Transcriptome analysis of the effect of C-C chemokine receptor 5 deficiency on cell response to Toxoplasma gondii in brain cells

Abstract

Background: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain.

Results: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway.

Conclusions: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.

Keywords: Astrocyte; Brain; C-C chemokine receptor 5; Microglia; Neuron; Toxoplasma gondii; Transcriptome.

Fig. 1

Expression of the top 20 DEGs in which upregulation during T. gondii infection was impaired by CCR5-deficiency. DEGs were ranked according to the fold-changes between the infected wild-type (WT) vs. infected CCR5-deficient (KO) mice. a, astrocytes; b, microglia; c, neurons. Each bar represents the fold-change between uninfected vs. infected mice. The values under the gene symbols are the ratio of the fold-change between uninfected KO vs. infected KO to that between uninfected WT vs. infected WT. The area of low fold-change is enlarged in panel A and B

Fig. 2

Survival rate and body weight of the infected wild-type (WT) and CCR5-deficient (KO) mice. a Survival rate was monitored until 30 dpi (infected WT, n = 10 + 8; infected CCR5KO, n = 9 + 6). Survival rate was compared between groups using the log-rank test (*p < 0.05). b Body weight change was monitored until 9 dpi, the day before the infected group started to die (uninfected WT, n = 4; uninfected CCR5KO, n = 4; infected WT, n = 10; infected CCR5KO, n = 9). Each point represents the mean ± SD. The weight difference between 0 dpi and 9 dpi was compared using two-way ANOVA. Effect of infection on body weight was significant (*p < 0.0001) but not the mouse genotype or the interaction between infection and mouse genotype (p > 0.05)

Fig. 3

Parasite numbers in the brain in the in vivo experiment. Parasite numbers in the brain tissue from the T. gondii-infected wild-type (WT) and CCR5-deficient (KO) mice were determined by real-time quantitative PCR on the T. gondii B1 gene. a, 30 dpi; b, 7 dpi. Parasite numbers were compared using the student’s unpaired t-test between the WT and CCR5KO mice (*p > 0.05). C_t values higher than those for the uninfected samples were considered indicative of nonspecific amplification and were excluded from the plot. Each symbol represents the data point for one mouse, and the bars represent the mean value for all the group data points (uninfected WT, n = 3; uninfected CCR5KO, n = 3; infected WT, n = 5; infected CCR5KO, n = 7)

Fig. 4

Expression of DEGs identified from the transcriptomic analysis was examined in the brain in the in vivo experiment. The brains of the T. gondii-infected wild-type (WT) and CCR5-deficient (KO) mice were prepared to determine their mRNA levels. a, 30 dpi; b, 7 dpi. The mRNA levels were normalized by the β-actin (Actb) mRNA level in the corresponding sample. * and # represent significant differences between the two groups in Tukey’s test after two-way ANOVA (**** and #### p < 0.0001, *** and ### p < 0.001, ** and ## p < 0.01, * and # p < 0.05). Each symbol represents the data point for one mouse, and the bars represent the mean value for all the group data points (uninfected WT, n = 4; uninfected CCR5KO, n = 4; infected WT, n = 5; infected CCR5KO, n = 5)