Beta interferon suppresses the development of experimental cerebral malaria

Abstract

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection, particularly in children. The pathogenesis of cerebral malaria involves parasitized red blood cell (RBC)-mediated vascular inflammation, immune stimulation, loss of blood-brain barrier integrity, and obstruction of cerebral capillaries. Therefore, blunting vascular inflammation and immune cell recruitment is crucial in limiting the disease course. Beta interferon (IFN-β) has been used in the treatment of diseases, such as multiple sclerosis (MS) but has not yet been explored in the treatment of CM. Therefore, we sought to determine whether IFN-β also limits disease progression in experimental cerebral malaria (ECM). Plasmodium berghei-infected mice treated with IFN-β died later and showed increased survival, with improved blood-brain barrier function, compared to infected mice. IFN-β did not alter systemic parasitemia. However, we identified multiple action sites that were modified by IFN-β administration. P. berghei infection resulted in increased expression of chemokine (C-X-C motif) ligand 9 (CXCL9) in brain vascular endothelial cells that attract T cells to the brain, as well as increased T-cell chemokine (C-X-C motif) receptor 3 (CXCR3) expression. The infection also increased the cellular content of intercellular adhesion molecule 1 (ICAM-1), a molecule important for attachment of parasitized RBCs to the endothelial cell. In this article, we report that IFN-β treatment leads to reduction of CXCL9 and ICAM-1 in the brain, reduction of T-cell CXCR3 expression, and downregulation of serum tumor necrosis factor alpha (TNF-α). In addition, IFN-β-treated P. berghei-infected mice also had fewer brain T-cell infiltrates, further demonstrating its protective effects. Hence, IFN-β has important anti-inflammatory properties that ameliorate the severity of ECM and prolong mouse survival.

FIG. 1.

IFN-β is protective in experimental cerebral malaria (ECM). (A) Survival. Mice were infected with Plasmodium berghei, and their survival was monitored. The values that were significantly different (P < 0.05) from the value for control, non-IFN-β-treated mice are indicated by an asterisk. (B) Blood-brain barrier (BBB). On day 5 postinfection, the mice were injected with Evans blue dye, and dye extravasation was measured (n = 5). Values are means plus standard errors of the means (SEMs) (error bars). The value for infected and IFN-β-treated mice was significantly different (P < 0.05) from the value for infected mice and is indicated by an asterisk. OD, optical density.

FIG. 2.

Brain pathology in the cerebral cortex. On day 5 postinfection, brains were collected from mice, and hematoxylin and eosin staining was performed. The arrows point to areas of vascular inflammation.

FIG. 3.

IFN-β suppresses ECM-associated inflammation. On day 5 postinfection, mouse plasma was isolated and evaluated by EIA. (A to E) The concentrations of CXCL9 (A), CXCL10 (B), sICAM-1 (C), TNF-α (D), and IFN-γ (E) in the three groups of mice are shown. (A) CXCL9 (n = 5). Values are means plus standard deviation (SDs) (error bars). The value that was significantly different (P < 0.05) from the value for infected mice is indicated by an asterisk. (B) CXCL10 (n = 4 or 5). The value that was significantly different (P < 0.01) from the value for infected mice is indicated by an asterisk. (C) sICAM-1 (n = 5). (D) TNF-α (n = 3). The value that was significantly different (P < 0.01) from the value for infected mice is indicated by an asterisk. (E) IFN-γ (n = 3). The value that was significantly different (P = 0.02) from the value for infected mice is indicated by an asterisk. Values in panels B, D, and E are means plus SEMs (error bars).

FIG. 4.

IFN-β alters brain CXCL9 production. (A) CXCL9 immunohistochemical analysis. The arrows point to blood vessels that are positive for CXCL9. (B) Quantification of CXCL9-positive vessels. Infected mice had approximately 14 CXCL9-positive (+ve) vessels per 20× field, whereas in IFN-β-treated mice, the number of such vessels was reduced to approximately 7 (5 fields per mouse; 5 mice). Values are means plus SDs (error bars). The value for infected and IFN-β-treated mice was significantly different (P < 0.01) from the value for infected mice and is indicated by an asterisk.

FIG. 5.

IFN-β alters brain CXCL10 production. (A) CXCL10 immunohistochemical analysis. The arrows point to CXCL10-positive astrocytes and glial cells. (B) Quantification of CXCL10-positive cells. Infected mice had approximately 40 CXCL10-positive cells per 20× field, whereas in IFN-β-treated mice, this number was increased to greater than 80 (5 fields per mouse; five mice). Values are means plus SDs (error bars). The value for infected and IFN-β-treated mice was significantly different (P < 0.01) from the value for infected mice and is indicated by an asterisk.

FIG. 6.

IFN-β alters expression of CXCL9, CXCL10, and ICAM-1 mRNA in the brain. (A) CXCL9 (n = 3 or 4). (B) CXCL10 (n = 3 or 4). The value for infected and IFN-β-treated mice was significantly different (P < 0.05) from the value for infected mice and is indicated by an asterisk. (C) ICAM-1 (n = 3 or 4). The value for infected and IFN-β-treated mice was significantly different (P < 0.02) from the value for infected mice and is indicated by an asterisk. Values are means plus SEMs (error bars). The mRNA levels for each primer pair were normalized to the housekeeping gene GAPDH rRNA level, and fold changes were calculated against control uninfected mice.

FIG. 7.

IFN-β reduces T-cell trafficking in ECM. T-cell trafficking to the brain in ECM was evaluated by immunohistochemical staining with anti-CD3 antibody.

FIG. 8.

T-cell trafficking in ECM is reduced by IFN-β. (A) Quantification of CD3-positive cells taken from Fig. 7. Infected mice had approximately 25 CD3-positive cells per 20× field, but the number was reduced to less than 10 in IFN-β-treated mice. There were 5 fields per mouse and 5 mice. The value for infected and IFN-β-treated mice was significantly different (P < 0.01) from the value for infected mice and is indicated by an asterisk. In panels A to C, values are means plus SDs (error bars). (B) Isolated T-cell subsets from mouse brains. Mononuclear cells were isolated from the brains of control mice, infected mice, and infected mice treated with IFN-β. T cells were quantified by FACS (n = 5). The value that was significantly different (P < 0.06) from the value for infected, CD8-positive cells is indicated by an asterisk. The value that was significantly different (P < 0.05) from the value for infected, CD4-positive cells is indicated by two asterisks. (C) T-cell CXCR3 expression. Splenocytes were isolated from control, infected, and infected mice treated with IFN-β. T-cell CXCR3 expression was determined by FACS (n = 5). The value that was significantly different (P < 0.05) from the value for infected mice is indicated by an asterisk. MFI, mean fluorescence intensity. (D) CXCR3 mRNA expression in the brain by qRT-PCR (n = 3 or 4). Values are means plus SEMs (error bars). The mRNA levels for each primer pair were normalized to the housekeeping gene GAPDH rRNA level, and fold changes were calculated against control uninfected mice.

FIG. 9.

Diagram of the hypothesized actions of IFN-β on the pathogenesis of cerebral malaria.