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Additional file 1. Additional figures and tables.

Immune-mediated pathology in interleukin-10 (IL-10)-deficient mice during blood-stage malaria infection typically manifests in nonlymphoid organs, such as the liver and lung. Thus, it is critical to define the cellular sources of IL-10 in these sensitive nonlymphoid compartments during infection. Moreover, it is important to determine if IL-10 production is controlled through conserved or disparate molecular programs in distinct anatomical locations during malaria infection, as this may enable spatiotemporal tuning of the regulatory immune response. In this study, using dual gamma interferon (IFN-γ)-yellow fluorescent protein (YFP) and IL-10-green fluorescent protein (GFP) reporter mice, we show that CD4(+) YFP(+) T cells are the major source of IL-10 in both lymphoid and nonlymphoid compartments throughout the course of blood-stage Plasmodium yoelii infection. Mature splenic CD4(+) YFP(+) GFP(+) T cells, which preferentially expressed high levels of CCR5, were capable of migrating to and seeding the nonlymphoid tissues, indicating that the systemically distributed host-protective cells have a common developmental history. Despite exhibiting comparable phenotypes, CD4(+) YFP(+) GFP(+) T cells from the liver and lung produced significantly larger quantities of IL-10 than their splenic counterparts, showing that the CD4(+) YFP(+) GFP(+) T cells exert graded functions in distinct tissue locations during infection. Unexpectedly, given the unique environmental conditions within discrete nonlymphoid and lymphoid organs, we show that IL-10 production by CD4(+) YFP(+) T cells is controlled systemically during malaria infection through IL-27 receptor signaling that is supported after CD4(+) T cell priming by ICOS signaling. The results in this study substantially improve our understanding of the systemic IL-10 response to malaria infection, particularly within sensitive nonlymphoid organs.

Experimental cerebral malaria (ECM) is a gamma interferon (IFN-γ)-dependent syndrome. However, whether IFN-γ promotes ECM through direct and synergistic targeting of multiple cell populations or by acting primarily on a specific responsive cell type is currently unknown. Here, using a panel of cell- and compartment-specific IFN-γ receptor 2 (IFN-γR2)-deficient mice, we show that IFN-γ causes ECM by signaling within both the hematopoietic and nonhematopoietic compartments. Mechanistically, hematopoietic and nonhematopoietic compartment-specific IFN-γR signaling exerts additive effects in orchestrating intracerebral inflammation, leading to the development of ECM. Surprisingly, mice with specific deletion of IFN-γR2 expression on myeloid cells, T cells, or neurons were completely susceptible to terminal ECM. Utilizing a reductionist in vitro system, we show that synergistic IFN-γ and tumor necrosis factor (TNF) stimulation promotes strong activation of brain blood vessel endothelial cells. Combined, our data show that within the hematopoietic compartment, IFN-γ causes ECM by acting redundantly or by targeting non-T cell or non-myeloid cell populations. Within the nonhematopoietic compartment, brain endothelial cells, but not neurons, may be the major target of IFN-γ leading to ECM development. Collectively, our data provide information on how IFN-γ mediates the development of cerebral pathology during malaria infection.

The murine model of experimental cerebral malaria (ECM) has been utilised extensively in recent years to study the pathogenesis of human cerebral malaria (HCM). However, it has been proposed that the aetiologies of ECM and HCM are distinct, and, consequently, no useful mechanistic insights into the pathogenesis of HCM can be obtained from studying the ECM model. Therefore, in order to determine the similarities and differences in the pathology of ECM and HCM, we have performed the first spatial and quantitative histopathological assessment of the ECM syndrome. We demonstrate that the accumulation of parasitised red blood cells (pRBCs) in brain capillaries is a specific feature of ECM that is not observed during mild murine malaria infections. Critically, we show that individual pRBCs appear to occlude murine brain capillaries during ECM. As pRBC-mediated congestion of brain microvessels is a hallmark of HCM, this suggests that the impact of parasite accumulation on cerebral blood flow may ultimately be similar in mice and humans during ECM and HCM, respectively. Additionally, we demonstrate that cerebrovascular CD8+ T-cells appear to co-localise with accumulated pRBCs, an event that corresponds with development of widespread vascular leakage. As in HCM, we show that vascular leakage is not dependent on extensive vascular destruction. Instead, we show that vascular leakage is associated with alterations in transcellular and paracellular transport mechanisms. Finally, as in HCM, we observed axonal injury and demyelination in ECM adjacent to diverse vasculopathies. Collectively, our data therefore shows that, despite very different presentation, and apparently distinct mechanisms, of parasite accumulation, there appear to be a number of comparable features of cerebral pathology in mice and in humans during ECM and HCM, respectively. Thus, when used appropriately, the ECM model may be useful for studying specific pathological features of HCM., Author summary Cerebral malaria (HCM) is the most severe complication of malaria infection. Despite this, we have an incomplete understanding of the cause (pathogenesis) of the syndrome. To improve our understanding of HCM pathogenesis, animal models of the syndrome have been developed. The most commonly used model is the murine experimental cerebral malaria (ECM) model. However, to date, there has not been a detailed investigation of the pathology of ECM using the same methodological approaches (histopathology) employed in the study of HCM. Thus, it has been unclear whether ECM is a valid model for HCM. In this histopathological study, we show that, as in HCM, cerebrovascular parasite accumulation is an important feature of ECM. However, unlike HCM, we did not observe large numbers of parasitised red blood cells (pRBCs) attached to the walls of cerebral blood vessels during ECM; instead individual pRBCs were trapped in narrow murine brain capillaries. Nevertheless, despite this, we showed that cerebrovascular parasites were still associated with disturbed blood flow, vascular leakage and impaired neuronal function in ECM, in a similar fashion to that reported in HCM. Therefore, our results define the specific aspects of HCM pathology that can potentially be studied within the ECM model.

There is significant evidence that brain-infiltrating CD8+ T cells play a central role in the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the mechanisms through which they mediate their pathogenic activity during malaria infection remain poorly understood. Utilizing intravital two-photon microscopy combined with detailed ex vivo flow cytometric analysis, we show that brain-infiltrating T cells accumulate within the perivascular spaces of brains of mice infected with both ECM-inducing (P. berghei ANKA) and non-inducing (P. berghei NK65) infections. However, perivascular T cells displayed an arrested behavior specifically during P. berghei ANKA infection, despite the brain-accumulating CD8+ T cells exhibiting comparable activation phenotypes during both infections. We observed T cells forming long-term cognate interactions with CX3CR1-bearing antigen presenting cells within the brains during P. berghei ANKA infection, but abrogation of this interaction by targeted depletion of the APC cells failed to prevent ECM development. Pathogenic CD8+ T cells were found to colocalize with rare apoptotic cells expressing CD31, a marker of endothelial cells, within the brain during ECM. However, cellular apoptosis was a rare event and did not result in loss of cerebral vasculature or correspond with the extensive disruption to its integrity observed during ECM. In summary, our data show that the arrest of T cells in the perivascular compartments of the brain is a unique signature of ECM-inducing malaria infection and implies an important role for this event in the development of the ECM-syndrome.

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs., Author Summary Cerebral malaria (CM) caused by the parasite Plasmodium sp. is a fatal disease, especially in children. Currently there is no effective treatment. We report here our investigation on the role of a recently discovered cytokine IL-33, in treating experimental cerebral malaria (ECM) in the susceptible C57BL/6 mice. IL-33 protects the mice against ECM. The protection is accompanied by a reduction of Th1 response and the enhancement of type 2 cytokine response. We also found that IL-33 mediates its protective effect by inducing a population of type 2 innate lymphoid cells (ILC2), which then polarize macrophages to alternatively-activated phenotypes (M2). M2 in turn expand regulatory T cells (Tregs) which suppress the deleterious Th1 response. Our report therefore reveals hitherto unrecognised mechanisms of the regulation of ECM and provide a novel function of IL-33.

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs.