Chemokine Mediators of Cerebral Malaria: A Comparative Study of the Role of Rantes in Murine and Human Cerebral Malaria

Abstract

Although the involvement of cytokines and adhesion molecules in malaria-induced brain inflammation has been established, the role of chemokines and chemokine receptors remain unclear. This unexplained component of cerebral malaria (CM) pathology may be responsible for the heterogeneity in the observed features of CM and the difficulty in its characterization. RANTES (regulated on activation normal T cell expressed and secreted), a chemokine involved in the generation of inflammatory infiltrates plays a special role in the modulation of inflammation. Trafficking of inflammatory T helper 1 (THL) cells into the brain is mediated partly by RANTES interactions with c-c chemokine receptor 5 (CCR5) receptor. The main hypothesis of this study are:(a) RANTES and corresponding receptors mediate malaria induced brain immunopathogenesis (b) Blocking RANTES which is upregulated during malaria infection will abrogate or minimize the outcome of the disease. Studies were directed at i) Characterizing and analyzing the expression of RANTES and corresponding receptors CCR1, CCR3 and CCR5 in a mouse model of CM (SW mice/P.yoelii 17X) using eDNA microarray. RT-PCR and Western Blot analyses ii) Evaluating effect of P.yoelii 17X infection on mouse brain by electron microscopy and immunohistological analyses iii) Evaluating expression of RANTES and receptors in cerebellum, cerebrum, brain stem and hippocampus of post-mortem CM and non-malaria (NM) tissue samples using RT-PCR and Western Blot analyses iv) Comparing and contrasting levels of RANTES in plasma of rodent malaria model and malaria-positive human subjects using ELISA v) Determine the functional role of RANTES by anti-RANTES antibody blocking experiment using P.yeolii 17X infected mice. Transcriptional analyses results indicate significant upregulation of chemokines; macrophage inflammatory protein-2a(MIP-2a.), monocyte chemotactic protein-1 (MCP-1) and RANTES chemokine receptors; CCR1, CCR3, and CCR5, adhesion molecules; platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule-l (VCAM-l) and cytokines; intereferon-gamma (INF-y), tumour necrosis factor-alpha (TNF-a) and interleukin-12 (IL-12) at peak parasitemia during P.yoelii 17X infection. Western Blot analysis revealed upregulation of RANTES protein in P.yoelii 17X infected mouse brain. Ultrastructural analysis showed that P.yoelii 17X infection induces perivascular oedema in cerebellum in mouse brain at peak parasitemia. lmmunohistological analysis demonstrates high immunoreactivity of glial fibrillary acidic protein (GFAP) ill P.yoelii l7X infected mouse brain. RANTES, CCR3 and CCR5 but not CCR1 mRNA are significantly upregulated in the cerebellum and cerebrum (P < 0.0001) in CM than NM samples. There were no changes in the expression of CCR1, CCR3 and CCR5 mRNA in brain stem and hippocampus of CM and NM. RANTES mRNA expression in cerebellum and cerebrum is highly significant(p < 0.0001) compared with the brain stem(p = 0.0018) and hippocampus (p=0.0027) in CM group. CCR5 and RANTES proteins were significantly upregulated in cerebellum (P < 0.0013 for CCR5, P < 0.0001 for RANTES) and cerebrum (P < 0.0124 for CCR5, P < 0.0001 for RANTES) but not brain stem and hippocampus of CM than in NM. Western Blot analysis could not detest CCR3 protein RANTES was significantly upregulated in plasma of murine malaria model and malaria positive subjects compared with controls. RANTES concentration in plasma correlated with P. falciparum infection. At day of sacrifice. level of parasitemia (4.2x 10^6/ml ±O.2) in mock antibody treated mice was higher (P < 0.05) than in mice in which RANTES was blocked with anti-RANTES antibody(1.2 x 10^6/ml ± 0.2). Anti-RANTES antibody treated mice survived longer (14 days) than mock antibody treated mice (10 days). This is the first temporal study of murine malaria associated RANTES and receptors CCR1, CCR3 and CCR5 expression. P.yoelii 17X murine malaria model is useful in characterizing differentially expressed genes associated with human clinical malaria. There is an association of RANTES expression in malaria-induced brain immunopathogenesis and endothelial lesions in infected mice. Cerebellum and cerebrum in humans were the focal points for increased malaria-induced RANTES and CCR5 expression. Active sequestration of infected red blood cells (IRBCs) and platelets in addition to leukocytes in these regions of the brain could exacerbate CM immnopathology. Blocking of RANTES decreased parasitemia and mortality associated with P.yoelii 17X infection.