Volume 145, Issue 6, Pages 1323-1333 (December 2013) Toll-Like Receptor 2 Regulates Intestinal Inflammation by Controlling Integrity of the Enteric Nervous System  Paola Brun, Maria Cecilia Giron, Marsela Qesari, Andrea Porzionato, Valentina Caputi, Chiara Zoppellaro, Serena Banzato, Alessia Rosaria Grillo, Lisa Spagnol, Raffaele De Caro, Daniela Pizzuti, Vito Barbieri, Antonio Rosato, Giacomo Carlo Sturniolo, Diego Martines, Giovanni Zaninotto, Giorgio Palù, Ignazio Castagliuolo  Gastroenterology  Volume 145, Issue 6, Pages 1323-1333 (December 2013) DOI: 10.1053/j.gastro.2013.08.047 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 Expression of functional TLR2 in the ileal smooth muscle layers of WT mice. (A) Western blots of TLR2 in protein extracts from full-thickness ileum, ileal mucosa and LMMP of WT mice (n = 5 per group). β-actin was used as loading control. (B) Dual-label immunohistochemistry showing expression of TLR2 and neural marker peripherin in ileal cryosections from WT and TLR2−/− mice. Nuclei were stained with TOTO-3. Scale bar = 50 μm. Enlarged view of peripherin+ and TLR2+ cells in LMMP from WT mice from boxed area in overlay (scale bar = 12.5 μm). (C) Expression of TLR2 in HuC/D+, GFAP+, α-smooth muscle actin+, CD31+, F4/80+, CD3+ cells harvested from LMMP of WT mice and analyzed by flow cytometry. For each LMMP, 104 cells were collected. Representative dot plots for each experiment are reported. The histogram shows the percentage of double-positive cells for each population subset (n = 6 per group). ∗P < .05 vs WT. (D) LMMP of WT mice (n = 5 per group) were incubated in the presence or absence of TLR2 ligand Pam3-CSK4 and protein lysates were immunoprecipitated with anti-TLR2. MyD88 in the immunoprecipitates was determined by Western blot. IP, immunoprecipitation. (E) Western blot analysis of phosphorylated nuclear factor-κB p65, IκBα, total p38 mitogen-activated protein kinase on proteins extracted from LMMP of WT mice (n = 5) after incubation in presence or absence of TLR2 ligand Pam3-CSK4. β-actin was used as loading control. (F) Protein lysates were obtained from full-thickness ileum, ileal mucosa, and LMMP of WT mice (n = 5 per group) and were immunoprecipitated with anti-TLR2. MyD88 in the immunoprecipitates was determined by Western blot. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 Absence of TLR2 signaling perturbs myenteric plexus architecture. (A−D) Immunofluorescence analysis of LMMP from WT and TLR2−/− mice, labeled for pan-neuronal marker HuC/D, glial marker S-100β, neurofilaments βIII-tubulin and peripherin, glial filament GFAP, and nNOS. Nuclei were stained with TOTO-3. Scale bars = 37.5 μm in (A) and 75 μm in (B−D). nNOS+ HuC/D+ neurons/mm2 in LMMP of WT and TLR2−/− mice. ∗P < .05 vs WT. (E) Western blot analysis of HuC/D, S-100β, βIII-tubulin, peripherin, GFAP, and nNOS in protein extracts from LMMP of WT and TLR2−/− mice (n = 5 per group). β-actin was used as loading control. Protein signals, quantified using densitometry analysis, are reported in Supplementary Figure 3. (F) Acetylcholine esterase (AChE)–stained neurons and fibers within the myenteric plexus of WT and TLR2−/− mice. Scale bar = 150 μm. Percentage change in the number of AchE+ fibers. ∗P < .05 vs WT. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 Impaired gastrointestinal motor function in TLR2−/− mice. (A) Amplitude and frequencies of spontaneous contractions of WT and TLR2−/− mice (n = 8 per group). ∗P < .05 vs WT. (B) Electric field stimulation (EFS)−elicited contractions in ileum segments of WT and TLR2−/− mice (n = 8 per group). ∗∗P < .01 compared with WT. Representative tracings showing EFS evoked responses in ileal segments of WT and TLR2−/− mice (20 Hz, 1-millisecond pulse duration, 10-second pulse trains, 40 V). (C) Amplitude of nonadrenergic, noncholinergic (NANC) responses of ileal segments of WT and TLR2−/− mice evoked by EFS (n = 6 per group). ∗P < .05 vs WT. (D) Relative distribution of nonabsorbable fluorescein isothiocyanate (FITC)−labeled dextran in the stomach, ileum, cecum, and colon in WT mice compared with TLR2−/− mice. (E) and (F) Percentage of residual nonabsorbable FITC-labeled dextran in the stomach (gastric emptying) and relative distribution of fluorescent probe in the intestine (geometric centre; n = 6−8 per group). ∗P < .05 vs WT. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 Loss of TLR2 signaling causes morphofunctional abnormalities in submucosal plexus. (A−C) Immunofluorescence analysis of submucosal plexus (SMP) from WT and TLR2−/− mice. SMP were labeled for the neurofilaments βIII-tubulin, peripherin and the glial filament GFAP. Nuclei were stained with TOTO-3. Scale bar = 75 μm. (D) Western blot analysis of βIII-tubulin, peripherin, and GFAP in protein extracts from SMP of WT and TLR2−/− mice (n = 5 per group). β-actin was used as loading control. Protein signals were quantified using densitometry analysis. (E) Measure of short-circuit current (Isc) in basal condition and after stimulation with carbachol (Cch) in ileal segments from WT and TLR2−/− mice mounted in Ussing's chambers (n = 6 per condition). Experiments were done with or without the neuronal blocker tetrodotoxin (TTX). ∗P < .05 vs WT without TTX. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 TLR2 pathway influences GDNF signaling. (A) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of Gdnf mRNA in LMMP from WT and TLR2−/− mice (n = 6 per group). *P < .05 vs WT. (B) GDNF immunostaining in ileum of WT and TLR2−/− mice. Scale bars = 75 μm. (C) Protein lysates from LMMP of WT and TLR2−/− mice were immunoprecipitated with anti-Ret. GFRα1, p38 mitogen-activated protein kinase (MAPK), RET phosphorylated tyrosine residues, extracellular signal–regulated kinase (ERK)1/2 in immunoprecipitates were determined by immunoblot analysis. IP, immunoprecipitation. (D) Quantitative RT-PCR of Gdnf mRNA in LMMP from WT mice incubated with lipopolysaccharide, Pam3-CSK4, FSL-1, or CpG. ∗P < .01 vs control (CTR). (E) Gdnf mRNA level in LMMP from WT mice preincubated with nuclear factor-κB or p38 MAPK inhibitors before exposure to Pam3-CSK4 or FSL-1. ∗P < .01 vs control. °P < .05 vs treatment with either Pam3-CSK4 or FSL-1. §P < .05 vs treatment with Pam3-CSK4 alone. (F) Terminal deoxynucleotidyl transferase−mediated deoxyuridine triphosphate nick-end labeling of ileum of WT and TLR2−/− mice. Arrows, apoptotic nuclei in myenteric ganglia. Scale bars = 40 μm. Cleaved caspase 7 determined by Western blot in protein lysates of LMMP from WT and TLR2−/− mice. β-actin was used as loading control. Signals, quantified using densitometry, are reported as arbitrary units below the blot. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 GDNF administration prevents ENS abnormalities in TLR2−/− mice and regulates mucosal inflammatory responses. (A) Protein lysates from LMMP of WT and TLR2−/− mice treated with GDNF or vehicle were immunoprecipitated with anti-Ret. GFRα1, p38 mitogen-activated protein kinase (MAPK), RET phosphorylated tyrosine residues, extracellular signal–regulated kinase (ERK)1/2 were determined by immunoblot. IP, immunoprecipitation. (B) Immunofluorescence of LMMP from WT and TLR2−/− mice treated with GDNF or vehicle were labeled for HuC/D and S-100β. Nuclei were stained with TOTO-3. Scale bar = 75 μm. Western blot of HuC/D and S-100β in protein extracts from LMMP of WT and TLR2−/− mice treated with GDNF or vehicle. β-actin was used as loading control. Protein signals were quantified using densitometry. (C) Electric field stimulation (EFS)−elicited contractions in ileum segments of WT and TLR2−/− mice treated with GDNF or vehicle (n = 6 per group). ∗∗P < .01 vs WT; °°P < .01 vs TLR2−/− treated with GDNF. (D) Immunofluorescence of SMP from TLR2−/− mice treated with GDNF or vehicle were labeled for βIII-tubulin. Nuclei were stained with TOTO-3. Scale bar = 75 μm. Western blot of βIII-tubulin in protein extracts from SMP of WT and TLR2−/− mice treated with GDNF or vehicle. β-actin was used as loading control. Protein signals were quantified using densitometry. (E) Basal and carbachol-induced short-circuit current (Isc) in ileal segments from TLR2−/− mice treated with GDNF mounted in Ussing's chambers (n = 6 per condition). Experiments were done with or without tetrodotoxin (TTX). ∗P < .05 vs stimulation with TTX. (F) Body-weight changes and H&E-stained ileal sections during dextran sulfate sodium (DSS) colitis. WT and TLR2−/− control (CTR) received tap water; WT and TLR2−/− DSS received 3% DSS; TLR2−/− DSS+GDNF were treated with GDNF from P14 to P21; GDNF treatment was then discontinued and 3% DSS administration started (n = 6 per group). Scale bar = 90 μm. ∗P < .05 vs CTR, °P < .05 vs TLR2−/− DSS, §P < .05 vs WT DSS. Gastroenterology 2013 145, 1323-1333DOI: (10.1053/j.gastro.2013.08.047) Copyright © 2013 AGA Institute Terms and Conditions