FICZ rescues activation of the microglia upon antibiotic treatment in vivo and improves cognitive function and GVHD without impairing the antitumor efficacies. (A) 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 antibiotics + vehicle (upper) or antibiotics + FICZ (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. (B) Scatter dot plot showing IMARIS-based quantification (FC MFI) of phospho–NF-κB p65 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 either 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. (C) Scatter dot plot showing IMARIS-based quantification (FC MFI) of phospho–NF-κB p65 nuclear 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. (D) 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 antibiotics + vehicle (upper) or antibiotics + FICZ (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. (E) Scatter dot plot showing IMARIS-based quantification (FC MFI) of 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 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. (F) Schematic overview of the murine model. C57BL/6 mice were treated with either vehicle or antibiotics and additionally with either vehicle (for FICZ) or FICZ for 14 days before and 14 days after transplantation. C57BL/6 mice were lethally irradiated and transplanted with allo-BM (5 × 10⁶ cells) and T cells (5 × 10⁵ cells) from BALB/c donor mice or did not receive a transplant (naïve). Behavior studies were performed on day 14 after transplantation. (G) Scatter dot plot showing the number of entries into open arm (%) made by naïve (nontransplanted) mice treated with antibiotics or vehicle and GVHD mice transplanted with allo-BM and T cells and treated with antibiotics as indicated in the elevated plus maze test. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P value was calculated using ordinary 1-way ANOVA. (H) Scatter dot plot showing the number of entries into open arm (%) made by GVHD mice transplanted with allo-BM and T cells and treated with antibiotics + vehicle or antibiotics + FICZ as indicated in the elevated plus maze test. Experiment was performed twice and results were pooled. Dots represent individual mice. Error bars showing mean ± SEM. P value was calculated using unpaired t test. (I) Schematic overview of the murine model. BALB/c mice were treated with antibiotics and either vehicle or FICZ for 14 days before and 14 days after transplantation. BALB/c mice were lethally irradiated and transplanted with allogeneic BM (5 × 10⁶ cells) and T cells (3 × 10⁵ cells) from C57BL/6 donor mice. Organs were analyzed on day 14 after transplantation. (J-L) Scatter dot plots showing GVHD pathology scores in the colon (J), small intestine (K), and liver (L) 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. (M) Schematic overview of the murine model. C57BL/6 mice were treated with either antibiotics or vehicle and additionally with either vehicle or FICZ for 14 days before and 14 days after transplantation. C57BL/6 mice were lethally irradiated and transplanted with allogeneic BM (5 × 10⁶ cells, BALB/c) and FLT3-ITD-MLL-PTD (acute myeloid leukemia [AML]) cells (5000 cells) from C57BL/6 background. On day 2, 5 × 10⁵ T cells were injected into allogeneic BALB/c donor mice. (N) Survival rates of C57BL/6 mice transplanted with AML (FLT3-ITD/MLL-PTD) cells and BALB/c (wild-type) BM and allogeneic T cells or no T cells. Mice were treated with antibiotics + vehicle, antibiotics + FICZ, or vehicle (no antibiotics) + vehicle as indicated. Experiment was performed twice and results were pooled. Tc, T cells.