Malaria primes the innate immune response due to interferon-gamma induced enhancement of toll-like receptor expression and function

Abstract

Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated "priming," we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a TLR9-, MyD88-, and IFNgamma-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while TLR9(-/-), IL12(-/-) and to a greater extent, IFNgamma(-/-) mice were protected. Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFNgamma responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.

Fig. 1.

Hyperresponsiveness of PBMCs from symptomatic malaria patients. ELISA analysis of cytokine levels in PBMC supernatants from uninfected health controls (open circles) or P. falciparum acutely infected subjects (closed triangles) after 20 h of stimulation with LPS (100 ng/ml), Pam2cysk4 (4 nM), Poly:IC (100 μg/ml), CL075 (100 ng/ml), and ODN 2007 (10 μM). All stimulations were performed with 2 other 10-fold different concentrations of TLR ligand, with similar results. Significant differences are indicated with p-values using unpaired t test with Welch correction or Mann-Whitney test when a normality test failed.

Fig. 2.

Cytokine response in symptomatic malaria patients is reversed after curative chemotherapy. (A) Levels of 6 cytokines (TNFα, IL1β, IL6, IL8, IL10, and IL12p70) were determined in the plasma of P. falciparum infected subjects before treatment (closed triangles) or 30 days after treatment (open boxes) using CBA. (B) PBMC isolated from P. falciparum infected individuals before (closed triangles) or after treatment (open boxes) were cultured in the presence of the indicated stimuli for 20 h. Levels of TNF-α, IL-1β, and IL12p40 were measured in culture supernatants by ELISA. Significant differences are indicated with p-values using paired t test or Willcoxon matched-pairs signed-ranks test when a normality test failed. (C) Results of the clusterization. Each row represents a gene and each column the Log2 of the ratio of gene expression level: “Before treatment”/“After treatment” for the gene in question. (D) Validation of the results by qPCR is shown for some of the genes in the Fig. 1C. Each row represents a gene tested and each column the log2 of the ratio of the quantity of cDNA “Before treatment”/“After treatment” standardized by the β2microglobuline cDNA.

Fig. 3.

Increased expression of TLR2 and TLR4 in monocytic cells from symptomatic malaria patients. PBMC were isolated from acute infected individuals before and 30 days after treatment and analyzed ex vivo through flow cytometry. The expression of TLR2 and TLR4 was evaluated in CD14⁺ and CD11c⁺ cells. Representative histogram showing fluorescence intensity of TLR2 in CD14⁺ (Left) and CD11c⁺ (Right) cells.

Fig. 4.

Hyperresponsiveness of spleen cells from mice undergoing acute malaria. C57BL/6 mice were challenged with 10⁵ iRBCs and followed every 3 days for (A) parasitemia and (B) levels of IFN measured in culture supernatants from mouse spleen cells harvested at various weeks post infection and stimulated with LPS (1 g/ml), Pam3cysk4ser (1 g/ml), CpG DNA (1 g/ml), or malaria extract (100 g/ml) for 48 h. As a control for cell viability, cells were stimulated with the mitogen Concanavalin A (5 μg/ml). The levels of IFNγ were measured in culture supernatants 48 sh post stimulation. The results are averages of 5 animals from a representative out of 2 experiments that yield similar results.

Fig. 5.

Over expression of TLRs during acute rodent malaria. (A) Total RNA was isolated from spleens of WT, TLR9^−/−, MyD88^−/−, and IFNγ^−/− mice before and after 6 and 9 days of infection with P. chabaudi. TLRs mRNA levels were measured by quantitative Real time PCR. The relative level of mRNA was determined by the comparative threshold cycle method, whereby data for each sample were normalized to β-actin and expressed as a fold change compared with uninfected controls. Bars are means + SEM from 4 animals per group performed in duplicate. Arrows indicate where differences were statistically significant (P < 0.05) compared to mRNA levels found in WT mice. (B). Spleen cells were harvested from WT, TLR9^−/− and IFNγ^−/− mice before and after 7 and 14 days of infection and cultivated in the presence of the indicated stimuli. IFNγ levels were measured in supernatants 48 h later. Results are means + SEM of twelve animals from 3 independent experiments. Arrows indicate levels of cytokines which differed statistically (P < 0.05) from those observed in WT mice.