Activated microglia contribute to neuronal apoptosis in Toxoplasmic encephalitis

Abstract

Background: A plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE.

Methods: A murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments.

Results: Microglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis.

Conclusions: Activated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.

Figure 1

Toxoplasma encephalitis model was established. Microscopic observation of the brain tissues of mice latently infected with cyst-forming T. gondii Wh6 strain (genotype Chinese 1) reactivated by cyclophosphamide administration. (A①) and (A③), a single cyst (arrows) in the cortex of chronically infected mice; (A②) necrosis and inflammatory infiltration noted in foci of recrudescence; (A④) free tachyzoites in TE (×400). The sections were incubated with mouse anti-Toxoplasma SAG1 monoclonal antibody and peroxidase-antiperoxidase-coupled secondary antibody, followed by DAB colorization. Figure B, hemiplegia and paraplegia in model mice with Toxoplasma encephalitis.

Figure 2

Neuronal apoptosis in the CNS of control, TE, and TE + M mice. (A) Double immunofluorescent staining of caspase-3 (a marker of cell death) and NeuN (a specific marker of neuron cell) demonstrated apoptosis of neuron cells. Brain sections from cortex of control, TE and TE + M animal groups were stained with polyclonal caspase-3(red) antibody and monoclonal NeuN antibody (green). Cells were counter-stained with Hoechst to show nuclei (blue). Double immunostaining showed caspase-3 expression in neurons. Increased number of caspase-3 positive cells in TE was reduced significantly in TE + M. (B) Western blotting assay was used for analysis of protein NeuN expression in control and TE and TE + M mice. It showed that the downregulated expression of NeuN in TE was increased significantly after treatment with minocyline (TE + M). Values represent the means ± SEM from six animals in each group (*, p < 0.01 vs. Control. #, p < 0.01 vs. TE).

Figure 3

TUNEL assay for analysis of apoptosis of mouse neuronal cells (N2a). A small number of cell death was observed when N2a was co-cultured with BV-2. The number of TUNEL positive cells increased when N2a was co-cultured with T.gondii tachyzoite-infected BV-2 cells. Treatment of BV-2 with minocycline before infection with T.gondii followed by co-culture with N2a for 24 h resulted in a dramatic reduction of TUNEL positive cells. Values represent the means ± SEM from three independent experiments. (*p < 0.01 vs. Control; #p < 0.01 vs. T. gondii).

Figure 4

Immunocytochemical analysis of microglia in the brain of TE and TE + M mice. Brain sections from control, TE and TE + M animal groups were stained with polyclonal antibodies against Iba-1(a specific microglial marker). Compared with control and TE + M mice, the number of Iba-1-positive cells in both hippocampal and cortex of TE mice was remarkably increased. Morphological characteristics of microglia activation were noted in TE. Minocycline treatment significantly inhibited microglial activation. Values represent the means ± SEM of six animals per group. (*P < 0.01 vs Control; #P < 0.01 vs TE).

Figure 5

Expression of pro-inflammatory cytokines (IL-1 β, IL-6 and TNF-α) in the brain tissues of control, TE, and TE + M mice. (A) Representative agarose-gel photographs show the expression level of IL-1 β, IL-6, and TNF-α mRNAs in brain tissues tested by quantitative RT-PCR. (B) Histograms represent densitometric quantitation of IL-1 β, IL-6, and TNF-α, normalized over GAPDH mRNA expression. (C) Expression of IL-1 β, IL-6, and TNF-α in brain tissues examined by ELISA. Values represent the means ± SEM of six animals per group. (*, p < 0.01 vs. Control. #, p < 0.01 vs. TE).

Figure 6

Pro-inflammatory cytokines (IL-1 β, IL-6, and TNF-α) produced by BV-2 cells. Mouse microglia BV-2 was either left uninfected, or T.gondii-infected (T.gondii) or T.gondii-infected and treated with minocycline (T.gondii + M) as described in the Methods section. (A) The mRNAs of pro-inflammatory cytokines were detected by quantitative RT-PCR. The relative levels of IL-1 β, IL-6, and TNF-α expression were compared with GAPDH. (B) Culture supernatants were collected after 24 h post-treatment and tested by ELISA to quantify IL-1 β, IL-6, and TNF-α secretion. The results represent the means ± SEM of three independent experiments. (*P < 0.01 vs Control; #P < 0.01 vs T. gondii).

Figure 7

Expression of iNOS in brain of control, TE, and TE + M mice. (A) Double immunofluorescent staining of iNOS with Iba-1 (a specific marker of microglia). In the inflammatory loci of hippocampus, the number of iNOS-immunoreactive cells increased and the iNOS was expressed in microglia. (B) Western blotting analysis of iNOS expression. Histograms show densitometric analyses that revealed the relative levels of iNOS normalized over β-actin. Values represent the means ± SEM of six animals per group. (*P < 0.01 vs Control; #P < 0.01 vs TE).

Figure 8

Expression of iNOS protein in BV-2 cells. The three groups include mouse microglia BV-2 cells (control), T.gondii-infected BV-2 cells (T.gondii) or BV-2 cells infected with T.gondii and treated with minocycline (T.gondii + M). (A) BV-2 cells were fixed and stained with antibodies against iNOS (red) and tachyzoite surface antigen SAG-1(green) 24 h after infection with T.gondii. Hoechst dye was used to stain nuclei (blue). Double immunofluorescence showed iNOS expression in parasite-infected BV-2 cells. (B) Western blotting analysis of iNOS. Histograms show densitometric analyses of the relative level of iNOS protein normalized over β-actin expression. Values represent the means ± SEM of six animals per group. (*P < 0.01 vs Control; #P < 0.01 vs TE).