Microglia display reactive phenotype upon antibiotic treatment in GVHD mice. (A) Heat map based on RNA sequencing depicts differentially expressed genes involved in TNF-α signaling via NF-κB pathway isolated from the microglia of syn-HCT (n = 4) or allo-HCT mice (n = 4) on day 14. BALB/c mice were transplanted with either syngeneic BM and T cells (syn-HCT) or allogeneic BM and T cells (allo-HCT). z score intensity (upper part) and log2 fold change (FC; lower part). Asterisks (∗) indicate significant changes (adjusted P < .05). (B) Representative images showing IF staining for Iba-1 (green), phospho–NF-κB p65 (red), and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with vehicle (upper panel) or antibiotics (lower panel). 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, 7 μm. (C) Scatter dot plot showing IMARIS-based quantification (FC mean fluorescence intensity [MFI]) of phospho–NF-κB p65 in the microglia. The quantification was based on a semiautomated 2-dimensional (2D) reconstruction of Iba-1⁺ cells. GVHD mice 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. (D) Scatter dot plot showing IMARIS-based quantification (FC MFI) of the nuclear phospho–NF-κB p65 expression in the microglia. The quantification was based on a semiautomated 2D reconstruction of Iba-1⁺ cells and DAPI for nuclei. GVHD mice 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. (E) Representative images showing IF staining for Iba-1 (green), IκBα (red), and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with vehicle (upper) or antibiotics (lower). 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, 7 μm. (F) Scatter dot plot showing IMARIS-based quantification (FC MFI) of the expression of IκBα. The quantification was based on a semiautomated 2D reconstruction of Iba-1⁺ cells. GVHD mice 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. (G) Scatter dot plot showing percentage of IκBα expressing microglia. GVHD mice were transplanted with allogeneic BM and T cells and treated with vehicle or antibiotics as indicated. Dots represent individual mice. Error bars showing mean ± SEM. P values were calculated using unpaired t test. (H) Representative images showing IF staining for Iba-1 (green), phospho-Src (red), and DAPI (blue) in the cortex of GVHD mice transplanted with allogeneic BM and T cells and treated with vehicle (upper) or antibiotics (lower). 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, 7 μm. (I) Scatter dot plot showing IMARIS-based quantification (FC MFI) of the phospho-Src expression in the microglia. The quantification was based on a semiautomated 2D reconstruction of Iba-1⁺ cells. GVHD mice 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. TNF-α, tumor necrosis factor α.