Volume 133, Issue 4, Pages 1219-1228 (October 2007) Activation of the Cholinergic Anti-Inflammatory Pathway Ameliorates Postoperative Ileus in Mice  Frans O. The, Guy E. Boeckxstaens, Susanne A. Snoek, Jenna L. Cash, Roel Bennink, Gregory J. LaRosa, Rene M. van den Wijngaard, David R. Greaves, Wouter J. de Jonge  Gastroenterology  Volume 133, Issue 4, Pages 1219-1228 (October 2007) DOI: 10.1053/j.gastro.2007.07.022 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 POI is ameliorated after treatment with alpha7-selective agonists AR-R17779. (A) Gastric retention 80 minutes after gavage of a semiliquid test meal in mice that had undergone the indicated treatment and L or IM 24 hours previously. (B) Quantitative analysis of IM-induced inflammatory cell recruitment 24 hours after indicated treatment and surgery. (C) Quantitative analysis of manipulation-induced inflammatory cell recruitment 24 hours after indicated treatment and surgery in VGX or sham-vagotomized animals. Data shown are mean values ± SEM of 6–8 mice. L, □; IM, ■. #P < .05 vs sham laparotomy. ##vs sham IM. *P < .05 vs saline laparotomy. **P < .05 vs saline IM. Gastroenterology 2007 133, 1219-1228DOI: (10.1053/j.gastro.2007.07.022) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Nicotinic agonists reduce cytokine and chemokine production in macrophages. (A) TNF, interleukin-6, or KC release from primary peritoneal macrophages stimulated with LPS (100 ng/mL) in the presence of nicotine (○) or AR-R17779 (•) at the indicated concentration. Data shown are mean percentages compared with vehicle/endotoxin treatment baseline concentration ± SEM of 5–7 mice measured in duplicate. *P < .05 vs 0 nmol/L nicotine or AR-R17779 baseline concentration. **P < .01 vs 0 nmol/L nicotine or AR-R17779 baseline concentration. (B) Nicotine suppresses the up-regulation of inflammatory mediator transcripts by activated macrophages via alpha7 nAChR. Peritoneal macrophages were pretreated with vehicle (media), nicotine (80 nmol/L), or nicotine (80 nmol/L) + methyllycaconitine (mla; 5 mmol/L) for 1 hour and then stimulated with LPS (100 ng/mL) for 15 hours. Cytokine transcript expression was normalized to hypoxanthine phosphoribosyltransferase messenger RNA. Data shown are mean ± SEM from 4 independent experiments using cells from different donors. Asterisks indicate significant differences (P < .05) relative to LPS-treated samples. Gastroenterology 2007 133, 1219-1228DOI: (10.1053/j.gastro.2007.07.022) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Nicotinic agonists reduce NF-κB transcriptional activity in macrophages. Macrophages (immortalized splenocytes Mf4/4) transiently transfected with a κB firefly luciferase reporter were treated with nicotine or AR-R17779 at the indicated concentrations, and then stimulated with medium () or zymosan (5 particles per cell), respectively; (■). Cells were co-transfected with a cytomegalovirus–renilla luciferase construct to normalize for transfection efficiency. Shown are normalized means ± SEM of 3–4 independent experiments in duplicate. Asterisks indicate significant differences (P < .05) of LPS vs vehicle. Gastroenterology 2007 133, 1219-1228DOI: (10.1053/j.gastro.2007.07.022) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Nicotinic agonists induce STAT3 activation in peritoneal macrophages. (A) Immunoblots showing a dose-dependent increase of phosphorylated STAT3 in peritoneal macrophages treated with nicotine (0–100 nmol/L) or AR-R17779 (0–1000 nmol/L). Blots shown are representative of 3 independent experiments. (B) Confocal images of peritoneal macrophages attached to glass slides and stimulated with LPS (10 ng/mL) with the addition of either vehicle: AR-R17779 (100 nmol/L), or nicotine (100 nmol/L). Treatment of cells with AR-R17779 (middle) or nicotine (lower) enhances nuclear staining of phosphorylated STAT3 in F4/80 (red)-positive macrophages. Gastroenterology 2007 133, 1219-1228DOI: (10.1053/j.gastro.2007.07.022) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 (A) TNF, (B) interleukin-6, and (C) KC release by peritoneal macrophages harvested from STAT3flox/flox(■) controls or LysMCre/STAT3flox/flox(□) mice and stimulated with LPS (100 ng/mL) in the presence of nicotine or AR-R17779 (0–1000 nmol/L). Data shown are mean percentages compared with vehicle/LPS treatment baseline concentration ± SEM of 6 assays. *P < .05 vs 0 nmol/L AR-R17779 baseline concentration. **P < .01 vs 0 nmol/L AR-R17779 baseline concentration. Gastroenterology 2007 133, 1219-1228DOI: (10.1053/j.gastro.2007.07.022) Copyright © 2007 AGA Institute Terms and Conditions