Volume 141, Issue 5, Pages 1887-1896.e2 (November 2011) Loss of Interleukin-10 or Transforming Growth Factor β Signaling in the Human Colon Initiates a T-Helper 1 Response Via Distinct Pathways  Anne Jarry, Céline Bossard, Guillaume Sarrabayrouse, Jean–François Mosnier, Christian L. Laboisse  Gastroenterology  Volume 141, Issue 5, Pages 1887-1896.e2 (November 2011) DOI: 10.1053/j.gastro.2011.08.002 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 IL-18, IL-12, and IFN-γ production in relation with IL-10 or TGF-β depletion. Mucosa explant cultures (at least 3 per condition) were incubated with neutralizing anti–IL-10 (1 μg/mL) or anti–TGF-βRII (5 μg/mL) antibodies or with appropriate isotype immunoglobulin (control) for 48 hours. Secretion of (A) IFN-γ, (B) mature IL-12p70, and (C) mature IL-18 was measured by ELISA in the culture supernatants. Horizontal lines represent mean values of 3 different experiments. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Time course of IL-18 production in relation with IFN-γ production on IL-10 depletion. Mucosa explant cultures were incubated with neutralizing anti–IL-10 antibodies or with the isotype immunoglobulin (control) for 24 or 48 hours. Mature (A) IL-18 and (B) IFN-γ secretion were measured by ELISA. Horizontal lines represent mean values of 2 separate kinetics performed at least in quadruplicate. (C and D) IL-18 mRNA were quantified by quantitative reverse-transcriptase PCR and expressed relative to β-actin levels in 48-hour explant cultures treated with (C) anti–IL-10 or (D) anti–TGF-βRII antibodies. Horizontal lines represent mean values of 3 to 4 different experiments, performed in triplicate or quadruplicate. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 The IFN-γ response is IL-18/caspase-1 dependent on IL-10 depletion. Mucosa explant cultures were treated with anti–IL-10 antibodies for 48 hours in the presence or absence of the pan-caspase inhibitor ZVAD (10 μmol/L), the specific caspase-1 inhibitor YVAD-FMK (3 μmol/L), the IL-18 inhibitor IL-18-BP (800 ng/mL), or the IL-1 inhibitor IL-1ra (100 ng/mL). (A and C) IFN-γ secretion was measured by ELISA in the culture supernatants, and (B) IFN-γ mRNA levels were quantified by quantitative reverse-transcriptase PCR. Results are expressed as percentage of IFN-γ level of explant cultures treated with anti–IL-10 only. Mean ± SEM of 3 separate experiments performed in triplicate. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 IL-10 depletion in colonic mucosa explant cultures leads to caspase-1 activation in intestinal epithelial cells and lamina propria macrophages. Mucosa explant cultures were treated or not with anti–IL-10 for 24 hours. (A) Double immunostaining of active caspase-1 (caspase-1 p10) (green) and CD68 (macrophages, red); nuclei appear in blue. Caspase-1 p10 is present in some lamina propria CD68+ macrophages (overlay images). In addition, caspase-1 p10 is present in some epithelial cells in anti–IL-10–treated explants but not in control explants. Arrows show caspase-1 p10–positive epithelial cells, and the inset shows a higher magnification of the circled epithelial cells. (B) Under IL-10 depletion, intestinal epithelial cells coexpress active caspase-1 (green) and IL-18 (red). Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 The IFN-γ response is dependent on IL-12 production in colonic mucosa explant cultures on TGF-β depletion. (A) IL-12p70 and (B) IFN-γ secretion were measured by ELISA in explant cultures (24 or 48 hours). Horizontal lines represent mean values of 2 separate kinetics performed at least in quadruplicate. (C) IL-12p35 or (D) IFN-γ mRNA levels were quantified by quantitative reverse-transcriptase PCR and expressed relative to β-actin levels. Horizontal lines represent mean values of 2 different experiments performed in triplicate or quadruplicate. (E) Effect of neutralizing anti–IL-12 antibodies on IFN-γ mRNA levels in 48-hour explant cultures. Horizontal lines represent mean values of 3 different experiments performed in triplicate. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Flow cytometry analysis of IL-12–positive cells in human peripheral blood monocytes. IL-12p40/p70 and IL-12p35/p70 production by peripheral blood monocytes on stimulation with IFN-γ and lipopolysaccharide, measured by intracellular cytokine labeling. In each histogram plot, the line indicates the percentage of IL-12–positive monocytes in the stimulated condition (solid line) compared with the nonstimulated condition (dashed line). Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Flow cytometry analysis of IL-12–positive cells in human colonic mucosa explant cultures. Cell suspensions, obtained after enzymatic dissociation of 24-hour explant cultures (TGF-β depleted or control cultures), were stained with anti–IL-12p40/p70 and one of the indicated phenotypic markers. (A and C) The dot plots shown are representative of experiments performed in 3 independent explant cultures after CD45 or CD3 membrane staining and intracellular IL-12p40/p70 staining. Numbers within the dot plots represent the percentage of IL-12–positive CD45+ cells. (B) The table shows the effects of TGF-β depletion on IL-12 intracellular production by the immune cell subsets. Results are expressed as fold increase percentage of double-positive cells or mean fluorescence intensity (MFI) for a given marker. Mean ± SEM of 3 independent experiments. (D) Cells were stained with ViViD and then with the membrane epithelial marker EpCAM and with IL-12. Numbers within the dot plots represent the percentage of IL-12–positive EpCAM-positive cells in gated ViViD-negative viable cells. These dot plots are representative of 3 independent experiments. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Phenotype of IFN-γ–positive cells in TGF-β–depleted explant cultures. Multi-color flow cytometry was performed on cell suspensions of TGF-β–depleted explant cultures (48h), as described in Materials and Methods. The CD3+-positive cells were assessed in gated ViViD-negative live cells. IFN-γ was assessed in the gated CD3+ cells, and then the percentage of IFN-γ–positive CD4+ and CD8α+ cells was determined. The IFN-γ–positive cells were only found within the CD3+ cell population. The table represents the mean ± SEM of 3 separate experiments. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 IL-17 and IL-23 production in relation to TGF-β or IL-10 depletion. Mucosa explant cultures were treated or not for 48 hours with anti–TGF-β or anti–IL-10. (A) IL-17 or (B) IL-23 secretion was measured by ELISA. Horizontal lines represent mean values of 5 experiments performed in quadruplicate. Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 IFN-γ and IL-17 production in mucosa explant cultures from active Crohn's disease. Mucosa explant cultures were maintained for 48 hours in standard medium. (A) IFN- γ or (B) IL-17 secretion was assessed by ELISA in the culture supernatants. Horizontal lines represent mean values of explant cultures from 8 different patients (at least 4 explants per patient). Gastroenterology 2011 141, 1887-1896.e2DOI: (10.1053/j.gastro.2011.08.002) Copyright © 2011 AGA Institute Terms and Conditions