Figure 7.
Indole-3-acetic acid is reduced in the blood and brain of patients with aGVHD. (A) Violin plots showing the abundance of different AhR ligands in blood samples of patients developing GVHD and patients without GHVD. (B) Heat map showing the column-scaled log abundance of different AhR ligands in blood samples of patients developing GVHD (red) and patients without GHVD (blue). (C) Scatterplot showing the correlation between indole-3-acetate concentration in blood samples and GVHD score of patients developing GVHD. Black line depicts the mean of a linear regression model with shaded areas indicating the 95% confidence interval. (D) Scatterplot showing the correlation between methyl indole-3-acetate concentration in blood samples and GVHD score of patients developing GVHD. Black line depicts the mean of a linear regression model with shaded areas indicating the 95% confidence interval. (E) Representative images showing IF staining for indole-3-acetic acid (red) and DAPI (blue) in the cortex of control patients (left), patients who had undergone allo-HCT without GVHD (middle), and patients who had received allo-HCT with GVHD (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus647 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, 100 μm. (F) Scatter dot plot showing quantification of the abundance of indole-3-acetic acid in human cortex of control patients, patients who had received allo-HCT without GVHD, and patients who had received allo-HCT with GVHD, as expressed MFI. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (G) Representative images showing IF staining for Iba-1 (green), phospho–NF-κB p65 (red), and DAPI (blue) in the cortex of control patients (left), patients who had received allo-HCT without GVHD (middle), and patients who had undergone allo-HCT with GVHD (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 and goat anti-rat IgG (H + L) Alexa Fluor 488 secondary antibodies were 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. (H) Scatter dot plot showing quantification of the expression of phospho–NF-κB p65 in the cortical microglia of control patients, patients who had received allo-HCT without GVHD, and patients who had undergone allo-HCT with GVHD, as expressed FC MFI. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. AUC, area under the curve; Max, maximum.

Indole-3-acetic acid is reduced in the blood and brain of patients with aGVHD. (A) Violin plots showing the abundance of different AhR ligands in blood samples of patients developing GVHD and patients without GHVD. (B) Heat map showing the column-scaled log abundance of different AhR ligands in blood samples of patients developing GVHD (red) and patients without GHVD (blue). (C) Scatterplot showing the correlation between indole-3-acetate concentration in blood samples and GVHD score of patients developing GVHD. Black line depicts the mean of a linear regression model with shaded areas indicating the 95% confidence interval. (D) Scatterplot showing the correlation between methyl indole-3-acetate concentration in blood samples and GVHD score of patients developing GVHD. Black line depicts the mean of a linear regression model with shaded areas indicating the 95% confidence interval. (E) Representative images showing IF staining for indole-3-acetic acid (red) and DAPI (blue) in the cortex of control patients (left), patients who had undergone allo-HCT without GVHD (middle), and patients who had received allo-HCT with GVHD (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus647 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, 100 μm. (F) Scatter dot plot showing quantification of the abundance of indole-3-acetic acid in human cortex of control patients, patients who had received allo-HCT without GVHD, and patients who had received allo-HCT with GVHD, as expressed MFI. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. (G) Representative images showing IF staining for Iba-1 (green), phospho–NF-κB p65 (red), and DAPI (blue) in the cortex of control patients (left), patients who had received allo-HCT without GVHD (middle), and patients who had undergone allo-HCT with GVHD (right). The primary antibody was incubated for 24 hours at 4°C. Goat anti-rabbit IgG (H + L) Alexa Fluor Plus 647 and goat anti-rat IgG (H + L) Alexa Fluor 488 secondary antibodies were 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. (H) Scatter dot plot showing quantification of the expression of phospho–NF-κB p65 in the cortical microglia of control patients, patients who had received allo-HCT without GVHD, and patients who had undergone allo-HCT with GVHD, as expressed FC MFI. Error bars showing mean ± SEM. P values were calculated using ordinary 1-way ANOVA. AUC, area under the curve; Max, maximum.

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