Volume 139, Issue 4, Pages 1277-1288 (October 2010) Interleukin-11 Reduces TLR4-Induced Colitis in TLR2-Deficient Mice and Restores Intestinal STAT3 Signaling  Deanna L. Gibson, Marinieve Montero, Mark J. Ropeleski, Kirk S.B. Bergstrom, Caixia Ma, Sanjoy Ghosh, Helen Merkens, Jingtian Huang, Lisa E. Månsson, Ho Pan Sham, Kelly M. McNagny, Bruce A. Vallance  Gastroenterology  Volume 139, Issue 4, Pages 1277-1288 (October 2010) DOI: 10.1053/j.gastro.2010.06.057 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 TLR4 is not required to protect against C rodentium colitis. Mice were infected 10 days with C rodentium. TLR4−/− infected mice do not display increased (A) morbidity or (B) damage. Histologic image: original magnification 200×. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Tissue resident cells mediate TLR2-dependent mucosal integrity. Bone marrow chimeric mice using either WT or TLR2−/− as recipients reconstituted with bone marrow from either WTLy or TLR2−/− mice were generated and infected over 10days with C rodentium. (A) TLR2−/−TR + WTLyH infected mice display 100% mortality, whereas WTTR + TLR2−/−H infected mice survive. The midcolons from the TLR2−/−TR + WTLyH infected mice display ulceration and bleeding (B) macroscopically (scale bar = 1 cm) and (C) microscopically (original magnification 200×) with (D) significantly higher damage scores for epithelial damage and mucodepletion when compared with WTTR + TLR2−/−H and WTTR + WTLyH mice. *Indicates significance higher score in TLR2−/−TR + WyLyH or WTTR + TLR2−/−H or TLR2−/− compared to WT. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Tissue resident cells are responsible for TLR2-dependent barrier function preventing C rodentium tissue invasion. (A) Chimeric mice deficient in TLR2 signaling in tissue resident cells but maintained in bone marrow cells (TLR2−/−TR + WTLyH) display increased FITC-dextran flux across the colonic epithelium after C rodentium 6 days postinfection compared with chimeric mice maintaining TLR2 signaling within the tissue resident cells (WTTR + WTLyH and WTTR + TLR2−/−H). This was similar to FITC-dextran flux in infected TLR2−/− mice compared with WT mice. *Significance = higher barrier dysfunction in TLR2 or TLR2−/−TR + WyLyH compared to WT mice. (B) Immunofluorescence of midcolonic tissues from infected bone marrow chimeric mice. C rodentium (Tir; green and arrow), neutrophil (myeloperoxidase; red), and nuclear staining (4′,6-diamidino-2-phenylindole; blue). Original magnification: top panel, 200×; bottom panel, 400×. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Compensatory TLR4 responses are responsible for the lethal colitis in the absence of TLR2. Mice were infected 10 days with C rodentium. (A) TLR2,4DKO infected mice do not (B) display increased morbidity or damage in the midcolon macroscopically (arrows indicate ulcers) or (C) score higher for microscopic damage (bottom panel, original magnification 200×). In A, *indicates significantly less weight loss in TLR2,4DKO compared to WT. In C, *indicates significantly less damage in TLR2,4DKO compared to WT. (D) Exaggerated chemokine and cytokine gene expression from midcolonic tissues from TLR2−/− infected mice is lost in the absence of TLR4. *Indicates significantly higher gene expression in TLR2−/− compared to TLR2,4DKO. (E) TLR2,4DKO infected mice displayed increased barrier permeability compared with WT and TLR4−/− mice, similar to TLR2−/− mice (Figure 3A). *Indicates significance higher barrier dysfunction in TLR2,4DKO compared to WT during infection. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 TLR4 signaling from hematopoietic cells is the primary source of inflammation-induced damage. Bone marrow chimeric mice using TLR2,4DKO as a recipient reconstituted with bone marrow from either TLR2−/− or TLR4−/− donors were infected 10 days with C rodentium. (A) TLR2,4DKOTR + TLR2−/−H infected mice display increased morbidity and damage in the midcolons (B) macroscopically and (C) microscopically (original magnification 200×) and (D) significantly increased damage scores compared with TLR2,4DKOTR + TLR4−/−H and TLR2,4DKO mice. In A, * indicates significantly more weight loss in TLR2,4DKOTR + TLR4−/−H or TLR2,4DKOTR + TLR2−/−H compared to TLR2,4DKO at specific time points. In D, * Indicates significantly more damage in TLR2,4DKOTR + TLR2−/−H compared to TLR2,4DKO. (E) IFN-γ transcripts are significantly higher in TLR2,4DKOTR + TLR2−/−H infected mice, whereas TNF-α transcripts are similarly increased when either TLR2 or TLR4 is active from hematopoietic cells compared with TLR2,4DKO mice. *Indicates significantly higher gene expression in TLR2,4DKOTR + TLR2−/−H compared to TLR2,4DKO. (F) TUNEL staining identified increased cell death in colons expressing TLR4 from hematopoietic cells. *Indicates significance increased cell death in TLR2,4DKOTR + TLR2−/−H compared to TLR2,4DKO and TLR2−/− compared to WT during infection. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 STAT3 activation in IECs is TLR2 dependent during C rodentium infection and associated with IL-11 expression from tissue resident cells in the muscularis mucosae. (A) p-STAT3 is increased in infected WT mice but not in infected TLR2−/− mice. Western blot analysis of pSTAT3, STAT3, and β-actin from colonic tissues from representative WT and TLR2−/− mice uninfected or infected for 10 days with C rodentium. Bar graph shows fold differences of mice infected to uninfected p-STAT3/STAT3 ratios. (B) IECs in transverse sections of midcolonic tissues from infected WT mice stained for nuclear p-STAT3 at day 10 postinfection, which was decreased in infected TLR2−/− mice (pSTAT3, red; nuclear stain with 4′,6-diamidino-2-phenylindole, blue). (C) Numerous cells in the muscularis mucosa were positive for IL-11 during C rodentium infection in WT and WTTR + TLR2−/−H mice, and this is impaired in the absence of TLR2 from tissue resident cells shown in both TLR2−/− and TLR2−/−TR + LyWTH infected mice (white arrows). The panel to the right shows with a white arrow the muscularis mucosa region (stains positive for smooth muscle actin) that is magnified (WT uninfected). IL-11, red; smooth muscle actin, green. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Cytoprotective IL-11 therapy prevents lethal colitis in susceptible hosts via STAT3 activation in IECs. TLR2−/− infected mice were treated with recombinant IL-11 for 10 days during C rodentium infection and displayed (A) survival similar to WT mice, (B) less weight loss, and less damage (C) histologically and (D) quantitatively than TLR2−/− mock controls (original magnification 200×), and (E) this corresponded with nuclear pSTAT3 in IECs (top panel: pSTAT3, red; bottom panel: merged p-STAT3 and nuclear stain with 4′,6-diamidino-2-phenylindole, blue). * Indicates significantly less damage in TLR2−/− + rIL-11 and WT compared to TLR2−/−. Gastroenterology 2010 139, 1277-1288DOI: (10.1053/j.gastro.2010.06.057) Copyright © 2010 AGA Institute Terms and Conditions