Although several mechanisms have been proposed to explain cerebral malaria immunopathogenesis, the precise mechanisms is still unknown. Dysregulation of homeostasis of pro- and anti- inflammatory factors has been linked to the severity of CM. For instance, over production of pro-inflammatory cytokines resulted in dysfunction of the blood-brain barrier (BBB) leading to CM complications [18–21].
Recently, a number of chemokines and their corresponding receptors were implicated in CM pathogenesis [2–4]. Studies conducted in our laboratory and elsewhere using human CM samples from Ghana and India revealed that excessive production of CXCL-10 in severe malaria patients is an important predictor of mortality risk in CM [4, 21]. These studies demonstrated that there were significant increase in pro-inflammatory markers including interleukin-8 (IL-8), interleukin-1 receptor agonist (IL-ra), interferon-inducible protein-10 (CXCL-10), macrophage inflammatory protein-1beta (MIP-1β), soluble tumor necrosis factor receptor-1 and 2 (sTNF-R1 and sTNF-R2) in patients who died of CM .
Interferon gamma inducible protein-10 (CXCL-10) levels in serum were independently associated with fatal CM [4, 21]. Thymus (T) cells produce CXCL-10, and, therefore, a significant up-regulation of CXCL-10 in sera of patients dying of CM indicates a high level of activation of T-cells associated with CM. CXCL-10 is a member of the CXC pro-inflammatory chemokines and binds the CXCR3 receptor which is expressed by activated T-cells . Thymus (T) cells play a major role in CM immunopathogenesis although the mechanism by which their regulation modulates CM severity is not understood [23, 24]. Mice without T-cells do not develop CM and CM is attenuated by blocking CD4+ and CD8+ T- cells . Thus it is logical that blocking T-cells or interfering with T-cell activation will decrease risk of fatal CM.
In this study CD4+CD25+Foxp3+ transcription factor and specific cell marker Foxp3 mRNA and protein in P. berghei-infected mice was up-regulated in mouse brain. Brain and PBMC Foxp3 expression induced by P. berghei were significantly up-regulated in CXCL-10-/- mice at Day 4 post-infection compared with WT. This suggests an elevated systemic expression of Foxp3 mRNA and protein in CXCL-10-/- than in WT mice during the infection. This observation is in line with recent human studies conducted in The Gambia in West Africa where individuals living in areas with high malaria transmission had higher levels of regulatory T-cells compared with those living in areas where malaria is rare . However this contrasts with other studies, which reported a minor increase in Foxp3 expression during P. berghei infection at Day 7 post-infection  (early expression of Foxp3 was not reported in this article).
Regulatory T cells are key cerebroprotective immunomodulators during acute strokes in murine model. Absence of CD4+CD25+Foxp3+ enhanced activation of resident and invading inflammatory cells (microglia and T-cells) to produce higher levels of TNF and IFN-γ, which are deleterious to the brain . In this study, it was determined that early up-regulation of Foxp3 (mRNA and protein) in the brain and PBMCs correlated positively with CM protection in CXCL-10-/- mice than in WT. Regulatory T-cells induced CD4+ T-cell apoptosis during the early stage of P. yoelii 17XL infection and this in effect regulates pro-inflammatory responses . Therefore, up-regulation of Foxp3 in the brain may inhibit over activation of microglia and T-cells which could prevent parasite associated complications in the brain. CXCL-10 itself has been associated with fatal CM and it absence has been linked to enhanced CD4+CD25+Foxp3+ expression and activation leading to its ability to dampen over activation and production of pathogenic inflammatory factors which cause CM. These taken together could be responsible for the inhibition of CM outcome in the CXCL-10-/- compared with the WT in our study, compared with the study in reference 29.
Some reports indicate that expansion of CD4+CD25+Foxp3+ during murine malaria did not prevent murine CM . However, other studies have shown that CD4+CD25+Foxp3+ suppressed CD4+ T-cell function and inhibited the development of murine CM in Plasmodium berghei-specific Th1 response [30, 31]. Increase in numbers and activation of CD4+CD25+Foxp3+ control the production of pro-inflammatory cytokines and mediate a counter-regulatory response to overwhelming inflammation during lethal Plasmodium chabaudi adami infection and did not contribute to parasite immune evasion . Significant up-regulation of Foxp3 mRNA and protein during P. berghei infection in CXCL-10-/- in this study is associated with protection against murine CM; a first report of the role of Foxp3 gene and protein expression in severity of CM in CXCL-10-/- mice.
It was observed in this study that IL-10 levels increased in plasma during P. berghei infection in CXCL-10-/- than in wild type mice. Interleukin-10 (IL-10) is an anti-inflammatory cytokine and thus increased production during parasite infection was necessary to ameliorate the disease outcome. Interleukin-10 (IL-10) levels in infected CXCL-10-/- at Days 2 and 4 were higher than in infected WT mice. Possibly, early induction of IL-10 and Foxp3 combined to provide anti-inflammatory responses to mediate the cerebroprotective effects against CM in CXCL-10-/- mice as evidenced in the absence of CM symptoms in those mice. Regulation of proinflammatory response during murine malaria infection is mediated by regulatory T- cells in an IL-10 dependent manner . Additionally, CD4+CD25+Foxp3+ have recently been reported as major cerebroprotective modulators of post-ischemic inflammatory brain damage with IL-10 as the predominant associated anti-inflammatory cytokine modulating this effect . Therefore, IL-10 producing CD4+CD25+Foxp3+ activated early in a robust inflammatory setting through a feedback mechanism would consequently reduce severity of CM.
In this study interleukin-2 cytokine was up-regulated in CXCL-10-/- mice than WT. Interleukin-2 (IL-2) is a Th1 cytokine produced by activated T lymphocytes and plays a key role in promoting the expansion of antigen-specific T cells and also mediates multiple immune responses on a variety of cell types including thymocytes, activated B cells, monocytes, natural killer cells and oligodendrocytes. IL-2 mediates signaling through IL-2Rα, and regulates Foxp3 expression in CD4+CD25+Foxp3+ [33, 34]. Induction of Foxp3 during Plasmodium falciparum infection is driven by IL-2 . The results showed that at days 2 and 4, IL-2 was significantly up-regulated in plasma of CXCL-10-/- than in WT, indicating that the absence of CXCL-10 promotes IL-2 expression during murine malaria and that it could play an important role in the pathogenesis of severe malaria.
Thymus (CD4+, and CD25+ T cells) were the major cellular source of IL-10 and TGF-b1 cytokines in malaria infected red blood cells (iRBC) when co-cultured with PBMCs . CD4+CD25+ and CD4+CD25- T-cells cultured with P. berghei antigens secreted more of IL-10 than cells cultured with non- P. berghei supernatant and RPMI 1640 medium alone confirming that P. berghei parasite antigens were inducing IL-10 production. It was observed that IL-10 production by CD4+CD25+ T-cells from CXCL-10-/- was significantly higher than wild type mice in vitro (Figures 6A, 6B and 6C), implying that CD4+CD25+ T-cells is a potential source of IL-10 during malaria infection.
Taking all of the above observation together, it could be concluded that, high levels of IL-2 in CXCL-10-/- mice during P. berghei ANKA infection induce CD4+CD25+Foxp3+ measured by levels of Foxp3 transcription and translation. Subsequently it seems that activated CD4+CD25+Foxp3+ produce high levels of IL-10, and are able to mount a robust anti-inflammatory response, which counter the inflammatory effects of P. berghei parasite thus preventing fatal CM in CXCL-10-/-.