Volume 138, Issue 3, Pages 1055-1067.e4 (March 2010) Apoptosis Signal-Regulating Kinase 1 Regulates Colitis and Colitis-Associated Tumorigenesis by the Innate Immune Responses  Yoku Hayakawa, Yoshihiro Hirata, Hayato Nakagawa, Kei Sakamoto, Yohko Hikiba, Motoyuki Otsuka, Hideaki Ijichi, Tsuneo Ikenoue, Keisuke Tateishi, Masao Akanuma, Keiji Ogura, Haruhiko Yoshida, Hidenori Ichijo, Masao Omata, Shin Maeda  Gastroenterology  Volume 138, Issue 3, Pages 1055-1067.e4 (March 2010) DOI: 10.1053/j.gastro.2009.11.015 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Increased colonic inflammation in ASK1−/− mice treated with 3% DSS. (A) Total colonic lysates were prepared from colons treated with 3% DSS. Lysates were immunoprecipitated with anti-ASK1 antibody and immunoblotted with anti-phospho-ASK1 and anti-ASK1 antibodies. (B) The colonic lysates from DSS-treated WT and ASK1−/− mice were immunoblotted with antibodies directed against the indicated proteins. (C) Relative weight losses during DSS colitis in WT (n = 10) and ASK1−/− (n = 10) mice. (D) Colon lengths after treatment with DSS. *P < .05 compared with WT mice. (E) Representative histologies of WT and ASK1−/− mice after termination of DSS (original magnification ×100). (F) GAPDH-normalized levels of TNF-α, IL-6, and Cox-2 mRNAs in colonic tissues from mice treated with DSS, as determined by real-time PCR analysis (n = 10 per group). Data are plotted as mean ± SE. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 ASK1 is associated with bacteria-induced colitis. (A) Body-weight curves of mice that received DSS plus antibiotics. WT and ASK1−/− mice (n = 7 per group) were administered 3% DSS in the drinking water together with broad-spectrum antibiotics (1.5 g/L neomycin sulfate and 1.5 g/L metronidazole). (B) Histologies of WT and ASK1−/− mice after DSS and antibiotic administration (original magnification ×100). (C) Colonization of C rodentium was assessed by measuring the number of colonies in the fecal samples of WT and ASK1−/− mice at 7 days after infection (n = 5 per group). (D) Typical histologies of WT and ASK1−/− mice at 14 days after infection (original magnification ×100). Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 The ASK1–p38 pathway regulates the bacterial killing ability. (A) BMDMs of WT and ASK1−/− mice infected with live C rodentium for 15 minutes. Total cell lysates were immunoprecipitated with anti-ASK1 antibody and immunoblotted with anti-phospho-ASK1 and anti-ASK1 antibodies. (B) Lysates of BMDMs stimulated with C rodentium were prepared at the indicated time points and immunoblotted with antibodies directed against the indicated proteins. (C) Bacterial killing abilities of BMDMs from WT and ASK1−/− mice. Cells were incubated with C rodentium. After 2- and 4-hour incubation, viable intracellular bacteria were quantitated. The results for the 4-hour time point are expressed as the percentage of killing compared with the 2-hour time point (in triplicate; mean ± SD). *P < .05 compared with WT. (D) Bacterial killing by BMDMs treated with SB203580 (20 μmol/L), SP600125 (20 μmol/L), or vehicle only (in triplicate; mean ± SD). *P < .05 compared with vehicle only. We analyzed 5 independent experiments. Representative data are shown. (E) WT mice treated with SB203580 (20 mg/kg, every 2 days) or vehicle alone were administrated C rodentium (n = 4 per group). Colonization of C rodentium was assessed by measuring the number of colonies in the fecal samples from mice at 7 days after infection. *P < .05 compared with mice treated with vehicle alone. (F) Histologies of treated mice at 14 days after infection. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 ASK1 deficiency promotes macrophage apoptosis. (A) TUNEL-stained colonic sections of C rodentium–infected WT and ASK1−/− mice at 14 days after infection (top) and at 7 days after the initiation of treatment with 3% DSS (bottom). (B) Average number of TUNEL-positive cells from mice treated with C rodentium and DSS (n = 5 per group). *P < .05 compared with WT mice. Data are plotted as mean ± SE. (C) TUNEL staining of colonic tissue sections after 5 days of DSS administration and immunostaining with F4/80-specific antibody. Apoptotic nuclei (green) and F4/80-positive (red) macrophages are presented together. (D) BMDMs from WT and ASK1−/− mice were stimulated with LPS (10 μg/mL) for 24 hours, and the levels of free DNA in the BMDM supernatants were measured. (E) IL-1β levels in the supernatants of LPS-treated WT and ASK1−/− BMDMs, as measured by ELISA. Data are plotted as mean ± SD. *P < .05 compared with the WT BMDMs. (F) Immunoblot analyses of processed IL-1β and activated caspase-1(p20) in the supernatants of LPS-stimulated and unstimulated BMDMs. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 The ASK1–p38 pathway prevents macrophage apoptosis through expression of antiapoptotic genes. (A) The levels of free DNA in the supernatants of WT and ASK1−/− LPS-stimulated BMDMs treated with SB203580 or vehicle were measured 24 hours after stimulation. Data are plotted as mean ± SD. *P < .05 compared with BMDMs treated with vehicle alone. (B–E) GAPDH-normalized mRNA levels of A1/Bfl1 (B), serpin B2 (C), cIAP1 (D), and cIAP2 (E) genes in BMDMs 4 hours after treatment with LPS (10 μg/mL) were determined by real-time PCR analysis (n = 4 per group). Data are plotted as mean ± SE. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 (A) Body-weight curves of bone marrow chimeric mice that received 3% DSS. Fifteen WT mice were transplanted with WT mouse-derived (n = 8) or ASK1−/− mouse-derived (n = 7) bone marrow cells. (B) Colon lengths after treatment with DSS. (C) Histologies of bone marrow chimeric mice after treatment with DSS (original magnification ×100). (D) Histologic scoring of mice treated with DSS. (E) GAPDH-normalized levels of TNF-α, IL-6, and Cox-2 mRNAs in colonic tissues from mice treated with DSS, as determined by real-time PCR analysis. Data are plotted as mean ± SE. *P < .05 compared with WT/WT mice. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 ASK1 deficiency promotes colitis-associated tumorigenesis. (A) Survival curves of WT (n = 21) and ASK1−/− (n = 16) mice in the CAC model (P =.02, log-rank test). (B) Typical examples of macroscopic tumorigenesis in the CAC model. Colons were cut open longitudinally, and the mucosal surface was stained with 1% methylene blue. The numbers (C) and maximum sizes (D) of the tumors in the mice were determined. We performed 2 independent experiments with a total of 31 mice (WT mice, n = 20; ASK1−/− mice, n = 11). *P < .05 compared with WT mice. (E) Typical examples of microscopic tumorigenesis in the CAC model; original magnification ×40. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 ASK1 deficiency has no effects on intestinal epithelial cell proliferation and apoptosis. (A) Immunohistochemical analysis of proliferative cell nuclear antigen and bromodeoxyuridine in non-tumor or tumor epithelium; original magnification ×100. (B) Expression levels of indicated proteins in DLD-1 cells 48 hours after transfection with or without ASK1 siRNA. (C) Numbers of DLD-1 cells counted 24, 48, and 72 hours after transfection. Data are plotted as mean ± SD. Immunohistochemical detection of F4/80 (D) and Cox-2 (E) in tumor sections. Representative images are shown (original magnification ×40; D and E, left and ×400; E, right). Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) Histologic scoring of mice that were treated with DSS (n ≥ 7 per group). (B) Colonic MPO levels in mice treated with or without DSS. *P < .05 compared with WT mice. Data are plotted as mean ± SE. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 (A) The crypt heights of the colons of mice that were treated with C rodentium or untreated were measured microscopically (n = 5 per group). (B) The F4/80-positive cells were counted (n = 5 per group). (C) MPO levels were measured in the colons of WT and ASK1−/− mice at 14 days after infection. (D) Immunofluorescent analysis of F4/80 in colon tissues 14 days after infections. *P < .05 compared with WT mice. Data are plotted as mean ± SE. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 (A) Phagocytic abilities of BMDMs from WT and ASK1−/− mice. Cells were incubated with C rodentium. After 1-hour incubation, viable intracellular bacteria were quantitated. Average of relative bacterial counts are shown (4 independent experiments, in triplicate, mean ± SE). (B) The crypt heights of the colons obtained from mice treated with SB203580 or vehicle at 14 days after infection were measured microscopically. *P < .05 compared with mice treated with vehicle alone. Data are plotted as mean ± SE. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 (A) Average ratio of TUNEL-positive to F4/80-positive macrophages. Data are expressed as percentages of TUNEL-positive plus F4/80-positive cells relative to the total number of F4/80-positive cells (n = 3 per group). *P < .05 compared with WT mice. Data are plotted as mean ± SE. (B) TUNEL staining of BMDMs from WT and ASK1−/− mice stimulated with LPS for 24 hours. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 Analyses of ERK, p38, JNK, and CREB activation, as well as IκBα degradation in the BMDMs of WT and ASK1−/− mice stimulated with LPS (time shown above the lanes). Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 (A) TUNEL staining of the colons of WT and ASK1−/− mice 2 days after DSS (3%) exposure. (B) Average number of TUNEL-positive cells from mice treated with DSS for 2 days (n = 3 per group). Data are plotted as mean ± SE. (C) Average number of PCNA-positive cells per field from mice treated with AOM plus DSS (n = 4 per group). Data are plotted as mean ± SE. (D) Immunoblot analysis for Cox-2 protein in colonic lysates from non-tumor epithelium and tumor of AOM plus DSS-treated WT and ASK1−/− mice. Gastroenterology 2010 138, 1055-1067.e4DOI: (10.1053/j.gastro.2009.11.015) Copyright © 2010 AGA Institute Terms and Conditions