Involvement of Toll-like receptor 2 in the cerebral immune response and behavioral changes caused by latent Toxoplasma infection in mice

Abstract

Subacute and chronic infections with the intracellular protozoan parasite Toxoplasma gondii are associated with an increased risk of psychiatric diseases like schizophrenia. However, little is known about the mechanisms involved in T. gondii-induced neuronal disorders. Recently, we reported that Toll-like receptor 2 (TLR2) was required to initiate the innate immune response in cultured mouse brain cells. However, how TLR2 contributes to latent infection with T. gondii remains unclear. Therefore, we examined the role of TLR2 in brain pathology and behavior using wild-type (TLR2+/+) and TLR2-deficient (TLR2-/-) mice. The behavioral analyses showed that TLR2 deficiency increased the anxiety state of the uninfected and infected animals alike, and TLR2 deficiency showed no relationship with the infection. In the contextual and cued fear-conditioning tests, T. gondii infection decreased the mouse freezing reaction while TLR2 deficiency increased it, but there was no interaction between the two factors. Our histopathological analysis showed that the TLR2+/+ and TLR2-/- mice had similar brain lesions at 30 days post infection (dpi) with T. gondii. Higher numbers of parasites were detected in the brains of the TLR2-/- mice than in those from the TLR2+/+ mice at 30 dpi, but not at 7 and 14 dpi. No significant differences were observed in the proinflammatory gene expression levels in the TLR2+/+ and TLR2-/- mice. Therefore, it appears that TLR2 signaling in the brain might contribute to the control of parasite growth, but not to brain pathology or the impaired fear memory response induced by infection with T. gondii.

Fig 1. Effects of T. gondii infection on the locomotor activity of mice.

Changes in mouse locomotor activity on day 30 post infection: (A) total distance travelled (cm), (B) average speed (cm/sec), and (C) latency to first response (sec). Data were summarized from two independent experiments (uninfected TLR2^+/+ mice, n = 16; uninfected TLR2^-/- mice, n = 13; infected TLR2^+/+ mice, n = 21; infected TLR2^-/- mice, n = 18). Significant differences among the four groups were analyzed by a two-way ANOVA followed by t tests (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001), significant main effects are shown for (A) T. gondii infection [F_(1,62) = 35.42, p < 0.0001], (B) T. gondii infection [F_(1,62) = 31.85, p < 0.0001], and (C) TLR2 deficiency [F_(1,63) = 7.596, p < 0.05]. The interaction between T. gondii infection and TLR2 deficiency was not statistically significant.

Fig 2. Effects of T. gondii infection on anxiety-related behavior in mice.

Changes in the exploratory behavior at 31 days post infection: (A) head-dip count (counts/5 min), (B) head-dip duration (sec/5 min), and (C) head dip latency (sec). Data were summarized from two independent experiments (uninfected TLR2^+/+, n = 16; uninfected TLR2^-/-, n = 13; infected TLR2^+/+, n = 21; infected TLR2^-/-, n = 18). Significant differences among the four groups were analyzed by a two-way ANOVA followed by t tests (*, p < 0.05; ***, p < 0.01; ****, p < 0.001), significant main effects are shown for (A) TLR2 deficiency [F_(1,62) = 25.30, p < 0.0001], (B) TLR2 deficiency [F_(1,62) = 8.996, p < 0.01], and (C) TLR2 deficiency [F_(1,60) = 13.83, p < 0.001]. The interaction between T. gondii infection and TLR2 deficiency was not statistically significant.

Fig 3. Effects of T. gondii infection on fear memory consolidation in mice.

The ordinate shows the percentage of time spent freezing. (A) Contextual conditioned freezing time. Significant differences among the four groups were analyzed by a two-way ANOVA followed by t tests (**, p < 0.01; ***, p < 0.001; ****, p < 0.0001), significant main effects are shown for TLR2 deficiency [F_(1,62) = 20.06, p < 0.0001] and T. gondii infection [F_(1,62) = 40.58, p < 0.0001]. The interaction between T. gondii infection and TLR2 deficiency was not statistically significant. (B) Tone-conditioned freezing time. Significant differences between habituation and tone sessions were determined by unpaired t tests (**p < 0.01). (C) Time course for fear extinction as revealed by the decreased freezing response in the extinction test. Statistical analysis between experimental group pairs including uninfected TLR2^+/+ mice and uninfected TLR2^-/- mice, uninfected TLR2^+/+ mice and infected TLR2^+/+ mice, and uninfected TLR2^-/- mice and infected TLR2^-/- mice were performed by a two-way ANOVA but no statistically significant main effects or interactions were observed. The average freezing response percentage across the 5 min intervals in the extinction test was normalized to the first 5 min. Freezing was calculated by dividing the freezing time by the observation times (300 s) in the context test, habituation (180 s), and tone (180 s) during the tone test, and over 5 min for every 5 min interval in the extinction test. Data represent the means ± SEMs. Data are summarized from two independent experiments (uninfected TLR2^+/+, n = 16; uninfected TLR2^-/-, n = 13; infected TLR2^+/+, n = 21; infected TLR2^-/-, n = 18).

Fig 4. Histopathological lesions and parasite burdens in the brains of T. gondii-infected mice.

On day 30 after infection, brain samples were collected. The total pathological score (A) and number of brain cysts (B) were calculated. Each circle represents the data for one mouse, and the bars represent the average values for all the data points in two independent experiments (TLR2^+/+ mice, n = 9; TLR2^–/–mice, n = 9). There was no statistically significant difference between the two groups in the unpaired t test (*p = 0.0628). (C) On day 30 after infection, brain samples were collected. DNA was extracted from each brain sample and the parasite numbers were quantified. Each circle represents the data for one mouse, and the bars represent the median value from all the data points in two independent experiments (TLR2^+/+ mice, n = 9; TLR2^–/–mice, n = 9). Statistically significant differences were determined by the Mann-Whitney test (*p = 0.0315). (D) TLR2^+/+ mice and TLR2^–/–mice were infected with T. gondii tachyzoites. On day 7 and 14 after infection, brain samples from all the mice were collected. DNA was extracted from each sample and the parasite numbers were quantified. Each circle represents the data for one mouse, and the bars represent the average values for all the data points in one experiment (TLR2^+/+ mice, n = 6; TLR2^–/–mice, n = 5). Significant differences were determined by unpaired t tests (*p < 0.05).

Fig 5. iNOS and cytokine mRNA expression in the brains of T. gondii-infected TLR2^+/+ and TLR2^–/–mice.

TLR2^+/+ and TLR2^–/–mice were infected with T. gondii tachyzoites. On day 30 after infection, brain samples from all the mice were collected. Total RNA was extracted from each sample and iNOS and cytokine mRNA expression was quantified by real-time PCR. Each circle and square represent the data for one mouse, and the bars represent the average values of all the data points in two independent experiments (TLR2^+/+ mice, n = 9; TLR2^–/–mice, n = 9). Significant differences were determined by unpaired t tests (*p < 0.05).