1. Volume 132, Issue 1, Pages 190-207 (January 2007)
Interleukin-10 Blocked Endoplasmic Reticulum Stress in Intestinal Epithelial Cells: Impact on Chronic Inflammation 
Anna Shkoda, Pedro A. Ruiz, Hannelore Daniel, Sandra C. Kim, Gerhard Rogler, R. Balfour Sartor, Dirk Haller 
Gastroenterology 
Volume 132, Issue 1, Pages (January 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Proteome analysis in IEC from E faecalis-monoassociated IL-10−/− mice. Germ-free IL-10−/− mice were monoassociated with the colitogenic E faecalis for 14 weeks. Primary IEC were purified from the large intestine (cecum + colon), and 2D SDS-PAGE was performed. The reference gel (big gel) was generated from pooled IEC samples of germ-free mice and then separately compared with each of the 5 IEC samples from E faecalis-monoassociated IL-10−/− mice (small gels). Identified protein spots with at least 2-fold changes in steady state expression levels confirmed in at least 3 out of the 5 mice were considered as significant. Protein spots 1–5 were up-regulated, and protein spots 6–14 were down-regulated.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
(A–C) Validation of grp-78 expression changes in primary IEC from E faecalis-monoassociated WT vs IL-10−/− mice. Proteome, Western blot, and RT-PCR analyses were performed from pooled IEC samples of the 5 E faecalis-monoassociated WT and IL-10−/− mice. (A) 2D SDS-PAGE was performed with 500 μg of total protein, and grp-78 protein spots were identified by MALDI-TOF MS. Expression intensities were given by ProteomeWeaver software. (B) Western blot analysis was performed with 50 μg of total protein using specific antibodies for grp-78 and β-actin. The expression intensities were calculated relative to β-actin using densitometric analysis. (C) Total RNA was extracted from primary IEC and reverse transcribed. Real-time PCR was performed using the Light Cycler system with specific primers for grp-78 and GAPDH. Expression changes (fold changes) of the specific mRNA were calculated relative to germ-free WT and IL-10−/− mice, using the crossing point of the log-linear portion of the amplification curve after normalization with GAPDH. The data represent mean fold changes ± SD from 5 different IEC samples.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
(A and B) Differential grp-78 protein expression in human IEC from noninflamed vs inflamed tissue sections under conditions of chronic intestinal inflammation. (A) Primary IEC were isolated from surgical specimens of 6 patients with active Crohn’s disease, ulcerative colitis, and sigmoid diverticulitis. Tissue specimens from 6 colon cancer patients were used as noninflamed controls. Western blot analysis was performed with 50 μg of total pooled protein using specific antibodies for grp-78 and β-actin. (B) H&E staining of paraffin-embedded tissue sections including noninflamed (left picture) vs inflamed regions (right picture) from 1 single patient with ulcerative colitis. Western blot analysis from IEC of noninflamed vs inflamed tissue regions was performed with 50 μg of total pooled protein using specific antibodies for grp-78 and β-actin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
(A and B) Purity of IEC isolations from murine and human intestinal sections. Western blot analysis was performed from primary IEC and leukocytes with 50 μg of total pooled protein using specific antibodies for CD3 and β-actin. (A) IEC from 5 germ-free and E faecalis-monoassociated WT as well as IL-10−/− mice. (B) IEC from 6 IBD and control patients.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
(A and B) IL-10 triggers p38 and STAT-3 phosphorylation in IL-10R reconstituted Mode-K cells. Mode-K cells were reconstituted with the murine IL-10R complex followed by stimulation with IL-10 at various concentrations (A) and times (B). Western blot analysis was performed with 20 μg of total protein using specific antibodies for phospho-p38, p38, phospho-STAT-3, and STAT3. Representative gels from at least 2 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
(A and B) IL-10 inhibits grp-78 mRNA and protein expression. Mode-K cells were reconstituted with the murine IL-10R complex followed by stimulation for 3, 6, 12, and 24 hours with IL-10 (50 ng/mL). (A) Western blot analysis was performed with 20 μg of total protein using specific antibodies for grp-78 and β-actin. Representative gels from at least 2 different experiments are shown. (B) Total RNA was extracted and reverse transcribed. Real-time PCR was performed using the Light Cycler system with specific primers for grp-78 and GAPDH. The expression changes (fold decrease) of the specific mRNA were calculated relative to unstimulated control cells, using the crossing point of the log-linear portion of the amplification curve after normalization with GAPDH. The data represent mean fold decrease ± SD from triplicate stimulations confirmed in at least 2 independent experiments. #Significant changes were indicated.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
(A and B) Induction of p38 phosphorylation in primary IEC from WT but not IL-10−/− mice after the colonization with E faecalis. Germ-free WT and IL-10−/− mice were monoassociated for 14 weeks with E faecalis. Western blot analysis was performed with 50 μg total protein derived from pooled IEC samples from 5 germ-free and E faecalis-monoassociated mice, using immunoreactive (A) phospho-p38, p38, phospho-STAT3, STAT3, and IL-10R and (B) phosphor-RelA, RelA, cleaved caspase-3, E-cadherin, β-actin antibodies. Representative gels from at least 2 different immunoblots are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
(A and B) Grp-78 knockdown inhibits TNF-induced RelA phosphorylation in Mode-K cells. (A) Mode-K cells were transfected for 72 hours with control siRNA followed by stimulation for 1 and 2 hours with TNF (10 ng/mL). (B) Mode-K cells were transfected with grp-78-specific as well as control siRNA for 78 hours followed by stimulation for 1 hour with TNF (10 ng/mL). Western blot analysis was performed with 20 μg of total protein using specific antibodies for phosphor-RelA, RelA, and grp-78. Representative gels from at least 2 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

10. Figure 9
(A and B) IL-10 inhibits TNF-induced grp-78 recruitment and TNF-induced RelA phosphorylation in the IKK complex of IL-10R reconstituted Mode-K cells. (A) Mode-K cells were reconstituted with the murine IL-10R complex followed by the stimulation for 20, 40, 60, and 90 minutes with TNF (10 ng/mL). (B) IL-10R reconstituted cells were pretreated for 24 hours with IL-10 (10 ng/mL) followed by stimulation for 20 and 60 minutes with TNF. Coimmunoprecipitation experiments were performed using anti-IKKα antibodies followed by Western blot analysis with specific antibodies for p-RelA, RelA, IKKα, and grp-78. Representative gels from at least 2 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

11. Figure 10
(A–C) TNF triggers relocation of grp-78 from the ER to the cytoplasmic compartment. IL-10R reconstituted Mode-K cells were stimulated with TNF (10 ng/mL) for various times. (A) Western blot analysis was performed with 20 μg of total protein using immunoreactive grp-78 and β-actin antibodies. (B) Cellular fractions from the mitochondrial (MC), ER, and cytoplasmic compartment (CP). Western blot analysis was performed with 20 μg of total acetone precipitated protein using immunoreactive grp-78, creatine kinase, and β-actin antibodies. (C) Immunofluorescence analysis was performed using DAPI for nuclear (blue) and rhodamine-coupled antibodies for grp-78-specific staining (red). Overlay pictures are shown in the lower row. The cells were stimulated with TNF for 1 hour. Representative gels from at least 2 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

12. Figure 11
(A–D) IL-10-mediated p38 signaling inhibits TNF-induced ATF-6 recruitment to the grp-78 promoter. IL-10R reconstituted Mode-K cells were pretreated with IL-10 for 24 hours followed by stimulation with TNF (10 ng/mL) for 1 and 24 hours. (A) Where indicated, Mode-K cells were stimulated with TNF for 24 hours. Total RNA was extracted and reverse transcribed. Real-time PCR was performed using the Light Cycler system with specific primers for grp-78 and GAPDH. The expression changes (fold decrease) of the specific mRNA were calculated relative to unstimulated control cells, using the crossing point of the log-linear portion of the amplification curve after normalization with GAPDH. The data represent mean fold decrease ± SD from triplicate stimulations. (B) Where indicated, Mode-K cells were stimulated with TNF for 1 hour. ChIP analysis was performed using anti-ATF-6, anti-c-fos, and antiacetylated/phosphorylated H3 antibodies for immunoprecipitation followed by ATF-6 promoter-specific PCR as described in the Materials and Methods section. (C) Where indicated, Mode-K cells were stimulated with TNF for 1 hour. Immunofluorescence analysis was performed using DAPI for nuclear (blue) and rhodamine-coupled antibodies for grp-78-specific staining (red). Overlay pictures are shown in the lower row. (D) Where indicated, the cells were pretreated with the pharmacological p38 inhibitor SB for 1 hour followed by 24 additional hours of stimulation with IL-10 (50 ng/mL). ChIP analysis was performed as described in the Materials and Methods section after stimulation of the cells with TNF for an additional 1 hour. Representative gels from at least 2 different experiments are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

13. Figure 12
Schematic illustration for the proposed mechanisms of IL-10 on TNF-induced ER stress responses.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions