NR2F6 as a Disease Driver and Candidate Therapeutic Target in Experimental Cerebral Malaria

Abstract

Cerebral malaria (CM) is the severe progression of an infection with Plasmodium falciparum, causing detrimental damage to brain tissue and is the most frequent cause of Plasmodium falciparum mortality. The critical role of brain-infiltrating CD8⁺ T cells in the pathophysiology of CM having been revealed, our investigation focuses on the role of NR2F6, an established immune checkpoint, as a candidate driver of CM pathology. We employed an experimental mouse model of CM based on Plasmodium berghei ANKA (PbA) infection to compare the relative susceptibility of Nr2f6-knock-out and wild-type C57BL6/N mice. As a remarkable result, Nr2f6 deficiency confers a significant survival benefit. In terms of mechanism, we detected less severe endotheliopathy and, hence, less damage to the blood-brain barrier (BBB), accompanied by decreased sequestered parasites and less cytotoxic T-lymphocytes within the brain, manifesting in a better disease outcome. We present evidence that NR2F6 deficiency renders mice more resistant to experimental cerebral malaria (ECM), confirming a causal and non-redundant role for NR2F6 in the progression of ECM disease. Consequently, pharmacological inhibitors of the NR2F6 pathway could be of use to bolster BBB integrity and protect against CM.

Keywords: CD8+ T cell brain infiltration; compromised blood–brain barrier (BBB) integrity; experimental cerebral malaria (ECM); group F; innovative pharmacological therapy solution for CM; member 6 (NR2F6); nuclear receptor subfamily 2.

Figure 1

Nr2f6 deficiency confers partial resistance against cerebral malaria. (A) Nr2f6^−/− mice show a survival benefit, (B) a slower disease progression according to the ICMS without clear clinical signs of ECM, (C) a trend to lower parasite load, as well as no severe hypothermia during the ECM vulnerable phase (D). n numbers: WT = 16, Nr2f6^−/− = 18. [B,D] The dashed line represents the cut-off point for terminating the experiment. Log-rank test [A], two-way ANOVA [B]. Data shown as mean ± SEM.

Figure 2

Nr2f6^−/− mice show a higher BBB integrity and less hemorrhagic lesions. (A) Colorimetric quantification of Evans Blue dye extravasation from isolated brains of PbA-infected mice on day 6 p.i. (wt n = 6, Nr2f6^−/− n = 5) and (B) determination of the amount of volatile liquid in the brain, as a measure of brain swelling, reveals a more intact BBB in Nr2f6-knock-out mice (n = 5) compared with WT control infected mice (n = 5). (C) Representative images of H&E-stained brain sections with (D) less abundant hemorrhagic lesions, smaller lesions, and therefore account for a markedly smaller total area of lesions in the brains of Nr2f6-knock-out animals compared with WT mice infected with PbA (day 6 p.i.). Size of scale bars: 200 µm; n numbers: wt = 3, Nr2f6^−/− = 4. Two-tailed unpaired Student’s t-test. Data shown as mean ± SEM.

Figure 3

Less parasites are detectable in Nr2f6^−/− brains. (A) Representative images of immuno-fluorescent staining of brain sections (20×) of WT (n = 3) and Nr2f6^−/− (n = 4) PbA-infected mice (day 6 p.i.). Intravasal CD8⁺ T cells (1), extravasal CD8⁺ T cells (2), and parasitized vessel (3) are shown (40×). (B) Decreased parasitized vessels manifesting less sequestration of GFP-expressing PbA parasites (green) in Nr2f6-knock-out mice. (C) The amount of intravasal, extravasal, as well as total CD8⁺ T cells (white) per image is lower in association with CD31⁺ ECs (red) of the brain microvasculature (nuclei are illustrated in blue) in Nr2f6-knock-out mice than in WT mice. Two-tailed unpaired Student’s t-test. Data shown as mean ± SEM. * p < 0.05, ** p < 0.01.

Figure 4

Lower number of CD8⁺ BILs in Nr2f6^−/− mice accompanied by higher PD-1 expression. Flow cytometric analysis of BILs reveals a lower amount of CD45⁺ leukocytes (p = 0.0073) of total lymphocytes (A) and decreased CD8⁺ T cells (p = 0.0041) (B,C) in the brains of PbA-infected Nr2f6-knock-out animals compared with WT mice (day 6 p.i.). (D,E) PD-1⁺Tim3⁺ CD8⁺ BILs were enriched in Nr2f6-knock-out mice (p = 0.0091). (E) Representative FACS blots of PD-1⁺Tim3⁺ T cells. (F) CD8⁺ BILs from Nr2f6^−/− mice showed augmented expression of PD-1 (p = 0.0003). Two-tailed unpaired Student’s t-test. Data shown as mean ± SEM.