Hyperinflammatory Experimental Cerebral Malaria: Coagulation and Glial Cell Activation in Plasmodium chabaudi Infection of IL-10-deficient Mice
Wilson, Kyle D
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Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitemia and inflammation. While mutations in inflammatory cytokine genes and the levels of cytokines found in serum and cerebrospinal fluid are correlated with the severity of human malaria infection, very little cellular infiltrate is identified on autopsy in fatal cerebral malaria cases, suggesting a novel mechanism of local inflammation drives this syndrome. Vascular congestion is a prominent feature of all malaria parasites, although some species promote a primarily leukocytic congestion while others promote parasite sequestration. The role coagulation plays in congestion is becoming an active area of research. Although parasite sequestration in the brain vasculature is a feature of the human malaria parasite P. falciparum, sequestering strains do not uniformly cause severe disease, suggesting interplay with other factors. Infection of mice with Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10 KO mice respond to the parasite with increased levels of the pro-inflammatory cytokines IFN-γ and TNF. While P. chabaudi sequesters during the schizont stage, as all malaria parasites do, this parasite does not accumulate in the brain vasculature. Nevertheless, these mice exhibit some cerebral symptoms similar to human cerebral malaria, including gross cerebral edema and hemorrhage, allowing study of these critical features of disease without the influence of brain-sequestered parasite. In these studies, IL-10 KO mice were found to suffer significant declines in behavioral and physical measures during infection compared to wildtype mice, suggesting that brain pathology contributes to the systemic consequences of infection. Elevated inflammatory monocyte and resident macrophage populations were identified in the IL-10 KO brain post-infection, and the activation state of monocytes and microglia was increased. CD4+ T cells making IFN-γ were also identified in the brain, but remained localized within the vasculature and did not enter the brain parenchyma. Theses T cells were also found to be contained within fibrinous thrombi. In order to understand the mechanisms of cerebral leukocyte accumulation in P. chabaudi infection, we investigated the relationship of vascular congestion with leukocytes and fibrinogen to astrocyte activation. As coagulation is known to cause localized ischemia and hypoxia that can lead to brain tissue damage, we investigated hypoxia within the brain, and found scattered hypoxic neurons in infected IL-10 KO mice. In order to determine the driving mechanisms of inflammation and congestion, we also investigated the roles of TNF, and the anti-coagulants heparin and low-molecular-weight heparin, on vascular coagulopathy and astrocyte activation in order to identify drivers of pathology in the brain parenchyma. Neutralization of TNF reduced mortality as previously reported, but also eliminated behavioral deficits, and significantly reduced both thrombus formation and astrocyte activation. Strikingly, anticoagulant treatment with low-molecular-weight heparin (LMWH) also rescued IL-10 KO mice from acute mortality, suggesting a role for coagulation in the lethal pathology. Glial cell activation near vessels in the brain was also partially-dependent on coagulation, as it was reduced with LMWH treatment. These studies demonstrate that neuroinflammation, driven by TNF and characterized by accumulation of leukocytes in the vasculature, is concurrent with the development of behavioral symptoms and glial cell activation in P. chabaudi infection of IL-10 KO mice. Our data also demonstrate that congestion of the vasculature is driven by coagulation and promotes lethal pathology. These findings support the contribution of cytokines, coagulation, and leukocytes within the brain vasculature, to neuropathology in malaria infection. In addition, localization of inflammatory leukocytes within intravascular clots suggests a mechanism by which cytokines can drive local inflammation without considerable cellular infiltration into the brain parenchyma.