Volume 129, Issue 3, Pages 913-927 (September 2005) CpG Motifs of Bacterial DNA Essentially Contribute to the Perpetuation of Chronic Intestinal Inflammation  Florian Obermeier, Nadja Dunger, Ulrike G. Strauch, Claudia Hofmann, Andre Bleich, Nicole Grunwald, Hans J. Hedrich, Elisabeth Aschenbrenner, Brigitte Schlegelberger, Gerhard Rogler, Jürgen Schölmerich, Werner Falk  Gastroenterology  Volume 129, Issue 3, Pages 913-927 (September 2005) DOI: 10.1053/j.gastro.2005.06.061 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 TLR9 mRNA detection in representative colonic sections of healthy mice and mice with chronic DSS-induced colitis. Hybridization with a sense probe and colonic tissue of a TLR9-deficient mouse served as negative controls. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 The role of TLR9 in chronic DSS-induced colitis. TLR9-deficient mice or wild-type littermates were used for chronic DSS colitis induction. Following induction of chronic colitis, mice were killed after 8 weeks. (A) The histologic score was determined as described in the Materials and Methods section. Data points were derived from 5 mice per group from 2 independent experiments and represent mean ± SEM; *significantly different from control group. (B) Colonic cytokine mRNA was quantified both from healthy (no colitis) and colitic TLR9-deficient mice and littermate controls using a Light Cycler (Roche, Molecular Systems). Data presented were derived from at least 5 mice per group. The result is representative of 2 independent experiments. Bars represent mean ± SEM. (C) Cytokine secretion from pooled mesenteral lymph node cells of both healthy (no colitis) and colitic TLR9-deficient mice and littermate controls. The result is representative of 2 independent experiments. Bars represent mean ± SEM. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effect of AV-ODN on cytokine secretion from CpG-ODN-stimulated mesenteral lymph node cells. MLCs were isolated from wt Balb/c (left panels) or TLR9-deficient (right panels) mice and incubated with CpG-ODN, AV-ODN, GpG-ODN control, or CpG-ODN plus AV-ODN (each at 10 μg/mL) and incubated in quadruplicate cultures for 48 hours. Supernatants were removed for (A) IFN-γ, (B) IL-10, and (C) TGF-β detection by ELISA. Bars represent mean ± SEM; **significantly different with P < .0001. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effect of AV-ODN on STAT1 and STAT3 phosphorylation and NF-κB activity from CpG-ODN-stimulated mesenteral lymph node cells. MLC were isolated from Balb/c mice and left unstimulated or incubated with CpG-ODN, AV-ODN, GpG-ODN control, or CpG-ODN plus AV-ODN (each at 10 μg/mL) and incubated for 4 hours to detect STAT1α (91 kilodaltons, upper signal) and β (86 kilodaltons, lower signal) and STAT3α (86 kilodaltons, upper signal) and β (79 kilodaltons, lower signal) phosphorylation (A) and for 12 hours to measure NF-κB activation (B). Cells were lysed, and protein or nuclear extracts were prepared and used for Western blotting (A) or ELISA measurement (B) as described in the Materials and Methods section. Bars represent mean ± SEM; *significantly different. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of a systemic AV-ODN treatment on histologic parameters on colonic cytokine expression and cytokine secretion of mesenteral lymph node cells in chronic DSS-induced colitis. Mice with chronic DSS-induced colitis received either AV-ODN-1 (10 μg or 2 μg) or GpG-ODN (control) per mouse and day for 5 days intraperitoneally. (A) The histologic score was determined as described in the Materials and Methods section. (B) Representative H&E-stained colonic sections of AV-ODN-treated and control mice. (C) Relative quantification of specific colonic mRNA. (D) Cytokine secretion from pooled mesenteral lymph node cells. Data points were derived from 10 mice per group from 2 independent experiments and represent mean ± SEM; *significantly different from control groups. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Effect of a systemic AV-ODN treatment on histologic parameters on colonic cytokine expression and cytokine secretion of mesenteral lymph node cells in chronic DSS-induced colitis. Mice with chronic DSS-induced colitis received either AV-ODN-1 (10 μg or 2 μg) or GpG-ODN (control) per mouse and day for 5 days intraperitoneally. (A) The histologic score was determined as described in the Materials and Methods section. (B) Representative H&E-stained colonic sections of AV-ODN-treated and control mice. (C) Relative quantification of specific colonic mRNA. (D) Cytokine secretion from pooled mesenteral lymph node cells. Data points were derived from 10 mice per group from 2 independent experiments and represent mean ± SEM; *significantly different from control groups. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Effect of an oral application of different AV-ODN on histologic parameters and cytokine secretion of mesenteral lymph node cells in chronic DSS-induced colitis. Mice with chronic DSS-induced colitis received either AV-ODN-1 (100 μg or 10 μg), AV-ODN-2 (100 μg), GpG-ODN (control, 100 μg), or PBS (control) per mouse over 2 weeks 3 times a week. (A) The histologic score was determined as described in the Materials and Methods section. (B) Cytokine secretion from pooled mesenteral lymph node cells. Data points were derived from at least 7 mice per group from 2 independent experiments and represent mean ± SEM; *significantly different from control groups. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 The role of IL-10 in AV-ODN-treated mice with chronic colitis. (A) Mice with chronic DSS-induced colitis received either rat IgG (100 μg), neutralizing anti-IL10 mAb (100 μg), AV-ODN (10 μg), GpG-ODN (control; 10 μg), or neutralizing anti-IL10 mAb (100 μg) plus AV-ODN (10 μg) for 5 days. The histologic score was determined as described in the Materials and Methods section. Each group consisted of 5–10 mice. The result is representative of 2 separate experiments. Bars represent mean ± SEM; *significantly different. (B) IL-10-deficient mice (>18 weeks old) received either AV-ODN (10 μg), GpG-ODN (control; 10 μg), or PBS for 5 days. The histologic score was determined as described in the Materials and Methods section, and IFN-γ secretion from mesenteric lymph node cells was detected by ELISA. Each group consisted of at least 5 mice from 2 independent experiments. Bars represent mean ± SEM; *significantly different. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Effect of AV-ODN on histologic parameters and cytokine secretion from mesenteral lymph node cells in the SCID-transfer colitis. At the onset of clinical symptoms (4 weeks after transfer), SCID recipients were treated with AV-ODN (10 μg) or GpG-ODN (10 μg, control) per mouse 3 times a week or left untreated. Mice were killed at week 6 after transfer. (A) The histologic score was determined as described in the Materials and Methods section. (B) Cells from pooled mesenteral lymph nodes were isolated and incubated in quadruplicate cultures for 24 hours. Supernatants were used for cytokine measurement by ELISA. Each group consisted of 5–10 mice. The result is representative of 2 independent experiments. Bars represent mean ± SEM; *significantly different. Gastroenterology 2005 129, 913-927DOI: (10.1053/j.gastro.2005.06.061) Copyright © 2005 American Gastroenterological Association Terms and Conditions