The Deubiquitinating Enzyme Cylindromatosis Dampens CD8+ T Cell Responses and Is a Critical Factor for Experimental Cerebral Malaria and Blood–Brain Barrier Damage

Abstract

Cerebral malaria is a severe complication of human malaria and may lead to death of Plasmodium falciparum-infected individuals. Cerebral malaria is associated with sequestration of parasitized red blood cells within the cerebral microvasculature resulting in damage of the blood–brain barrier and brain pathology. Although CD8+ T cells have been implicated in the development of murine experimental cerebral malaria (ECM), several other studies have shown that CD8+ T cells confer protection against blood-stage infections. Since the role of host deubiquitinating enzymes (DUBs) in malaria is yet unknown, we investigated how the DUB cylindromatosis (CYLD), an important inhibitor of several cellular signaling pathways, influences the outcome of ECM. Upon infection with Plasmodium berghei ANKA (PbA) sporozoites or PbA-infected red blood cells, at least 90% of Cyld−/− mice survived the infection, whereas all congenic C57BL/6 mice displayed signatures of ECM, impaired parasite control, and disruption of the blood–brain barrier integrity. Cyld deficiency prevented brain pathology, including hemorrhagic lesions, enhanced activation of astrocytes and microglia, infiltration of CD8+ T cells, and apoptosis of endothelial cells. Furthermore, PbA-specific CD8+ T cell responses were augmented in the blood of Cyld−/− mice with increased production of interferon-γ and granzyme B and elevated activation of protein kinase C-θ and nuclear factor “kappa light-chain enhancer” of activated B cells. Importantly, accumulation of CD8+ T cells in the brain of Cyld−/− mice was significantly reduced compared to C57BL/6 mice. Bone marrow chimera experiments showed that the absence of ECM signatures in infected Cyld−/− mice could be attributed to hematopoietic and radioresistant parenchymal cells, most likely endothelial cells that did not undergo apoptosis. Together, we were able to show that host deubiqutinating enzymes play an important role in ECM and that CYLD promotes ECM supporting it as a potential therapeutic target for adjunct therapy to prevent cerebral complications of severe malaria.

The Glycosylphosphatidylinositol Transamidase Complex Subunit PbGPI16 of Plasmodium berghei Is Important for Inducing Experimental Cerebral Malaria

“…CD8 ϩ T cells promote the pathology through the cytotoxic effector molecule granzyme B (47,48). The CD8 ϩ T cell response in the brain is a critical factor for ECM and BBB damage (49). Consistent with Plasmodium GPI anchors as an inducer of Th1 responses in the hosts (50,51), our study showed significantly less proliferation of Th1 cells in the spleens and CD3 ϩ CD8 ϩ granzyme B ϩ T cells in the brains of ⌬pbgpi16 mutant-infected mice than in WT P. berghei ANKA-infected mice.…”

Regulatory interplay between deubiquitinating enzymes and cytokines

“…CYLD also interacted with IPS-1 to negatively regulate it, but did not deubiquitinate it [27]. Schmid et al found that in brain and peripheral blood of C57BL/6, the mRNA level of IFN-γ gene decreased with the knockdown of CYLD, while the serum concentration of IFN-γ increased [28]. A study conducted using human kidney mesangial cells (MC) showed slightly different results: silencing CYLD in MC cells and stimulating them with poly IC increased the toll-like receptor 3 (TLR3)-induced activation of RIG-I and MDA5 [26]; however, the level of mRNA of RIG-I and MDA5 actually decreased [26].…”

Blood–Brain Barrier in Cerebral Malaria: Pathogenesis and Therapeutic Intervention

“…Hayashi et al [62] have shown that contact with astrocytes increased the expression of tight junctions by the ECs. We and others have shown that astrocytes are activated during PbA infection, further pointing towards a role in ECM [35,38]. Medana et al [63] identified that astrocytes retract their processes during ECM and are unevenly distributed, leading to reduced wrapping of the blood vessels.…”

“…Microglia are also the major producers of intracerebral NO [70], which could preserve the BBB by preventing the activation of ECs, as discussed above [53,55]. Several studies, including ours, have shown that microglia are activated early during ECM [35,38]. During ECM, microglia retract their ramified processes and migrate towards the venous side of the circulation [63].…”

“…Neuregulin-1 treatment protected from ECM by reducing the intracerebral levels of the proinflammatory cytokines TNF, IL-6, and IL-1α and the chemokine CXCL10, thus decreasing leukocyte accumulation in brain [37]. It is well documented that CD8 + T cells play an important role in the pathology of ECM [35]; they mediate the disruption of the BBB by fostering leukocyte accumulation, and by inducing apoptosis of ECs through granzyme B and perforin-mediated cytotoxicity ( Figure 4) [35,38]. The chemokine receptor CXCR3, expressed on leukocytes, is responsible for their intracerebral trafficking.…”