Murine cerebral malaria development is independent of toll-like receptor signaling - PubMed (original) (raw)

Murine cerebral malaria development is independent of toll-like receptor signaling

Dieudonnée Togbe et al. Am J Pathol. 2007 May.

Abstract

Malaria pigment hemozoin was reported to activate the innate immunity by Toll-like receptor (TLR)-9 engagement. However, the role of TLR activation for the development of cerebral malaria (CM), a lethal complication of malaria infection in humans, is unknown. Using Plasmodium berghei ANKA (PbA) infection in mice as a model of CM, we report here that TLR9-deficient mice are not protected from CM. To exclude the role of other members of the TLR family in PbA recognition, we infected mice deficient for single TLR1, -2, -3, -4, -6, -7, or -9 and their adapter proteins MyD88, TIRAP, and TRIF. In contrast to lymphotoxin alpha-deficient mice, which are resistant to CM, all TLR-deficient mice were as sensitive to fatal CM development as wild-type control mice and developed typical microvascular damage with vascular leak and hemorrhage in the brain and lung, together with comparable parasitemia, thrombocytopenia, neutrophilia, and lymphopenia. In conclusion, the present data do not exclude the possibility that malarial molecular motifs may activate the innate immune system. However, TLR-dependent activation of innate immunity is unlikely to contribute significantly to the proinflammatory response to PbA infection and the development of fatal CM.

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Figures

Figure 1

Figure 1

Cytokine production by macrophages derived from MyD88- and TLR9-deficient mice. Bone marrow-derived macrophages from WT, MyD88-deficient (MyD88), or TLR9-deficient (TLR9) mice were stimulated with LPS (100 ng/ml), CpG (0.125 μmol/L), and noninfected (RBC NI) or parasitized red blood cells (PbA at 3 or 10 × 105 cells per ml). After 20 hours, the culture supernatants were analyzed for concentrations of TNF (A–D) and IL-6 (E, F). Results are from one representative experiment (n = 2 mice per genotype) of three independent experiments; the differences in response to LPS, CpG, and PbA between WT and MyD88-deficient mice were all statistically significant whereas statistically significant differences between WT controls and TLR9-deficient mice were seen merely for CpG and PbA 10× stimulated TNF (P < 0.05).

Figure 2

Figure 2

Survival from PbA-infected TLR-deficient mice. WT mice or those deficient for TLR9 or LT-α (A); TLR1 or TLR6 (B); TLR2 alone or plus TLR4 (C); TLR4 or CD14 (D); TLR3 (E); or MyD88, TIRAP, or TRIF (F) were infected with 106 PbA_-_parasitized red blood cells and the survival monitored daily. Results are given as Kaplan-Meyer curves of n = 7 mice per group pooled from two independent experiments. There was no statistically significant difference between the WT controls and the genetically deficient mice, except for LT-α-deficient mice (P < 0.001).

Figure 3

Figure 3

Cerebral microvascular leak and lesion in mice developing CM. Vascular leak as assessed macroscopically (A) by the blue discoloration of the brain of control WT, MyD88-deficient, and LT-α-deficient mice injected intravenously with 1% Evans blue on day 7 with severe CM or quantified after extravasation in formamide (C). Microvascular damage with mononuclear cell adhesion and perivascular hemorrhage (B) and ICAM-1 expression by immunohistochemistry on microvessels (D) in WT, MyD88-deficient, and LT-α-deficient mice. The data represent the mean ± SD of n = 5 mice per group pooled from two independent experiments (**P < 0.01); n.s., not significant. NI, noninfected.

Figure 4

Figure 4

Acute pulmonary inflammation, hemorrhage, and parasite sequestration in mice developing CM. A: Pulmonary inflammation, edema, and hemorrhage in the alveolar space was evaluated in H&E-stained microscopic sections of WT mice and those deficient for TLR9, MyD88, or LT-α 7 days after infection with 106 PbA-parasitized red blood cells. B: Parasite sequestration in the lung was analyzed by flow cytometry of WT, MyD88-deficient, and LT-α-deficient mice 7 days after infection with 106 enhanced green fluorescent protein (EGFP)_-_PbA parasitized red blood cells. C: Pulmonary vascular leak was quantified by lung Evans blue extravasation in formamide on day 7 with severe CM. The data represent the mean ± SD of n = 5 mice per group, from one of two independent experiments (**P < 0.01; n.s., not significant). NI, noninfected mice. Original magnifications, ×40.

Figure 5

Figure 5

Parasitemia and hematological alterations are TLR-independent. The percentage of parasitized erythrocytes in the peripheral blood was determined by analyzing GFP fluorescent erythrocytes using flow cytometry (A) on day 7 for all TLR pathway-deficient mice and on days 7, 11, 18, and 22 after infection for resistant LT-α-deficient mice (B). Total platelet, leukocyte, lymphocyte, neutrophil, and erythrocyte counts were determined in the peripheral blood of control WT and MyD88-, TLR2-, and LT-α-deficient mice (C–G). The data represent the mean ± SD of n = 5 mice per group from two independent experiments (*P < 0.05; **P < 0.01). NI, noninfected mice. n.s., not significant.

Figure 6

Figure 6

Hepatic parasite sequestration and hemoglobin-derived pigment deposition is TLR-independent. A: Microscopic analysis of liver sections from control WT and MyD88- and TLR9-deficient mice at day 7 of infection, H&E staining. B: Sequestration of GFP-labeled parasites in the liver by flow cytometry analysis of liver homogenates. The data represent the mean ± SD of n = 5 mice per group from two independent experiments (NI, noninfected; n.s., not significant). Original magnifications, ×40.

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