Volume 19, Issue 13, Pages 2756-2770 (June 2017) NOD2 Suppresses Colorectal Tumorigenesis via Downregulation of the TLR Pathways  S.M. Nashir Udden, Lan Peng, Jia-Liang Gan, John M. Shelton, James S. Malter, Lora V. Hooper, Md. Hasan Zaki  Cell Reports  Volume 19, Issue 13, Pages 2756-2770 (June 2017) DOI: 10.1016/j.celrep.2017.05.084 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Nod2-Deficient Mice Are Susceptible to Colorectal Tumorigenesis WT (n = 19) and Nod2−/− (n = 19) mice were treated with AOM (10 mg/kg). After 5 days of DSS administration, mice were treated with 2.5% DSS in drinking water for 5 days. DSS cycle was repeated two more times. Mice were sacrificed on day 80 after tumor induction. (A) Representative image of tumor-bearing colons from WT and Nod2−/− mice. (B) The number of tumors per colon was counted. (C) Size of the tumor was measured. Colon tissues collected at day 80 after tumor induction were stained with H&E. (D) Representative pictures of H&E staining of tumor-bearing colons. Arrows indicate invasive carcinoma. (E) Histopathological analyses for tumor grading of H&E-stained colon tissue sections. (F) Distribution of animals having low-grade dysplasia, high-grade dysplasia, and invasive carcinoma based on histological analysis. Data represent means ± SEM. ∗p < 0.05; ∗∗∗p < 0.0001. See also Figure S1. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 The CRC Susceptibility of Nod2-Deficient Mice Is Independent of Microbial Dysbiosis (A) Genomic DNA was isolated from stools of separately housed WT (n = 20) and Nod2−/− (n = 20) mice. 16S rDNA of the indicated bacteria was analyzed by real-time PCR and normalized to universal bacterial 16S rDNA. (B and C) WT (n = 8) and Nod2−/− (n = 7) mice were co-housed for 4 weeks before treatment with AOM/DSS as described in the Experimental Procedures. Mice were sacrificed on day 80 after tumor induction. (B) Representative image of colon after tumor induction. (C) The number of tumors per colon was counted. (D–F) Littermate (litt) Nod2+/+ (n = 5) and Nod2−/− (n = 5) mice were treated with AOM/DSS at 8 weeks after birth and sacrificed at day 80 following AOM injection. (D) Representative image of the colon after tumor induction. (E) The number of tumors per colon was counted. (F) Representative H&E staining of tumor-bearing colons. Arrows indicate invasive carcinoma. (G–J) GF mice were co-housed with either conventionally raised WT mice (GF-WT) or Nod2−/− mice (GF-Nod2−/−) for 7 days. Two weeks after colonization, colorectal tumorigenesis was induced in mice with an AOM/DSS regimen. (G) Body weight changes were monitored for 10 days following 2.5% DSS administration. Clinical scores for (H) stool consistency and (I) occult bleeding were measured at days 3, 4, and 5 after DSS treatment. (J) Tumor numbers were counted at 80 days after tumor induction. Data represent means ± SEM. ∗p < 0.05; ∗∗p < 0.001. See also Figure S2 and Table S1. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 NOD2 Deficiency Induces Higher Inflammatory Responses in the Colon during Tumorigenesis Colorectal tumorigenesis was induced in WT, Nod2−/−, and Rip2−/− mice with AOM/DSS treatment as described in the Experimental Procedures. Mice were sacrificed on day 80 after tumor induction. (A) RNA was isolated from whole colons, and expression of the indicated genes was analyzed by real-time PCR. (B) Homogenates of whole colon from WT and Nod2−/− mice were analyzed for the activation of ERK, NF-κB, and STAT3 by western blotting. (C) Densitometric analysis of band intensity of western blots as shown in (B). (D) Homogenates of tumor from WT and Nod2−/− mice were analyzed for the activation of ERK, NF-κB, and STAT3 by western blotting. (E) Densitometric analysis of band intensity of western blots as shown in (D). (F) Homogenates of whole colon from WT and Rip2−/− mice were analyzed for the activation of ERK, NF-κB, and STAT3 by western blotting. (G) Densitometric analysis of band intensity of western blots as shown in (F). Data represent means ± SEM. ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001. See also Figure S3 and Table S2. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 NOD2 Suppresses Inflammatory Signaling Pathways during Colitis WT and Nod2−/− mice were allowed to drink 3% DSS for 5 days (d). Mouse colons were collected at days 3, 5, and 10 after DSS administration. Colons collected from healthy mice are designated as day 0. (A) Whole-colon homogenates were analyzed for the activation of ERK, NF-κB, and STAT3 by western blotting. (B) Densitometric analyses of P-p65, P-ERK, and P-STAT3 bands relative to β-actin. (C) mRNA isolated from whole colons was analyzed for inflammatory and tumorigenic mediators by real-time PCR. (D) Representative images of H&E staining of colon sections collected at day 10 following DSS administration. Images were captured at 40× magnification. (E) Histopathological analysis of H&E-stained colon sections collected at day 10 following DSS administration. (F and G) Lamina propria cells were isolated from colons of WT and Nod2−/− mice at day 10 after DSS administration and stained for CD11b, F4/80, and IL-6. (F) Representative image of flow cytometry analysis of lamina propria cells. (G) Frequency of CD11b+F4//80+ and CD11b+IL-6+ cells in the lamina propria of WT and Nod2−/− mice. Data represent means ± SEM. ∗p < 0.05; ∗∗∗p < 0.0001. See also Figure S4 and Table S2. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 NOD2 Negatively Regulates TLR Signaling Pathways (A–E) BMDMs from WT and Nod2−/− mice were stimulated with MDP (10 μg/mL), LPS (1 μg/mL), or poly(I:C) (1 μg/mL). (A) Cell lysates collected at indicated times were analyzed for P-IκB, IκB, P-ERK, ERK, P-p65, and P-STAT3 by western blotting. (B and C) Densitometric analyses of P-IκB and P-ERK bands, relative to IκB and ERK, respectively, in (B) LPS and (C) poly(I:C)-treated cells. Data represent means ± SD of three independent experiments. (D) Cytoplasmic and nuclear fractions were isolated from LPS-treated BMDM cell lysates and analyzed for P-p65 by western blotting. α-tubulin and Lamin b1 were used as loading controls of cytoplasmic and nuclear fractions, respectively. (E) mRNA was isolated from LPS-stimulated WT and Nod2−/− macrophages at indicated times and analyzed for the expression of IL-1β, IL-6, and IL-12p40. (F and G) WT and Nod2−/− BMDCs were stimulated with MDP (10 μg/mL), LPS (1 μg/mL), or poly(I:C) (1 μg/mL). (F) Western blot analysis of P-IκB, IκB, P-ERK, ERK, P-p65, and P-STAT3 in MDP-, LPS-, and poly(I:C)-stimulated BMDCs. (G) mRNA was isolated at 0, 2, and 4 hr after stimulation with LPS and analyzed for the expression of IL-1β, IL-6, and IL-12p40 by real-time PCR. Data represent means ± SEM. ∗p < 0.05; ∗∗p < 0.001. (H) WT and Rip2−/− BMDMs were stimulated with LPS (1 μg/mL), and the cell lysates were analyzed for P-IκB, IκB, P-ERK, ERK, P-p65, and P-STAT3 by western blotting. See also Figure S5 and Table S2. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 NOD2 Downregulates TLR-Induced NF-κB Activation via Induction of IRF4 (A–F) WT and Nod2−/− BMDMs were stimulated with MDP (10 μg/mL) or LPS (1 μg/mL). Expression of IRF4 was measured by real-time RT-PCR (A and D) and western blotting (B and E) at indicated times. (C and F) IRF4 band intensity relative to β-actin was analyzed. Data represent means ± SD of three independent experiments. ∗∗p < 0.001; ∗∗∗p < 0.0001. (G–I) WT and Nod2−/− BMDCs were stimulated with LPS (1 μg/mL). Expression of IRF4 was measured by (G) real-time RT-PCR and (H) western blotting at indicated times. (I) Densitometric analysis of IRF4 band intensity relative to β-actin. Data represent means ± SD of three independent experiments. ∗p < 0.05; ∗∗p < 0.001. (J–L) Nod2 was cloned into pcDNA4/TO vector and transfected into RAW264.7 cells. Nod2- or GFP (mock)-transfected cells were stimulated with LPS. (J) The cell lysates collected at the indicated times were analyzed for P-ERK, ERK, P-p65, and IRF4 by western blotting. Expression of Nod2 was measured by anti-Nod2 and anti-FLAG antibodies. (K) Densitometric analysis of IRF4 and P-p65 band intensities relative to β-actin. Data represent means ± SD of three independent experiments. ∗p < 0.05; ∗∗p < 0.001. (L) Nod2- or GFP-transfected RAW264.7 cells were stimulated with LPS (1 μg/mL) or MDP (10 μg/mL) for 4 hr. mRNA was isolated and analyzed for the expression of IRF4, IL-6, Nod2 by real-time PCR. Data represent means ± SD of triplicate wells. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (M) Expression of IRF4 was measured in the colons of WT and Nod2−/− mice at different days after DSS (3%) administration. (N and O) Colon lysates collected at day 0, day 3, and day 10 following DSS administration were analyzed for IRF4 by western blotting. (O) Densitometric analysis of IRF4 band relative to β-actin. Data represent means ± SEM. ∗p < 0.05; ∗∗p < 0.001. See also Figure S6 and Table S2. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Increased IL-6 Production in Nod2-Deficient Mouse Colon Leads to Hyperproliferation of Tumor Epithelium (A–D) Colorectal tumorigenesis was induced in WT and Nod2−/− mice with AOM/DSS treatment as described in the Experimental Procedures. Colons collected at day 80 after tumor induction were processed for immunostaining of Ki67. (A) Representative images of Ki67 staining in non-tumor and tumor area of colon sections. (B) Ki67+ cells were counted in tumor and non-tumor tissue under 20× microscopic lenses (n = 3 mice per group). (C) mRNA was isolated from whole colons and analyzed for Ki67 by real-time PCR (n = 8 per group). Data represent means ± SEM. ∗∗p < 0.001. (D) MODE-K cells were stimulated with IL-6 (20 ng/mL), and the expression of Ki67 mRNA was analyzed by real-time PCR. Data represent means ± SD. ∗p < 0.05; ∗∗p < 0.001. (E) The cell lysates from IL-6-stimulated MODE-K cells were analyzed for P-STAT3, P-p65, P-ERK, and ERK by western blotting. (F) Proposed pathway for NOD2-mediated regulation of inflammation and tumorigenesis in the colon. See also Figure S7 and Table S2. Cell Reports 2017 19, 2756-2770DOI: (10.1016/j.celrep.2017.05.084) Copyright © 2017 The Author(s) Terms and Conditions