Therapeutical targeting of nucleic acid-sensing Toll-like receptors prevents experimental cerebral malaria

Abstract

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.

Fig. 1.

Structure of the E6446 compound. E6446 is a small, water-soluble, aromatic organic compound. Its bioactive structure is composed of benzoxazole with two-sided pyrrolidine rings.

Fig. 2.

Targeting inhibition of nucleic acid-sensing TLRs by E6446. (A) (Upper) HEK293 cells stably transfected with plasmids carrying TLR4/MD2, TLR7, or TLR9 genes and the NF-κB reporter gene ELAM-1–luciferase were stimulated with the appropriate ligand (LPS with soluble CD14, R848, or oligo 2006) overnight. Next, Steady-Glo reagent (Promega, Inc.) was added to the wells, and the amount of luciferase activity in each sample was quantified in a Wallac Envision counter. (Lower) Ficoll-separated mononuclear cells were isolated from healthy volunteer donors, washed, and plated with stimulatory oligonucleotide CpG ODN 2216, R848, or ssRNA in the presence of the lyposomal trasfection reagent DOTAP in complete RPMI for 72 h. IL-6 levels in supernatant were quantified by ELISA (R&D Systems) and expressed as the percentage of levels found in nonstimulated cell supernatants. (B) (Upper) BALB/c mice were predosed with 60 mg/kg of E6446 1.5 h before challenge with the indicated dose of Oligo 1668 (Oligos, Etc.). At 2 h postchallenge, serum was collected and assayed for IL-6 by ELISA. (Lower) Spleen cells from mice were incubated with CpG ODN 1401 (1 μg/mL) or R818 (1 μM) for 72 h in the presence of increasing concentrations of E6446. (C) Mice were orally treated with 20 mg·kg⁻¹·d⁻¹ of E6446 during 5 d. Two hours after the last dose, spleen cells were harvested and challenged with the indicated stimuli. Cytokine levels in culture supernatants were analyzed by Searchlight multiplex. *P < 0.05.

Fig. 3.

E6446 prevents priming of TLR proinflammatory responses. C57BL/6 mice were treated with 60 mg·kg⁻¹·d⁻¹ of E6446 or vehicle 1 d before and during 6 d postinfection with 10⁵ P. chabaudi iRBCs. (A) At 7 d postinfection, spleens were harvested and cultured in the presence of LPS (1 μg/mL), Pam3cysK4 (1 μg/mL), Poly:IC (100 μg/mL), CL075 (100 ng/mL), CpG ODN (1 μg/mL), or P. chabaudi extract (100 μg/mL) for 48 h. Cytokine levels in cell culture supernatant or in sera were assessed by ELISA. P.c., P. chabaudi. (B) Mice were treated with 120 mg·kg⁻¹·d⁻¹ during 6 d postinfection with 10⁵ P. chabaudi iRBCs. Mice were challenged i.v. at 7 d postinfection with 10 μg of LPS. Survival was monitored from 12–48 h after LPS challenge. An asterisk marks where comparisons reached statistically significant (P < 0.05).

Fig. 4.

E6446 protects mice against PbA-mediated CM. C57BL/6 mice were treated with 120 mg·kg⁻¹·d⁻¹ of E6446 1 d before and during 12 d postinfection with 10⁵ PbA-infected erythrocytes (iRBCs). Mice treated with vehicle (acidified water) were infected and used as a control group. (A) Survival and parasitemia were compared between groups of mice at various days postinfection. (B) Effect of emergency treatment with E6446 on ECM. Mice were infected with 10⁵ PbA iRBCs and treated with E6446 beginning at different days postinfection. p.i., postinfection. Survival was monitored throughout infection. (C) Cytokine levels were assessed in the sera (10 mice per group) or in supernatants of splenocytes cultured in the presence of LPS (1 μg/mL) or CpG ODN (1 μg/mL). (D) Analysis of intracellular staining of cytokines in CD11c⁺ MHCII⁺ splenic DCs from vehicle- or E6446-treated mice at 6 d postinfection. Results are from a sum of four different experiments that yielded similar results. An unpaired two-tailed Student's t test or Mann–Whitney U test was used to compare means between cytokine levels detected in the vehicle-treated or E6446-treated group of mice. A P value <0.05 was considered to be statistically significant.

Fig. 5.

E6446 prevents breakdown of the BBB and neurological signs of malaria. (A) Each of the classic CM symptoms (ruffled fur, abnormal posture, unbalancing, limb paralysis, convulsion, coma, and death) was given a score (0, 2, 4, 8, or 10). Mice were then graphically ranked based on symptoms presented at each time point. Open and closed circles represent mice treated with either E6446 or vehicle. (B) After 7 d of infection, mice were injected i.v. with 0.2 mL of 1% Evans blue (Sigma–Aldrich) shortly before the death of vehicle-treated mice (120 mg·kg⁻¹·d⁻¹). One hour later, mice were killed and brain coloration was assessed. Naive mice were also injected with Evans blue and used as a control. (C) Brains from vehicle- or E6446-treated mice were harvested at day 7 postinfection and fixed in formalin; brain sections were then stained with H&E. Brain lesions were identified over oil immersion 1,000× magnification (Left) and counted over 40× magnification (Right) on an optical microscope. Statistical analysis was performed using an unpaired two-tailed Student's t test. A P value <0.05 was considered to be statistically significant.