IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain.

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection

Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.

Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings

Memory CD8⁺ T cells exhibit tissue imprinting and non-stable exposure-dependent reactivation characteristics following blood-stage Plasmodium berghei ANKA infections

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8(+) T‐cell accumulation within the brain. Whilst the dynamics of CD8(+) T‐cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite‐specific CD8(+) T cells upon resolution of ECM is not understood. In this study, we show that memory OT‐I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA‐OVA infection. Whereas memory OT‐I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT‐I cells within the brain post‐PbA‐OVA infection displayed an enriched CD69(+)CD103(−) profile and expressed low levels of T‐bet. OT‐I cells within the brain were excluded from short‐term intravascular antibody labelling but were targeted effectively by longer‐term systemically administered antibodies. Thus, the memory OT‐I cells were extravascular within the brain post‐ECM but were potentially not resident memory cells. Importantly, whilst memory OT‐I cells exhibited strong reactivation during secondary PbA‐OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA‐OVA infection. Overall, our results demonstrate that memory CD8(+) T cells are systemically distributed but exhibit a unique phenotype within the brain post‐ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8(+) T‐cell populations within the brain and other tissues during repeat Plasmodium infections

Parasite-Derived Plasma Microparticles Contribute Significantly to Malaria Infection-Induced Inflammation through Potent Macrophage Stimulation

There is considerable debate as to the nature of the primary parasite-derived moieties that activate innate pro-inflammatory responses during malaria infection. Microparticles (MPs), which are produced by numerous cell types following vesiculation of the cellular membrane as a consequence of cell death or immune-activation, exert strong pro-inflammatory activity in other disease states. Here we demonstrate that MPs, derived from the plasma of malaria infected mice, but not naive mice, induce potent activation of macrophages in vitro as measured by CD40 up-regulation and TNF production. In vitro, these MPs induced significantly higher levels of macrophage activation than intact infected red blood cells. Immunofluorescence staining revealed that MPs contained significant amounts of parasite material indicating that they are derived primarily from infected red blood cells rather than platelets or endothelial cells. MP driven macrophage activation was completely abolished in the absence of MyD88 and TLR-4 signalling. Similar levels of immunogenic MPs were produced in WT and in TNF−/−, IFN-γ−/−, IL-12−/− and RAG-1−/− malaria-infected mice, but were not produced in mice injected with LPS, showing that inflammation is not required for the production of MPs during malaria infection. This study therefore establishes parasitized red blood cell-derived MPs as a major inducer of systemic inflammation during malaria infection, raising important questions about their role in severe disease and in the generation of adaptive immune responses

IL-27 receptor signaling regulates CD4+ T cell chemotactic responses during infection.

IL-27 exerts pleiotropic suppressive effects on naive and effector T cell populations during infection and inflammation. Surprisingly, however, the role of IL-27 in restricting or shaping effector CD4(+) T cell chemotactic responses, as a mechanism to reduce T cell-dependent tissue inflammation, is unknown. In this study, using Plasmodium berghei NK65 as a model of a systemic, proinflammatory infection, we demonstrate that IL-27R signaling represses chemotaxis of infection-derived splenic CD4(+) T cells in response to the CCR5 ligands, CCL4 and CCL5. Consistent with these observations, CCR5 was expressed on significantly higher frequencies of splenic CD4(+) T cells from malaria-infected, IL-27R-deficient (WSX-1(-/-)) mice than from infected wild-type mice. We find that IL-27 signaling suppresses splenic CD4(+) T cell CCR5-dependent chemotactic responses during infection by restricting CCR5 expression on CD4(+) T cell subtypes, including Th1 cells, and also by controlling the overall composition of the CD4(+) T cell compartment. Diminution of the Th1 response in infected WSX-1(-/-) mice in vivo by neutralization of IL-12p40 attenuated CCR5 expression by infection-derived CD4(+) T cells and also reduced splenic CD4(+) T cell chemotaxis toward CCL4 and CCL5. These data reveal a previously unappreciated role for IL-27 in modulating CD4(+) T cell chemotactic pathways during infection, which is related to its capacity to repress Th1 effector cell development. Thus, IL-27 appears to be a key cytokine that limits the CCR5-CCL4/CCL5 axis during inflammatory settings

Treatment with specific and pan-plasma membrane calcium ATPase (PMCA) inhibitors reduces malaria parasite growth in vitro and in vivo

BACKGROUND: Rapid emergence of Plasmodium resistance to anti-malarial drug mainstays has driven a continual effort to discover novel drugs that target different biochemical pathway (s) during infection. Plasma membrane Calcium + 2 ATPase (PMCA4), a novel plasma membrane protein that regulates Calcium levels in various cells, namely red blood cell (RBC), endothelial cell and platelets, represents a new biochemical pathway that may interfere with susceptibility to malaria and/or severe malaria. METHODS: This study identified several pharmacological inhibitors of PMCA4, namely ATA and Resveratrol, and tested for their anti-malarial activities in vitro and in vivo using the Plasmodium falciparum 3D7 strain, the Plasmodium berghei ANKA strain, and Plasmodium yoelii 17XL strain as model. RESULTS: In vitro propagation of P. falciparum 3D7 strain in the presence of a wide concentration range of the inhibitors revealed that the parasite growth was inhibited in a dose-dependent manner, with IC(50)s at 634 and 0.231 µM, respectively. RESULTS: The results confirmed that both compounds exhibit moderate to potent anti-malarial activities with the strongest parasite growth inhibition shown by resveratrol at 0.231 µM. In vivo models using P. berghei ANKA for experimental cerebral malaria and P. yoelii 17XL for the effect on parasite growth, showed that the highest dose of ATA, 30 mg/kg BW, increased survival of the mice. Likewise, resveratrol inhibited the parasite growth following 4 days intraperitoneal injection at the dose of 100 mg/kg BW. CONCLUSION: The findings indicate that the PMCA4 of the human host may be a potential target for novel anti-malarials, either as single drug or in combination with the currently available effective anti-malarials

Exhausted CD4+ T Cells during Malaria Exhibit Reduced mTORc1 Activity Correlated with Loss of T-bet Expression

CD4<sup>+</sup> T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4<sup>+</sup> T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (<i>Ag-exp</i>) CD4<sup>+</sup> T cell exhaustion during <i>Plasmodium yoelii</i> nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4<sup>+</sup> T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted <i>Ag-exp</i>CD4<sup>+</sup> T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4<sup>+</sup> T cells. Consistent with this, <i>Ag-exp</i>Th1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) <i>Ag-exp</i>T cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4<sup>+</sup> T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4<sup>+</sup> T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in <i>Ag-exp</i>CD4<sup>+</sup> T cells but that reduction in mTOR activity may not directly underpin <i>Ag-exp</i>Th1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4<sup>+</sup> T cells during malaria infection and other chronic conditions

Memory CD8 + T cells exhibit tissue imprinting and non‐stable exposure‐dependent reactivation characteristics following blood‐stage Plasmodium berghei ANKA infections

From Wiley via Jisc Publications RouterHistory: received 2020-11-02, rev-recd 2021-08-09, accepted 2021-08-13, pub-electronic 2021-08-27Article version: VoRPublication status: PublishedFunder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100000265; Grant(s): G0900487, MR/R010099/1Abstract: Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8+ T‐cell accumulation within the brain. Whilst the dynamics of CD8+ T‐cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite‐specific CD8+ T cells upon resolution of ECM is not understood. In this study, we show that memory OT‐I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA‐OVA infection. Whereas memory OT‐I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT‐I cells within the brain post‐PbA‐OVA infection displayed an enriched CD69+CD103− profile and expressed low levels of T‐bet. OT‐I cells within the brain were excluded from short‐term intravascular antibody labelling but were targeted effectively by longer‐term systemically administered antibodies. Thus, the memory OT‐I cells were extravascular within the brain post‐ECM but were potentially not resident memory cells. Importantly, whilst memory OT‐I cells exhibited strong reactivation during secondary PbA‐OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA‐OVA infection. Overall, our results demonstrate that memory CD8+ T cells are systemically distributed but exhibit a unique phenotype within the brain post‐ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8+ T‐cell populations within the brain and other tissues during repeat Plasmodium infections

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

From Springer Nature via Jisc Publications RouterHistory: received 2021-03-03, accepted 2021-06-24, registration 2021-06-24, pub-electronic 2021-07-02, online 2021-07-02, collection 2021-12Publication status: PublishedFunder: Medical Research Council; doi: http://dx.doi.org/10.13039/501100000265; Grant(s): MR/P015816/1Abstract: Background: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. Methods: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. Results: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. Conclusions: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs