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

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Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation Figure 4. Nicotine-induced STAT3 phosphorylation occurs through activation of Jak2 that is recruited to the α7 nAChR subunit. (a) Immunoblot of phosphorylated STAT3 (PY-STAT3) and STAT3 in peritoneal macrophages incubated with AG 490 (concentrations, above blots). Blot is representative of three independent experiments. (b) Immunoblots of peritoneal macrophages treated with 1 μM nicotine (lanes 4 and 5) or with 1 μM nicotine plus 100 μM AG 490 (lane 5). Cell lysates were immunoprecipitated (IP) with anti-α7 (top), anti-Jak2 (middle) or anti−phosphorylated Jak2 (PY-Jak2; bottom), followed by immunoblot (IB; antibodies, left margin). Lane 2, coprecipitate in the absence of lysate (negative control). IgH, immunoglobulin heavy chain. Blots are representative of four independent experiments. 51
Chapter 3 reports 2 . We next incubated intestinal segments with the nicotinic receptor blocker hexamethonium before intestinal manipulation combined with stimulation of the vagus nerve. In intestinal segments treated with hexamethonium, stimulation of the vagus nerve failed to prevent inflammation, in contrast to incubation with vehicle (supplementary figure 1), demonstrating that the anti-inflammatory effect of vagus stimulation acted through local activation of nicotinic receptors. Stimulation of the vagus nerve activates STAT3 in vivo To further investigate whether macrophages mediated the anti-inflammatory effect of stimulation of the vagus nerve, we analyzed the expression of transcripts of macrophage-derived inflammatory mediators in muscularis tissue 3 h after surgery. Stimulation of the vagus nerve reduced the expression of Cxcl2 mRNA (Fig. 6a,b) and Ccl3 mRNA (data not shown) but did not notably alter the expression of Tnf transcripts in muscularis tissue, confirming earlier reports2,4 . However, when we analyzed peritoneal lavage fluid for the presence of macrophage inflammatory mediators 3 h after intestinal manipulation, we found that stimulation of the vagus nerve significantly reduced the secretion of TNF, IL-6, MIP-2 (Fig. 6c) and MIP-1α (data not shown) in the peritoneal cavity. This reduction was not due to enhanced expression of IL-10, as stimulation of the vagus nerve was similarly potent in reducing intestinal manipulation−induced inflammation in IL-10-deficient mice (Fig. 6d). Moreover, the peritoneal IL-10 in wild-type mice did not reach the limit of detection (31 pg/ml) at 1, 3 or 6 h after intestinal manipulation (data not shown). Expression of Socs3 (Fig. 6a) but not Socs1 (data not shown) was increased in muscularis tissue after stimulation of the vagus nerve even in mice that underwent this stimulation without manipulation of the bowel. Given the short half-life of acetylcholine, cholinergic regulation of macrophage activation most likely requires that cholinergic nerves be in close proximity to intestinal macrophages. To investigate this, we immunohistochemically doublelabeled vesicular acetylcholine transporter−positive vagal efferent fibers and macrophages in rat intestinal musclaris tissue. Macrophages were in close proximity to nerve terminals in the myenteric plexus in the ileum (Fig. 7a) and circular muscle of gastric corpus (data not shown). Hence, acetylcholine released from efferent nerve terminals could easily reach macrophages in the nanomolar concentration range. To verify that the enhanced SOCS3 expression reflected increased STAT3 activation in vivo, we immunohistochemically analyzed intestinal tissues for the presence of phosphorylated STAT3 in mice that underwent control laparotomy surgery, intestinal manipulation alone or intestinal manipulation plus stimulation of the vagus nerve (Fig. 7b,c). We found phosphorylated STAT3−positive nuclei in mice that underwent control laparotomy (Fig. 7b). Intestinal manipulation resulted in the appearance of phosphorylated STAT3−positive cells adhering to the serosal site of the bowel wall, 52
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 and 38: Chapter 3 ABSTRACT Acetylcholine re
Page 39 and 40: Chapter 3 by bowel manipulation 12
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: Chapter 3 macrophage cell lysates s
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
Page 91 and 92: Chapter 5 REFERENCE LIST 96 1. Boro
Page 93 and 94: Thesis E.P.M. van der Zanden The va
Page 95 and 96: 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
Page 103 and 104:
Nederlandse samenvatting
Page 105 and 106:
Chapter 7 van STAT3 in de ‘cholin
Page 107 and 108:
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
Page 113 and 114:
Shizuo Akira Department of Host Def
Page 115 and 116:
List of publications
Page 117 and 118:
Dankwoord
Page 119 and 120:
om de ene keer olijfolie te drinken
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Curriculum Vitae Curriculum Vitae E
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The vagus nerve as a modulator of intestinal inflammation - TI Pharma

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