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. 2021 Mar 9;22(5):2764.
doi: 10.3390/ijms22052764.

Reduction of Amyloid Burden by Proliferated Homeostatic Microglia in Toxoplasma gondii-Infected Alzheimer's Disease Model Mice

Ji-Hun Shin  1 , Young Sang Hwang  1 , Bong-Kwang Jung  2 , Seung-Hwan Seo  1 , Do-Won Ham  1 , Eun-Hee Shin  1   3
Affiliations

Reduction of Amyloid Burden by Proliferated Homeostatic Microglia in Toxoplasma gondii-Infected Alzheimer's Disease Model Mice

Ji-Hun Shin et al. Int J Mol Sci. .

Abstract

In this study, we confirmed that the number of resident homeostatic microglia increases during chronic Toxoplasma gondii infection. Given that the progression of Alzheimer's disease (AD) worsens with the accumulation of amyloid β (Aβ) plaques, which are eliminated through microglial phagocytosis, we hypothesized that T. gondii-induced microglial proliferation would reduce AD progression. Therefore, we investigated the association between microglial proliferation and Aβ plaque burden using brain tissues isolated from 5XFAD AD mice (AD group) and T. gondii-infected AD mice (AD + Toxo group). In the AD + Toxo group, amyloid plaque burden significantly decreased compared with the AD group; conversely, homeostatic microglial proliferation, and number of plaque-associated microglia significantly increased. As most plaque-associated microglia shifted to the disease-associated microglia (DAM) phenotype in both AD and AD + Toxo groups and underwent apoptosis after the lysosomal degradation of phagocytosed Aβ plaques, this indicates that a sustained supply of homeostatic microglia is required for alleviating Aβ plaque burden. Thus, chronic T. gondii infection can induce microglial proliferation in the brains of mice with progressed AD; a sustained supply of homeostatic microglia is a promising prospect for AD treatment.

Keywords: 5XFAD mouse; Alzheimer’s disease; Toxoplasma gondii; chronic infection; disease-associated microglia; homeostatic microglia; lysosomal digestion; plaque-associated microglia; plaque-free microglia.

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Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1
Proliferation and activation of resident homeostatic microglia over 36 weeks after Toxoplasma gondii infection. (A) T. gondii infection was confirmed by the presence of cysts in brain tissue (H&E staining; scale bar, 20 µm). (B) Microglia in the hippocampal formation were stained with Iba1 (red; scale bar, 100 µm). (C) Microglia in the hippocampal formation were stained with TMEM119 (red; scale bar, 100 µm). (D) Activated microglia were co-stained with CD11b and Iba1 (green and red, respectively; scale bar, 50 µm). (E,F) Mean fluorescence intensity (MFI) was calculated from fluorescence-stained images (B,C) using ImageJ. The fold changes of MFI at 3, 6, 12, and 36 weeks PI were compared with those of the control (0 weeks). H, hippocampus; C, cortex. (G) The number of activated microglia (Iba1+/CD11b+) was designated by cell number per mm2 of the brain tissue. (H) Ki67-stained proliferating microglia. The yellow arrow head shows microglial cells in the mitotic phase (sky blue) co-stained with DAPI (blue) and Ki67 (green), and the number of proliferating microglia (Iba1+/Ki67+) was designated by cell number/mm2 brain tissue. Scale bar; 20 µm. Quantification of MFI intensity and co-stained cell counting; two brain sections per mouse. (I) Microarray analysis of genes encoding trophic factors, homeostatic markers, and M1 and M2 markers in T. gondii-infected brain. Data are represented as the mean ± SEM. * Statistical significance compared with the control (* p < 0.05).
Figure 2
Figure 2
Reduction of Aβ plaques and microglial proliferation in T. gondii-infected 5XFAD mouse brain. (A) A T. gondii cyst (black arrow) and stained amyloid plaque (blue arrow head) in a brain section (H&E and Congo red staining) at 40 weeks PI. Scale bar; 20 µm. (B) Dense core plaques (black spots) stained using Congo red dye (scale bar; 100 µm) and acidophilic neurons (black quadrangle) in cortical layer V (scale bar; 20 µm). (C) The number of dense core plaques counted in Congo red-stained brain. (D) Concentration of Aβ in brain tissue lysate analyzed by ELISA. (E) DAB-color immunohistochemistry of microglia around Aβ plaques. Scale bar; 20 µm. (F) Microglial cells accumulated; Iba1-stained microglia in the brain. The fold change of MFI in the AD + Toxo group compared with the AD group in the Iba1-stained brain. Scale bar; 100 µm. H, hippocampus; C, cortex. (G) TMEM119-stained homeostatic microglia accumulation. The fold changes of MFI in the TMEM119-stained brain. Scale bar; 100 µm. H, hippocampus; C, cortex. (H) Protein expression of microglial trophic factors (IL-1β and TNF-α) and microglial polarization inducers (IFN-γ and IL-4) examined by ELISA. (I) Microarray analysis for microglial trophic factors (Il1β, Tnfα, Mcsf, and NFKB1), homeostatic microglial markers (P2ry13, Cx3cr1, and Tmem119), and inducers and markers of M1 and M2 polarized microglia (Ifnγ, Il4, Cd86, and Cd206) in AD and AD + Toxo groups compared with the gene expression in wild-type (WT) mice. Data are represented as the mean ± SEM. * Statistical significance compared with the control (* p < 0.05). # Statistical significance compared with each experimental group (# p < 0.05).
Figure 3
Figure 3
Plaque-associated patterns of microglia and Ly6C+ monocytes. (A) Iba-1-stained microglia (red) around methoxy-XO4-stained amyloid β (Aβ) plaques (white). The counting result of microglia (plaque-associated or plaque-free) found around 60 amyloid plaques distributed in the brain ((10 randomly selected plaques per mouse) × 6 mice per group). Scale bar; 25 µm. (B) Plaque-associated homeostatic microglia (yellow, due to co-staining of Iba-1 (red) and TMEM119 (green) and indicated by an arrow). Number of plaque-associated homeostatic microglial cells. Scale bar; 20 µm. (C) Ly6C+ monocytes (green) accumulated around Aβ plaque in brain tissues of both AD and AD + Toxo groups. (Ly6C+ (green) monocytes indicated by white arrow). Number of plaque-associated Ly6C+ monocytes. Scale bar; 20 µm. Data are represented as the mean ± SEM. * Statistical significance compared with the control (* p < 0.05).
Figure 4
Figure 4
Disease-associated microglia (DAM) phenotype of microglia and microglial phagocytosis. (A) DAM- or homeostatic markers of microglia investigated by gene array analysis. (B) qPCR results for Cst7 and Tmem119. (C) The TREM2 signal is a predictor of the DAM phenotype. (yellow, due to co-staining of Iba-1 (red) and TREM2 (green) and indicated by an arrow); Scale bar; 20 µm. (D) Lipoprotein lipase (LPL) signal as a stage two marker in DAM. The number of plaque-associated and LPL-expressing microglia. The counting result of microglia found around 60 amyloid plaques distributed in the brain ((10 randomly selected plaques per mouse) × 6 mice per group). (yellow, due to co-staining of Iba-1 (red) and LPL (green) and indicated by an arrow); Scale bar; 20 µm. (E) LAMP1 expression, and colocalization of LAMP1 and Iba1 in microglia around the amyloid β (Aβ) plaque. Lysosomal degradation of Aβ plaque as seen by the internalized puncta per microglial cell. Both “a” and “b” indicate LAMP1 positive lysosomes (white and yellow arrow) with the colocalization of methoxy-X04 stained amyloid β (*). Scale bar; 20 µm. (F) Internalized puncta per microglial cell indicate lysosomal degradation of the Aβ plaque (yellow arrow). Scale bar; 20 µm. Data are represented as the mean ± SEM. * Statistical significance compared with the control (* p < 0.05). # Statistical significance compared with each experimental group (# p < 0.05).
Figure 5
Figure 5
Apoptosis of plaque-associated microglia. TUNEL+-microglial cells surrounding the amyloid β (Aβ) plaque. Arrows represent Iba1 and TUNEL double-positive cells (light blue). Number of Iba1 and TUNEL double-positive cells in plaque-associated microglia. The counting result of plaque-associated apoptotic microglia found around 60 amyloid plaques ((randomly selected 10 plaques per mouse) × 6 mice per group). Scale bar; 20 µm. Data are represented as the mean ± SEM.
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