Volume 141, Issue 2, Pages 642-652.e4 (August 2011) Mycobacterium bovis Bacillus Calmette-Guérin Killed by Extended Freeze-Drying Reduces Colitis in Mice  Micheline Lagranderie, Christoph Kluge, Helene Kiefer–Biasizzo, Mohammad Abolhassani, Marie–Anne Nahori, Catherine Fitting, Michel Huerre, Antonio Bandeira, Herve Bercovier, Gilles Marchal  Gastroenterology  Volume 141, Issue 2, Pages 642-652.e4 (August 2011) DOI: 10.1053/j.gastro.2011.05.002 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Prophylactic EFD BCG treatment of C57Bl/6 mice reduced inflammation in acute DSS-induced colitis. (A) Body weight loss and clinical scores in PBS- and EFD BCG–treated mice. (B) Colon length and (C) representative H&E-stained colon sections showing inflammatory infiltrate and loss of normal architecture in PBS-treated mice and few inflammatory cells in EFD BCG–treated mice. Original magnification 4× (upper panel) and 10× (lower panel). (D) Foxp3 expression on gated CD4+ T cells from the spleen, MLNs, and lamina propria on day 5 (upper panel) and day 10 (lower panel) after DSS exposure. (E) Absolute number of CD4+Foxp3+ cells in the spleen, MLNs, and lamina propria on days 5 and 10. Data are representative of 2 independent experiments. n = 6 to 8 mice per group. *P < .05, **P < .01, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Therapeutic EFD BCG treatment of C57Bl/6 mice reduced inflammation. (A and B) Body weight loss in acute and chronic model of DSS-induced colitis. (C) Inflammatory cytokines in spleen cell supernatants. (D) NF-κB and (E) PPAR-γ transcription factor levels determined in nuclear extracts of individual colon cells. Acute colitis, left panel; chronic colitis, right panel. n = 6 mice per group. *P < .05, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Antibodies against TGF-β, IL-10, and CD25 blocked the protective effect of EFD BCG. Prophylactic EFD BCG treatment was administered in an acute model of DSS-induced colitis, and blocking antibodies and an isotype control were injected 2 days after the start of DSS treatment. (A) Body weight loss. (B) MPO activity expressed as arbitrary units per milligram of colon. (C) IL-10, TGF-β, and IL-17 levels in individual serum and (D) transcripts from 2 pools of 3 colons expressed as ratio versus GADPH. (E) Foxp3, T-bet, GATA-3, and RORγt expression in the spleen after injection of blocking antibodies. Transcription factors were analyzed by Western blotting and expressed as ratios of the normalized intensities of the bands (target proteins/β-actin). For each transcription factor, we analyzed 2 pools of spleen cell extracts from 3 mice. Black bars represent the isotype control. Data are representative of 2 independent experiments. n = 6 mice per group. **P < .01, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Prophylactic EFD BCG treatment reduced Th2-type inflammation. (A) Weight loss measured from 8 days before oxazolone (OXA) sensitization to day 4 after intrarectal OXA challenge. (B) Colon length and distal lesions (insets) in PBS- and EFD BCG–treated mice. (C) Representative H&E-stained colon sections showing necrotic lesion with bleeding 4 days after OXA challenge in PBS-treated mice and only mild inflammation in EFD BCG–treated mice. Original magnification 10×, white arrow showing hemorrhage. (D) Cytokine levels in the individual serum. *P < .05, **P < .01, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 EFD BCG treatment at the time of naïve CD4+ T cells transfer to RAG2−/− mice reduced colitis features. Naïve CD4+CD45highFoxp3− (3 × 105) T cells were transferred to RAG2−/− mice at the time of PBS or EFD BCG treatment. (A) Body weight loss, (B) colon length, and MPO activity in the colon (arbitrary units/mg) were measured on day 50. (C) Representative H&E-stained colon sections showing inflammatory loss of normal architecture and ulceration in PBS-treated mice and few inflammatory cells in EFD BCG–treated mice. Original magnification 10×. (D) Cytokine levels in the individual serum. (E) NF-κB and PPAR-γ levels measured in individual colon cell extracts and RXRα phosphorylation measured by Western blotting in 2 pools of 3 colons. Data are representative of 2 independent experiments. n = 6 mice per group. *P < .05, **P < .01, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 EFD BCG treatment converted CD4+Foxp3− cells into CD4+Foxp3+ cells in RAG2−/− mice. (A) Fluorescence-activated cell sorter analysis of CD4+Foxp3 GFP+ spleen cells. (B) Foxp3 expression analyzed by Western blotting in spleen and colon extracts. (C) Transcription factors analyzed by Western blotting at day 50 in 2 pools of 3 spleen cell extracts and expressed as ratios of the normalized intensities of the bands (target proteins/β-actin). Data are representative of 2 independent experiments. n = 6 mice per group. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Isotype control (rat immunoglobulin G2a). No significant positive cells stained with Foxp3 isotype control were found in the gate of CD4+Foxp3+ cells shown in Figure 1. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 EFD BCG treatment reduced the number of inflammatory cells in the gut in DSS-induced colitis. (A) Total number of cells and (B) absolute number of CD4+ T cells in the spleen, MLNs, and lamina propria 5 and 10 days after DSS treatment. n = 8 mice per group. *P < .05, **P < .01, ***P < .001 Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 CD4+Foxp3+ cells isolated from EFD BCG–treated mice produced large amounts of IL-10. (A) Fluorescence-activated cell sorter analysis of cells producing IL-10 isolated 10 days after DSS treatment from the MLNs and lamina propria, and stimulated in vitro with PMA/ionomycin. (B) Absolute number of CD4+Foxp3+ cells producing IL-10. (C) Relative expression levels of IL-10 and TGF-β in Foxp3GFP+ cells isolated from MLNs of PBS- and EFD BCG–treated mice 10 days after DSS treatment. n= 5 to 8 mice per group. **P < .01, ***P < .001, Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 4 EFD BCG treatment had no effect on the development of colon cancer. OF1 Swiss mice were treated with PBS or EFD BCG and were then submitted to 3 cycles of treatment with 5% DSS 21 days later. (A) Clinical scores after the last DSS cycle. (B) NF-κB and PPAR-γ transcription factor levels determined in nuclear extracts from individual colons. (C) Colon length and (D) indices of ulceration or erosion, polynuclear infiltration, and dysplasia. n = 25 mice per group. *P < .05, **P < .01, ***P < .001. Gastroenterology 2011 141, 642-652.e4DOI: (10.1053/j.gastro.2011.05.002) Copyright © 2011 AGA Institute Terms and Conditions