Figure 3.
Nuclear translocation of AhR is reduced upon antibiotic treatment owing to the depletion of bacterial AhR ligands in the brain. (A) Representative images showing IF staining for indole-3-acetic acid (red, upper), kynurenine (red, lower), and DAPI (blue) in the cortex of mice transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 or donkey anti-mouse IgG (H + L) Alexa Fluor Plus 647 secondary antibodies were 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, 100 μm. (B) Scatter dot plot showing QuPath-based quantification (MFI) of the abundance of indole-3-acetic acid in the cortex. The MFI was calculated by DAPI-based cell segmentation with 2 μm cytoplasm thickness. Mice were transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (C) Scatter dot plot showing QuPath-based quantification (MFI) of the abundance of kynurenine in the cortex. The MFI was calculated by DAPI-based cell segmentation with 2 μm cytoplasm thickness. Mice were transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (D) Representative images showing IF staining for AhR (red) and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with vehicle (left) or antibiotics (right). Antibody was incubated for 24 hours 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, 30/10 μm. (E) Scatter dot plot showing quantification of AhR translocation as nuclear-to-cytoplasmic expression ratio in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics 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. (F) Representative images showing IF staining for Iba-1 (green) and AhR (red) in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle (left) or antibiotics (right). White arrows indicating AhR-expressing Iba-1+ cells. The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) AlexaFluor488 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. (G) Scatter dot plot showing the number of AhR-expressing cells in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics 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.

Nuclear translocation of AhR is reduced upon antibiotic treatment owing to the depletion of bacterial AhR ligands in the brain. (A) Representative images showing IF staining for indole-3-acetic acid (red, upper), kynurenine (red, lower), and DAPI (blue) in the cortex of mice transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 or donkey anti-mouse IgG (H + L) Alexa Fluor Plus 647 secondary antibodies were 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, 100 μm. (B) Scatter dot plot showing QuPath-based quantification (MFI) of the abundance of indole-3-acetic acid in the cortex. The MFI was calculated by DAPI-based cell segmentation with 2 μm cytoplasm thickness. Mice were transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (C) Scatter dot plot showing QuPath-based quantification (MFI) of the abundance of kynurenine in the cortex. The MFI was calculated by DAPI-based cell segmentation with 2 μm cytoplasm thickness. Mice were transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT) and treated with vehicle or antibiotics as indicated. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (D) Representative images showing IF staining for AhR (red) and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with vehicle (left) or antibiotics (right). Antibody was incubated for 24 hours 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, 30/10 μm. (E) Scatter dot plot showing quantification of AhR translocation as nuclear-to-cytoplasmic expression ratio in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics 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. (F) Representative images showing IF staining for Iba-1 (green) and AhR (red) in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle (left) or antibiotics (right). White arrows indicating AhR-expressing Iba-1+ cells. The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) AlexaFluor488 secondary antibody was incubated for 90 minutes at 4°C. Nuclei were stained using DAPI. (G) Scatter dot plot showing the number of AhR-expressing cells in the cortex of GVHD mice that were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics 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.

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