 Production by Tr1 Cells Regulates TNF-Mediated Tissue Pathology.

Tumor necrosis factor (TNF) is critical for controlling many intracellular infections, but can also contribute to inflammation. It can promote the destruction of important cell populations and trigger dramatic tissue remodeling following establishment of chronic disease. Therefore, a better understanding of TNF regulation is needed to allow pathogen control without causing or exacerbating disease. IL-10 is an important regulatory cytokine with broad activities, including the suppression of inflammation. IL-10 is produced by different immune cells; however, its regulation and function appears to be cell-specific and context-dependent. Recently, IL-10 produced by Th1 (Tr1) cells was shown to protect host tissues from inflammation induced following infection. Here, we identify a novel pathway of TNF regulation by IL-10 from Tr1 cells during parasitic infection. We report elevated Blimp-1 mRNA levels in CD4+ T cells from visceral leishmaniasis (VL) patients, and demonstrate IL-12 was essential for Blimp-1 expression and Tr1 cell development in experimental VL. Critically, we show Blimp-1-dependent IL-10 production by Tr1 cells prevents tissue damage caused by IFNγ-dependent TNF production. Therefore, we identify Blimp-1-dependent IL-10 produced by Tr1 cells as a key regulator of TNF-mediated pathology and identify Tr1 cells as potential therapeutic tools to control inflammation.

IL-17A-Producing γδ T Cells Suppress Early Control of Parasite Growth by Monocytes in the Liver.

Intracellular infections, such as those caused by the protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis (VL), require a potent host proinflammatory response for control. IL-17 has emerged as an important proinflammatory cytokine required for limiting growth of both extracellular and intracellular pathogens. However, there are conflicting reports on the exact roles for IL-17 during parasitic infections and limited knowledge about cellular sources and the immune pathways it modulates. We examined the role of IL-17 in an experimental model of VL caused by infection of C57BL/6 mice with L. donovani and identified an early suppressive role for IL-17 in the liver that limited control of parasite growth. IL-17-producing γδ T cells recruited to the liver in the first week of infection were the critical source of IL-17 in this model, and CCR2(+) inflammatory monocytes were an important target for the suppressive effects of IL-17. Improved parasite control was independent of NO generation, but associated with maintenance of superoxide dismutase mRNA expression in the absence of IL-17 in the liver. Thus, we have identified a novel inhibitory function for IL-17 in parasitic infection, and our results demonstrate important interactions among γδ T cells, monocytes, and infected macrophages in the liver that can determine the outcome of parasitic infection.

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions.

BACKGROUND & AIMS: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied. METHODS: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types. RESULTS: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite. CONCLUSIONS: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology. LAY SUMMARY: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist. DATA REPOSITORY: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).

Spatiotemporal requirements for IRF7 in mediating type I IFN-dependent susceptibility to blood-stage Plasmodium infection.

Type I IFN signaling suppresses splenic T helper 1 (Th1) responses during blood-stage Plasmodium berghei ANKA (PbA) infection in mice, and is crucial for mediating tissue accumulation of parasites and fatal cerebral symptoms via mechanisms that remain to be fully characterized. Interferon regulatory factor 7 (IRF7) is considered to be a master regulator of type I IFN responses. Here, we assessed IRF7 for its roles during lethal PbA infection and nonlethal Plasmodium chabaudi chabaudi AS (PcAS) infection as two distinct models of blood-stage malaria. We found that IRF7 was not essential for tissue accumulation of parasites, cerebral symptoms, or brain pathology. Using timed administration of anti-IFNAR1 mAb, we show that late IFNAR1 signaling promotes fatal disease via IRF7-independent mechanisms. Despite this, IRF7 significantly impaired early splenic Th1 responses and limited control of parasitemia during PbA infection.  Finally, IRF7 also suppressed antiparasitic immunity and Th1 responses during nonlethal PcAS infection. Together, our data support a model in which IRF7 suppresses antiparasitic immunity in the spleen, while IFNAR1-mediated, but IRF7-independent, signaling contributes to pathology in the brain during experimental blood-stage malaria.

Plasmodium berghei bio-burden correlates with parasite lactate dehydrogenase: application to murine Plasmodium diagnostics.

BACKGROUND: The spectrum of techniques to detect malaria parasites in whole blood is limited to measuring parasites in circulation. One approach that is currently used to enumerate total parasite bio-burden involves the use of bio-luminescent parasites. As an alternative approach, this study describes the use of a commercial ELISA human parasite lactate dehydrogenase (pLDH) detection kit to estimate total parasite bio-burden in murine malaria models. METHODS: The cross reactivity of pLDH in a commercial human malaria pLDH diagnostic kit was established in different components of blood for different murine malaria models. The use of pLDH as a measure of parasite bio-burden was evaluated by examining pLDH in relation to peripheral blood parasitaemia as determined by microscopy and calculating total parasite bio-burden using a bio-luminescent Plasmodium berghei ANKA luciferase parasite. RESULTS: The pLDH antigen was detected in all four murine Plasmodium species and in all components of Plasmodium-infected blood. A significant correlation (r = 0.6922, P value <0.0001) was observed between total parasite bio-burden, measured as log average radiance, and concentration of pLDH units. CONCLUSIONS: This high throughput assay is a suitable measure of total parasite bio-burden in murine malaria infections. Unlike existing methods, it permits the estimation of both circulating and sequestered parasites, allowing a more accurate assessment of parasite bio-burden.

Tissue requirements for establishing long-term CD4+ T cell-mediated immunity following Leishmania donovani infection.

Organ-specific immunity is a feature of many infectious diseases, including visceral leishmaniasis caused by Leishmania donovani. Experimental visceral leishmaniasis in genetically susceptible mice is characterized by an acute, resolving infection in the liver and chronic infection in the spleen. CD4+ T cell responses are critical for the establishment and maintenance of hepatic immunity in this disease model, but their role in chronically infected spleens remains unclear. In this study, we show that dendritic cells are critical for CD4+ T cell activation and expansion in all tissue sites examined. We found that FTY720-mediated blockade of T cell trafficking early in infection prevented Ag-specific CD4+ T cells from appearing in lymph nodes, but not the spleen and liver, suggesting that early CD4+ T cell priming does not occur in liver-draining lymph nodes. Extended treatment with FTY720 over the first month of infection increased parasite burdens, although this associated with blockade of lymphocyte egress from secondary lymphoid tissue, as well as with more generalized splenic lymphopenia. Importantly, we demonstrate that CD4+ T cells are required for the establishment and maintenance of antiparasitic immunity in the liver, as well as for immune surveillance and suppression of parasite outgrowth in chronically infected spleens. Finally, although early CD4+ T cell priming appeared to occur most effectively in the spleen, we unexpectedly revealed that protective CD4+ T cell-mediated hepatic immunity could be generated in the complete absence of all secondary lymphoid tissues.

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.

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.