Chemokine receptors CCR2 and CX3CR1 regulate viral encephalitis-induced hippocampal damage but not seizures

Abstract

Viral encephalitis is a major risk factor for the development of seizures, epilepsy, and hippocampal damage with associated cognitive impairment, markedly reducing quality of life in survivors. The mechanisms underlying seizures and hippocampal neurodegeneration developing during and after viral encephalitis are only incompletely understood, hampering the development of preventive treatments. Recent findings suggest that brain invasion of blood-born monocytes may be critically involved in both seizures and brain damage in response to encephalitis, whereas the relative role of microglia, the brain's resident immune cells, in these processes is not clear. CCR2 and CX3CR1 are two chemokine receptors that regulate the responses of myeloid cells, such as monocytes and microglia, during inflammation. We used Ccr2-KO and Cx3cr1-KO mice to understand the role of these receptors in viral encephalitis-associated seizures and neurodegeneration, using the Theiler's virus model of encephalitis in C57BL/6 mice. Our results show that CCR2 as well as CX3CR1 plays a key role in the accumulation of myeloid cells in the CNS and activation of hippocampal myeloid cells upon infection. Furthermore, by using Cx3cr1-cre^ER+/-tdTomato^St/Wt reporter mice, we show that, with regard to CD45 and CD11b expression, some microglia become indistinguishable from monocytes during CNS infection. Interestingly, the lack of CCR2 or CX3CR1 receptors was associated with almost complete prevention of hippocampal damage but did not prevent seizure development after viral CNS infection. These data are compatible with the hypothesis that CNS inflammatory mechanism(s) other than the infiltrating myeloid cells trigger the development of seizures during viral encephalitis.

Keywords: Theiler’s virus; epilepsy; hippocampus; monocytes; myeloid cells.

Fig. 1.

Decreased accumulation of myeloid cells in the brains of CCR2-deficient but not CX3CR1-deficient mice after intracranial TMEV infection. C57BL/6 WT, Ccr2-KO, and Cx3cr1-KO animals were mock infected or infected with TMEV intracranially. Animals were killed at 2 or 7 dpi. After enzymatic digestion, immune cells were isolated using Percoll gradient and were immunolabeled, and flow cytometry was performed. Myelin debris and dead cells were excluded by FSC/SSC gating, and singlet populations were analyzed. (A) Representative flow cytometry data of immune cells isolated from the brains at 7 dpi. (B) Quantification of CD45^highCD11b^high myeloid cells in the brain at 2 dpi (red-marked population in A). Shown are combined data of two independent experiments; n = 7. (C) Quantification of CD45^highCD11^high myeloid cells in the brain at 7 dpi (red-marked population in A). Shown are combined data of two independent experiments; n = 6–14). (D) Quantification of CD45⁺CD11b⁻ cells in the brain at 7 dpi (green-marked population in A). Shown are combined data of two independent experiments; n = 6–13). The data in B–D are shown as mean ± SEM (plus individual data). Analysis of data in C by two-way ANOVA indicated a significant effect of infection [F (1, 57) = 13.91; P = 0.0004], genotype [F (2, 57) = 3.98; P = 0.0241], and interaction [F (2, 57) = 3.601; P = 0.0337]. Similar, analysis of data in D indicated a significant effect of infection [F (1, 52) = 36.29; P < 0.0001], genotype [F (2, 52) = 7.054; P = 0.0019], and interaction [F (2, 52) = 7.034; P = 0.002]. Post hoc results in B–D are indicated by asterisks: **P < 0.01; ***P < 0.001); ns, not significant.

Fig. 2.

Genetic deficiency of CCR2 but not CX3CR1 leads to decreased activation of myeloid cells in the hippocampus after intracranial TMEV infection. Animals were treated as described in Fig. 1. On day 7 after perfusion brains were removed, and immunohistology was performed. (A) Representative examples of Iba-1– and Mac-3–stained hippocampal sections. (Scale bars: 50 µm.) (B) Area of the hippocampus from which the sections shown in A were taken. (C) Quantification of hippocampal Iba-1⁺ cells shown in A. (D) Semiquantitative data for Mac-3 staining shown in A. The data in C and D are shown as mean ± SEM (plus individual data). Analysis of data in C by two-way ANOVA indicated a significant effect of infection [(F (1, 36) = 20.89; P < 0.0001] but not genotype [F (2, 36) = 0.4272; P = 0.6556] or interaction [F (2, 36) = 0.03501; P = 0.9656]. Analysis of data in D indicated a significant effect of infection [F (1, 41) = 38.21; P < 0.0001], genotype [F (2, 41) = 3.088; P ≤ 0.05], and interaction [F (2, 41) = 8.251; P = 0.0010]. Post hoc results are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 3.

Genetic deficiency of CCR2 and CX3CR1 prevents microglia proliferation in the hippocampus after intracranial TMEV infection. Animals were treated as described in Fig. 1; on day 7 after perfusion, brains were removed, and immunohistology was performed. (A) Representative examples of Iba-1 and Ki67 double-stained cells in hippocampal sections. Slice thickness was only 2 µm (Methods), so processes of Iba-1⁺ microglial cells (green) are often cut from the cell bodies. Cell nuclei were stained using DAPI (blue); Ki67⁺ is shown in magenta. A Iba-1⁺/Ki67⁺ microglial cell (arrow) can be seen in this example of an infected B6 WT mouse, while Iba-1⁺ cells in Ccr2-KO and Cx3cr1-KO mice are mainly negative for Ki67 (see also C). (Scale bar: 10 µm.) (B) Area of the hippocampus from which the sections shown in A were taken. (C) Quantification of Iba-1⁺/Ki67⁺ double-labeled cells shown in A in the ipsilateral hippocampus. (D) Percent of Iba-1⁺ cells that are also positive for Ki67 (proliferation index). The data in C and D are shown as mean ± SEM (plus individual data). Analysis of data in C by two-way ANOVA indicated a significant effect of infection [F (1, 38) = 9.88; P = 0.0032], genotype [F (2, 38) = 9.664; P = 0.0004], and interaction [F (2, 38) = 7.038; P = 0.0025]. Analysis of data in D indicated a significant effect of infection [F (1, 38) = 5.243; P = 0.0277] and genotype [F (2, 38) = 11.2; P = 0.0002] but not interaction [F (2, 38) = 2.186; P = 0.1263]. Post hoc results are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.

A population of microglia up-regulates CD45 and becomes indistinguishable from infiltrating monocytes after intracranial TMEV infection. Tamoxifen-treated Cx3cr1-cre^ER+/−tdTomato^St/Wt mice were infected, and CNS immune cells were isolated as described in Fig. 1. (A) Representative flow cytometry data of immune cells isolated from brain. (B) Representative flow cytometry data of tdTomato expression on CD45^lowCD11b^low cells (black-marked population in A) and CD45^highCD11b^high cells (red-marked population in A). (C) Expression of CD86 and CD206 on the CD45^highCD11b^hightdTomato⁺ cells (orange-marked population in B). (D) Quantification of CD45^highCD11^high myeloid cells in the brain at 2 and 7 dpi (red-marked population in A). Shown are the combined data of two independent experiments; n = 3–8. (E) Quantification of tdTomato-expressing cells in the CD45^highCD11b^high population in A (orange-marked population in B); n = 3–6; shown are combined data of two independent experiments. (F) Quantification of tdTomato⁻ cells in the CD45^highCD11b^high population in A (green-marked population in B; n = 3–6; combined data from two independent experiments are shown). The data in D–F are shown as mean ± SEM (plus individual data); open symbols represent mock-infected controls, filled symbols represent infected mice. Analysis of data in D by two-way ANOVA indicated a significant effect of infection [F (1, 18) = 25.23; P < 0.0001] but not time [F (1, 18) = 1.891; P = 0.1859] or interaction [F (1, 18) = 1.931; P = 0.1816]. Similar, analysis of data in E and F indicated only a significant effect of infection [E: F (1, 13) = 27.66; P = 0.0002; F: F (1, 13) = 17.58; P = 0.0011]. Post hoc results are indicated by asterisks: **P < 0.01; ***P < 0.001.

Fig. 5.

Genetic deficiency of CCR2 and CX3CR1 leads to decreased neuronal death in the hippocampus after TMEV infection. Animals were treated as described in Fig. 1. Brains were removed on day 7 after perfusion, and histology was performed on sections of the dorsal hippocampus. Section levels (−2.06 ± 0.26 mm from bregma) were similar in all groups. (A) Representative photomicrographs illustrating NeuN⁺ neurons in mock-infected and TMEV-infected B6, Ccr2-KO, and Cx3cr1-KO mice. [Scale bars: 200 µm (overview, Top and Center Rows) or 50 µm (magnification, Bottom Row).] (B) Semi-quantitative data on NeuN⁺ neurons in the CA1/CA2 regions of the hippocampus. Data are shown as boxplots with whiskers indicating minimum and maximum values; the horizontal line in the boxes represents the median value. In addition, individual data are shown (n = 5–13). Analysis of data by two-way ANOVA indicated a significant effect of infection [F (1, 41) = 5.33; P = 0.0261], genotype [F (2, 41) = 6.348; P = 0.0040], and interaction [F (2, 41) = 7.35; P = 0.0019]. Significant differences from post hoc testing of mock-infected mice are indicated by the hash sign: ^#P < 0.05; significant differences between infected groups are indicated by asterisks: **P < 0.01. (C) The number of FJC⁺ neurons in infected mice (n = 6–14). No FJC staining was observed in controls. Analysis of data by one-way ANOVA indicated a significant difference between groups [F (2, 28) = 5.174; P = 0.0122]. Significant differences between infected groups are indicated by asterisks: *P < 0.05; **P < 0.01. (D) Representative photomicrographs illustrating FJC staining in infected mice. [Scale bars: 200 µm (Top Row) or 50 µm (Bottom Row).]

Fig. 6.

Genetic deficiency of CCR2 and CX3CR1 does not decrease seizure incidence after TMEV infection. Mice were treated as described in Fig. 1, and the incidence, frequency, and severity of seizures were calculated over the 7-d monitoring period. Group sizes were 28 B6-WT, 31 Ccr2-KO, and 33 Cx3cr1-KO mice. (A) Temporal profile of seizure incidence following infection; analysis of data by Fisher’s exact test did not indicate significant intergroup differences. (B) Cumulative seizure incidence calculated from the data shown in A. Seizure incidence is indicated by the blue part of the columns; exact percentages are indicated within the columns. No seizures were observed in mock-infected controls (not shown; n = 28–33); analysis of data by Fisher’s exact test did not indicate significant intergroup differences. (C) Raw Racine scores for each seizure in each animal recorded over the 7 d following infection. Each tick mark on the x axis represents a mouse with observed seizure(s); each symbol represents a single seizure. Ccr2-KO mice exhibited significantly fewer generalized convulsive (stage 5) seizures than B6-WT (P < 0.05) or Cx3cr1-KO mice (P < 0.01; Fisher’s exact test). (D) The number of seizures counted in each mouse, i.e., seizure frequency recorded over the 7 dpi. Data are shown as boxplots with whiskers indicating minimum and maximum values; the horizontal lines in the boxes represent the median value. In addition, individual data are shown. Analysis of data by ANOVA did not indicate significant differences (P = 0.7178). (E) Cumulative seizure burden at each day postinfection in the three groups of mice. Cumulative seizure burden at each day postinfection for a mouse was calculated by summing all its seizure scores up to that day. No significant intergroup differences were observed.