Figure 5.
AhR ligand FICZ treatment reverses microglia expansion, T-cell infiltration, and morphological changes of the microglia in GVHD mice. (A) Schematic overview of the murine model. BALB/c mice were treated with antibiotics and either vehicle or FICZ (200 μg/kg body weight FICZ [MedChemExpress, catalog no. HY-12451] in 5% DMSO + 40% polyethylene glycol (PEG) 300 + 5% Tween 80 + 50% phosphate-buffered saline) for 14 days before and 14 days after transplantation. BALB/c mice were lethally irradiated and transplanted with allo-BM (5 × 106 cells) and T cells (3 × 105 cells) from C57BL/6 donor mice. Organs were analyzed on day 14 after transplantation. (B) Representative images showing immunohistochemistry staining for CD3 in meninges of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). Sections were imaged with Zeiss Axio Imager M2m fluorescence microscope with Plan-Apochromat 20×/0.8 M27 objective. Scale bar, 50 μm. (C-D) Scatter dot plots showing numbers of CD3+ cells per mm2 meninges (C) and cortex (D) of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (E) Representative images showing IF staining for Iba-1 in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with either antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss Axio Imager M2m fluorescence microscope with Plan-Apochromat 20×/0.8 M27 objective. Scale bar, 50 μm. (F-G) Scatter dot plots showing numbers of Iba-1+ cells per mm2 in the cortex (F) and cerebellum (G) of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (H) Representative images showing IF staining for VCAM1 (red) and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss LSM710 (Plan-Apochromat 63×/1.4 Oil DIC M27) or Zeiss LSM880 (Plan-Apochromat 63×/1.4 Oil DIC M27) confocal laser scanning microscopes. Scale bar, 10 μm. (I) Scatter dot plot showing quantification (area per 20× HPF) of VCAM1 expression in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (J) Representative images showing IMARIS-based 3D reconstruction of the microglia (upper) and IF staining for Iba-1 (red) and DAPI (blue; lower) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 48 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor 568 secondary antibody was incubated for 48 hours at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss LSM710 (Plan-Apochromat 63×/1.4 Oil DIC M27) or Zeiss LSM880 (Plan-Apochromat 63×/1.4 Oil DIC M27) confocal laser scanning microscopes. Scale bar, 5 μm. (K-P) Scatter dot plots showing IMARIS-based semiautomated quantification of the morphological parameters including filament dendrite length (K), filament number of terminal points (L), filament number of dendrite branch points (M), filament number of dendrite segments (N), filament dendrite volume (O), and filament dendrite area (P). GVHD mice were transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. HPF, high power field.

AhR ligand FICZ treatment reverses microglia expansion, T-cell infiltration, and morphological changes of the microglia in GVHD mice. (A) Schematic overview of the murine model. BALB/c mice were treated with antibiotics and either vehicle or FICZ (200 μg/kg body weight FICZ [MedChemExpress, catalog no. HY-12451] in 5% DMSO + 40% polyethylene glycol (PEG) 300 + 5% Tween 80 + 50% phosphate-buffered saline) for 14 days before and 14 days after transplantation. BALB/c mice were lethally irradiated and transplanted with allo-BM (5 × 106 cells) and T cells (3 × 105 cells) from C57BL/6 donor mice. Organs were analyzed on day 14 after transplantation. (B) Representative images showing immunohistochemistry staining for CD3 in meninges of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). Sections were imaged with Zeiss Axio Imager M2m fluorescence microscope with Plan-Apochromat 20×/0.8 M27 objective. Scale bar, 50 μm. (C-D) Scatter dot plots showing numbers of CD3+ cells per mm2 meninges (C) and cortex (D) of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (E) Representative images showing IF staining for Iba-1 in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with either antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss Axio Imager M2m fluorescence microscope with Plan-Apochromat 20×/0.8 M27 objective. Scale bar, 50 μm. (F-G) Scatter dot plots showing numbers of Iba-1+ cells per mm2 in the cortex (F) and cerebellum (G) of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (H) Representative images showing IF staining for VCAM1 (red) and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss LSM710 (Plan-Apochromat 63×/1.4 Oil DIC M27) or Zeiss LSM880 (Plan-Apochromat 63×/1.4 Oil DIC M27) confocal laser scanning microscopes. Scale bar, 10 μm. (I) Scatter dot plot showing quantification (area per 20× HPF) of VCAM1 expression in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (J) Representative images showing IMARIS-based 3D reconstruction of the microglia (upper) and IF staining for Iba-1 (red) and DAPI (blue; lower) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle (left) or antibiotics + FICZ (right). The primary antibody was incubated for 48 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor 568 secondary antibody was incubated for 48 hours at 4°C. Nuclei were stained using DAPI. Sections were imaged with Zeiss LSM710 (Plan-Apochromat 63×/1.4 Oil DIC M27) or Zeiss LSM880 (Plan-Apochromat 63×/1.4 Oil DIC M27) confocal laser scanning microscopes. Scale bar, 5 μm. (K-P) Scatter dot plots showing IMARIS-based semiautomated quantification of the morphological parameters including filament dendrite length (K), filament number of terminal points (L), filament number of dendrite branch points (M), filament number of dendrite segments (N), filament dendrite volume (O), and filament dendrite area (P). GVHD mice were transplanted with allogeneic BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. HPF, high power field.

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