J. Brian de Souza

Locally Up-regulated Lymphotoxin α, Not Systemic Tumor Necrosis Factor α, Is the Principle Mediator of Murine Cerebral Malaria

Cerebral malaria (CM) causes death in children and nonimmune adults. TNF-α has been thought to play a key role in the development of CM. In contrast, the role of the related cyto-kine lymphotoxin α (LTα) in CM has been overlooked. Here we show that LTα, not TNFα, is the principal mediator of murine CM. Mice deficient in TNFα (B6.TNFα−/−) were as susceptible to CM caused by Plasmodium berghei (ANKA) as C57BL/6 mice, and died 6 to 8 d after infection after developing neurological signs of CM, associated with perivascular brain hemorrhage. Significantly, the development of CM in B6.TNFα−/− mice was not associated with increased intracellular adhesion molecule (ICAM)-1 expression on cerebral vasculature and the intraluminal accumulation of complement receptor 3 (CR3)-positive leukocytes was moderate. In contrast, mice deficient in LTα (B6.LTα−/−) were completely resistant to CM and died 11 to 14 d after infection with severe anemia and hyperparasitemia. No difference in blood parasite burden was found between C57BL/6, B6.TNFα−/−, and B6.LTα−/− mice at the onset of CM symptoms in the two susceptible strains. In addition, studies in bone marrow (BM) chimeric mice showed the persistence of cerebral LTα mRNA after irradiation and engraftment of LTα-deficient BM, indicating that LTα originated from a radiation-resistant cell population.

IL-10 from CD4(+)CD25(-)Foxp3(-)CD127(-) adaptive regulatory T cells modulates parasite clearance and pathology during malaria infection

The outcome of malaria infection is determined, in part, by the balance of pro-inflammatory and regulatory immune responses. Failure to develop an effective pro-inflammatory response can lead to unrestricted parasite replication, whilst failure to regulate this response leads to the development of severe immunopathology. IL-10 and TGF-β are known to be important components of the regulatory response, but the cellular source of these cytokines is still unknown. Here we have examined the role of natural and adaptive regulatory T cells in the control of malaria infection and find that classical CD4+CD25hi (and Foxp3+) regulatory T cells do not significantly influence the outcome of infections with the lethal (17XL) strain of Plasmodium yoelii (PyL). In contrast, we find that adaptive IL-10-producing, CD4+ T cells (which are CD25−, Foxp3−, and CD127− and do not produce Th1, Th2, or Th17 associated cytokines) that are generated during both PyL and non-lethal P. yoelii 17X (PyNL) infections are able to down-regulate pro-inflammatory responses and impede parasite clearance. In summary, we have identified a population of induced Foxp3− regulatory (Tr1) T cells, characterised by production of IL-10 and down regulation of IL-7Rα, that modulates the inflammatory response to malaria.

Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis

Cerebral malaria is a serious and often fatal complication of Plasmodium falciparum infections. The precise mechanisms involved in the onset of neuropathology remain unknown, but parasite sequestration in the brain, metabolic disturbances and host immune responses are all thought to be involved. This review outlines the current state of knowledge of cerebral disease in humans, and discusses the contribution of studies of animal models to elucidation of the underlying mechanisms.

Liposome-mediated DNA vaccination

Numerous reports have indicated that intramuscular injection of antigen‐coding naked plasmid DNA can trigger humoral and cell‐mediated protective immunity against infection. This follows DNA uptake by muscle fibres, leading to the expression and extracellular release of the antigen. Here it is shown for the first time that intramuscular immunization of mice with pRc/CMV HBS (encoding the S region of hepatitis B antigen; HBsAg) entrapped into positively charged (cationic) liposomes leads to greatly improved humoral and cell‐mediated immunity. These cationic liposome‐entrapped DNA vaccines generate titres of anti‐HBsAg IgG1 antibody isotype in excess of 100‐fold higher and increased levels of both IFN‐γ and IL‐4 when compared with naked DNA or DNA complexed with preformed similar (cationic) liposomes. It is likely that immunization with liposome‐entrapped plasmid DNA involves antigen‐presenting cells locally or in the regional draining lymph nodes.

Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease.

Cerebral malaria is a life-threatening complication of malaria infection. The pathogenesis of cerebral malaria is poorly defined and progress in understanding the condition is severely hampered by the inability to study in detail, ante-mortem, the parasitological and immunological events within the brain that lead to the onset of clinical symptoms. Experimental murine models have been used to investigate the sequence of events that lead to cerebral malaria, but there is significant debate on the merits of these models and whether their study is relevant to human disease. Here we review the current understanding of the parasitological and immunological events leading to human and experimental cerebral malaria, and explain why we believe that studies with experimental models of CM are crucial to define the pathogenesis of the condition.

Differential induction of TGF-beta regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections.

Transforming growth factor-β is an essential moderator of malaria-induced inflammation in mice. In this study, we show that the virulence of malaria infections is dependent upon the cellular source of TGF-β and the timing of its production. C57BL/6 mice infected with a nonlethal (Py17X) strain of Plasmodium yoelii produce TGF-β from 5 days postinfection; this correlates with resolution of parasitemia, down-regulation of TNF-α, and full recovery. In contrast, infection with the lethal strain Py17XL induces high levels of circulating TGF-β within 24 h; this is associated with delayed and blunted IFN-γ and TNF-α responses, failure to clear parasites, and 100% mortality. Neutralization of early TGF-β in Py17XL infection leads to a compensatory increase in IL-10 production, while simultaneous neutralization of TGF-β and IL-10R signaling leads to up-regulation of TNF-α and IFN-γ, prolonged survival in all, and ultimate resolution of infection in 40% of Py17XL-infected animals. TGF-β production can be induced in an Ag-specific manner from splenocytes of infected mice, and by cross-linking surface CTLA-4. CD25+ and CD8+ cells are the primary source of TGF-β following Py17X stimulation of splenocytes, whereas Py17XL induces significant production of TGF-β from adherent cells. In mice immunized against Py17XL, the early TGF-β response is inhibited and is accompanied by significant up-regulation of IFN-γ and TNF-α and rapid resolution of challenge infections.

Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice.

Investigation of the role of regulatory T cells (Treg) in model systems is facilitated by their depletion using anti-CD25 Abs, but there has been considerable debate about the effectiveness of this strategy. In this study, we have compared the depletion and repopulation of CD4+CD25+Foxp3+ Treg in uninfected and malaria-infected mice using 7D4 and/or PC61 anti-CD25 Abs. We find that numbers and percentages of CD25high cells, but not Foxp3+ cells, are transiently reduced after 7D4 treatment, whereas treatment with PC61 alone or in combination with 7D4 (7D4 plus PC61) reduces but does not eliminate Foxp3+ cells for up to 2 wk. Importantly, all protocols fail to eliminate significant populations of CD25−Foxp3+ or CD25lowFoxp3+ cells, which retain potent regulatory capacity. By adoptive transfer we show that repopulation of the spleen by CD25highFoxp3+ cells results from the re-expression of CD25 on peripheral populations of CD25−Foxp3+ but not from the conversion of peripheral Foxp3− cells. CD25highFoxp3+ repopulation occurs more rapidly in 7D4-treated mice than in 7D4 plus PC61-treated mice, reflecting ongoing clearance of emergent CD25+Foxp3+ cells by persistent PC61 Ab. However, in 7D4 plus PC61-treated mice undergoing acute malaria infection, repopulation of the spleen by CD25+Foxp3+ cells occurs extremely rapidly, with malaria infection driving proliferation and CD25 expression in peripheral CD4+CD25−Foxp3+ cells and/or conversion of CD4+CD25−Foxp3− cells. Finally, we reveal an essential role for IL-2 for the re-expression of CD25 by Foxp3+ cells after anti-CD25 treatment and observe that TGF-β is required, in the absence of CD25 and IL-2, to maintain splenic Foxp3+ cell numbers and a normal ratio of Treg:non-Treg cells.

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.

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.

Essential Role for IL-27 Receptor Signaling in Prevention of Th1-Mediated Immunopathology during Malaria Infection

Successful resolution of malaria infection requires induction of proinflammatory immune responses that facilitate parasite clearance; however, failure to regulate this inflammation leads to immune-mediated pathology. The pathways that maintain this immunological balance during malaria infection remain poorly defined. In this study, we demonstrate that IL-27R–deficient (WSX-1−/−) mice are highly susceptible to Plasmodium berghei NK65 infection, developing exacerbated Th1-mediated immune responses, which, despite highly efficient parasite clearance, lead directly to severe liver pathology. Depletion of CD4+ T cells—but not CD8+ T cells—prevented liver pathology in infected WSX-1−/− mice. Although WSX-1 signaling was required for optimal IL-10 production by CD4+ T cells, administration of rIL-10 failed to ameliorate liver damage in WSX-1−/− mice, indicating that additional, IL-10–independent, protective pathways are modulated by IL-27R signaling during malaria infection. These data are the first to demonstrate the essential role of IL-27R signaling in regulating effector T cell function during malaria infection and reveal a novel pathway that might be amenable to manipulation by drugs or vaccines.