The vagus nerve as a modulator of intestinal inflammation - TI Pharma

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INTRODUCTION Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation The innate immune response has been increasingly recognized as being under substantial neuronal control 1 . For example, acetylcholine or nicotine effectively attenuates the activation of macrophages 2 . This so-called ‘cholinergic antiinflammatory pathway’ is characterized by a nicotine dose−dependent decrease in the production of proinflammatory mediators, including high-mobility group box 1 proteins 3 , tumor necrosis factor (TNF), interleukin 1β (IL-1β), IL-6 and IL-18 (ref 2), by macrophages stimulated with endotoxin. Consistently, stimulation of the efferent vagus nerve dampens macrophage activation in rodent models of endotoxemia and shock 1,2 . Two nicotinic acetylcholine receptor (nAChR) subtypes are involved in the nicotine-induced decrease in proinflammatory cytokine production by stimulated human and mouse macrophages: the α7 homopentamer expressed by monocytederived human and mouse macrophages 4 , and the α4β2 heteropentamer expressed by alveolar macrophages 5 . Activation of the α7 homopentamer nAChR inhibits transactivational activity of the transcription factor NF-ĸB p65 (ref 3). However, the subcellular mechanism explaining the deactivating effect of acetylcholine on macrophages has remained unknown. Here we evaluated the involvement of the transcription factor STAT3 in this process, because STAT3 is a potential negative regulator of inflammatory responses 6,7 . STAT3 and the tyrosine kinase Jak2, which phosphorylates STAT3, are required for both IL-6 receptor (IL-6R) and IL-10R signaling. IL-6 contributes to the progression of many inflammatory diseases, whereas IL-10 is an anti-inflammatory cytokine that suppresses the activation of macrophages. IL-6R signaling is inhibited by the Src homology 2 domain protein SOCS3, whose expression is induced by STAT3 activation 8,9 . SOCS3 binds to the glycoprotein 130 (gp130) subunit of the IL-6R, leading to inhibited activation of STAT3 by IL-6R ligands 8,9 . Consistent with that finding, in LPS-stimulated macrophages deficient in SOCS3, IL-6R ligands induce a sustained STAT3 activation, which leads to the reduced production of proinflammatory cytokines such as TNF 10 . Here we demonstrate that nicotine exerts its anti-inflammatory effect on peritoneal macrophages via Jak2 and STAT3 signaling in vitro and in vivo. In isolated peritoneal macrophages, nicotine activated nAChRs, leading to phosphorylation of STAT3 via Jak2. Jak2 was recruited to the α7 subunit of the nAChR and was phosphorylated after nicotine binding. We further studied the effect of cholinergic inhibition of macrophage activity in vivo on the occurrence of post-surgical intestinal inflammation in a mouse model of postoperative ileus 11,12 . Postoperative ileus is characterized by general hypomotility of the gastrointestinal tract and delayed gastric emptying 13 and is a pathological condition commonly noted after abdominal surgery with intestinal manipulation. This condition is the result of inflammation of the intestinal muscularis due to activation of resident macrophages 14,15 that are triggered 41
Chapter 3 by bowel manipulation 12 . We show here that perioperative stimulation of the vagus nerve prevented manipulation-induced inflammation of the intestinal muscularis externa and ameliorated postoperative ileus. The effectiveness of stimulation of the vagus nerve in reducing intestinal inflammation depended on STAT3 activation in macrophages in the intestinal muscularis. Hence, our data demonstrate the molecular pathway responsible for cholinergic inhibition of macrophage activation and suggest that stimulation of the vagus nerve or administration of cholinergic agents may be effective anti-inflammatory therapy for the treatment of postoperative ileus and other inflammatory diseases. MATERIALS AND METHODS Reagents and antibodies. Nicotine, Hexamethonium, α-Bungarotoxin, Methyllycaconitine citrate, (+)-Tubocurarine chloride hydrate, dihydro-β-erythroidine, AG490, cyclohexamin, actinomycin-D, and monoclonal rat anti-β2 were from Sigma- Aldrich. Polyclonal rabbit antibodies against Jak-2, phosphorylated Jak-2 (PY 1007-8 Jak-2), Socs-3 and α7 were obtained from Abcam (Cambridge, UK), goat polyclonal anti-actin, rabbit polyclonal anti-Stat-1, and anti-Stat-3 were from Santa Cruz Biotechnology (Santa Cruz, California), and rabbit polyclonal against phosphorylated Stat-1 and Stat-3 (PY 705 ) were from Cell Signaling Technology (Beverly, Maryland). ELISAs for IL-6, IL-10, MIP-1α, MIP-2 and TNF were from R&D Systems, (Minneapolis, Michigan). Cell culture and transient transfection. Resident peritoneal macrophages were harvested from Balb/C mice by flushing the peritoneal cavity with 5 mL of ice-cold Hank’s Balanced Salt Solution containing 10 U/mL heparin. Peritoneal cells (1*10 6 per cm 2 ) were plated in RPMI medium supplemented with 10% FCS and macrophages were left to adhere for 2 h in a humidified atmosphere at 37 °C with 5% CO 2 . Cells were washed and remaining macrophages were left for 16-20 h. Subsequently, cells were pre-incubated with the appropriate concentration of nicotine for 15 min, followed by LPS (1-100 ng/mL) challenge for 3 h. NachR blockers were added 30 min before nicotine, and no toxicity was observed after 4 h incubation of any blocker as assessed by tryphan blue exclusion test. Cells were lysed 30 min following nicotine/ LPS exposure for immunoblotting. Transfection of peritoneal macrophages was performed using Effectine reagent (Qiagen, Cambridge, UK) according to the manufacturer’s instructions. A CMV driven Renilla luciferase reporter plasmid was co-transfected to assess transfection efficiency. The pCAGGS-neo expression vectors encoding wild type hemagluttinin (HA)-tagged Stat-3, or the dominant negative mutant HA-Stat-3D cDNA 17 were kindly provided by Drs I. Touw and T. Hirano. In HA-Stat-3D, glutamic acids 434 and 435 were replaced by alanines 17 . Following 42
Page 1 and 2: The vagus nerve as a modulator ofin
Page 3 and 4: The vagus nerve as a modulator of i
Page 5 and 6: Promotiecommissie Promotores: Prof.
Page 7 and 8: Table of contents Chapter 1 Introdu
Page 9 and 10: Introduction and outline The aim of
Page 11 and 12: Introduction and outline Hence, the
Page 13 and 14: Introduction and outline 18. van de
Page 15 and 16: Chapter 2 ABSTRACT The cholinergic
Page 17 and 18: Chapter 2 neurotransmitter ACh with
Page 19 and 20: Chapter 2 THE VAGUS NERVE AND CHOLI
Page 21 and 22: Chapter 2 nicotine treatment impair
Page 23 and 24: Chapter 2 Figure 1. Hypothetical sc
Page 25 and 26: Chapter 2 unclear whether this hybr
Page 27 and 28: Chapter 2 reduction of NF-κB activ
Page 29 and 30: Chapter 2 CONCLUDING REMARKS The hy
Page 31 and 32: Chapter 2 34 17. The FO, Boeckxstae
Page 33 and 34: Chapter 2 55. Moser N, Mechawar N,
Page 35 and 36: Chapter 2 93. Shafique S, Dalsing M
Page 37: Chapter 3 ABSTRACT Acetylcholine re
Page 41 and 42: Chapter 3 for preparation of whole
Page 43 and 44: Chapter 3 Figure 1. Nicotine attenu
Page 45 and 46: Chapter 3 Figure 2. Inhibition of m
Page 47 and 48: Chapter 3 macrophage cell lysates s
Page 49 and 50: Chapter 3 reports 2 . We next incub
Page 51 and 52: Chapter 3 Figure 5. Perioperative e
Page 53 and 54: Chapter 3 Figure 7. Stimulation of
Page 55 and 56: Chapter 3 Activation of the STAT3 c
Page 57 and 58: Chapter 3 REFERENCE LIST 60 1. Trac
Page 59 and 60: 4 CHAPTER Deciphering STAT3 signali
Page 61 and 62: INTRODUCTION Deciphering STAT3 sign
Page 63 and 64: Deciphering STAT3 signaling in chol
Page 69 and 70: DISCUSSION Deciphering STAT3 signal
Page 71 and 72: REFERENCES Deciphering STAT3 signal
Page 73 and 74: Chapter 5 ABSTRACT Background and A
Page 75 and 76: Chapter 5 METHODS Reagents and anti
Page 77 and 78: Chapter 5 described previously 6 .
Page 79 and 80: Chapter 5 Figure 1 Cholinergic agon
Page 81 and 82: Chapter 5 Figure 3 Cholinergic agon
Page 83 and 84: Chapter 5 we analyzed whether nicot
Page 85 and 86: Chapter 5 Zymosan induced formation
Page 87 and 88: Chapter 5 marker CD11 c (Fig. 7E),
Page 89 and 90:
Chapter 5 Our data demonstrate that
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Chapter 5 REFERENCE LIST 96 1. Boro
Page 93 and 94:
Thesis E.P.M. van der Zanden The va
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Summary and conclusions The goal of
Page 97 and 98:
Summary and conclusions whether the
Page 99 and 100:
Summary and conclusions exposure co
Page 101 and 102:
REFERENCE LIST Summary and conclusi
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Nederlandse samenvatting
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Chapter 7 van STAT3 in de ‘cholin
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Chapter 7 De bevinding dat nicotine
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Chapter 7 macrofagen aan te tonen (
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Chapter 7 18. Yu Z, Zhang W, Kone B
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Shizuo Akira Department of Host Def
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List of publications
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Dankwoord
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om de ene keer olijfolie te drinken
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Curriculum Vitae Curriculum Vitae E
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