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

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The macrophage α7 nAChR recruits Jak2 Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation STAT3 phosphorylation normally requires activity of the cytoplasmic tyrosine kinase Jak2 (ref. 8). Therefore, we investigated whether STAT3 phosphorylation depended on Jak2 activity and whether nAChRs expressed on macrophages recruit Jak2. Phosphorylation of STAT3 after nicotine treatment of peritoneal macrophages was effectively blocked by AG 490, a selective inhibitor of Jak2 phosphorylation 19,20 (fig. 4a). In agreement with that finding, nicotine failed to reduce IL-6 release by LPS-stimulated peritoneal macrophages treated with AG 490 (data not shown). Binding studies have distinguished two main categories of nAChRs based on their affinity for either α-bungarotoxin (α7-containing homopentamers) or nicotine (α4β2 pentamers) 18 . Because our blocking studies suggested involvement of the α7 nAChR subtype, we analyzed putative associations of α7 with Jak2 (ref. 20) by immunoprecipitation (Fig. 4b). The α7 (56-kilodalton) 21 receptor was expressed in primary peritoneal macrophage lysates. Immunoprecipitation of Jak2 from peritoneal Figure 3. STAT3 phosphorylation by nicotine is prevented by α7-selective nAChR antagonists.(a,b) Peritoneal macrophages were pretreated with the nAChR blockers d-tubocurarin (d-TC), α-bungarotoxin (αBgt), hexamethonium (Hexa) or -methyllycaconitine (MLA) and were incubated with nicotine (concentration, above lanes). Lysates were collected for immunoblot of phosphorylated STAT3 (PY- STAT3), STAT3 and actin (a) and IL-6 was measured in supernatants (b). (a) Blots are representative of three independent experiments. (b) Filled squares, hexamethonium; open squares, methyllycaconitine; open circles, d-tubocurarine). Data are presented as the percentage of inhibition of IL-6 release measured without the addition of an nAChR blocker and represent mean values +/- s.e.m. of three independent experiments done in triplicate. 49
Chapter 3 macrophage cell lysates showed a weak association of Jak2 with the α7 receptor after culture in the absence of nicotine. To investigate whether Jak2 is recruited to the nAChR and is phosphorylated after binding of its ligand, we preincubated cells with nicotine. Nicotine exposure increased the amount of α7 nAChR detected in Jak2 and phosphorylated Jak2 immunoprecipitates (fig. 4b). To further demonstrate that Jak2 is phosphorylated after nAChR activation, we pretreated cells with the Jak2 phosphorylation blocker AG 490 before adding nicotine. Cells treated with AG 490 had reduced phosphorylated Jak2 in α7 immunoprecipitates, whereas Jak2 recruitment to the α7 receptor was not affected (Fig. 4b). The latter finding demonstrates that Jak2 is recruited and phosphorylated after nicotine binding. Stimulation of the vagus nerve ameliorates inflammation We next evaluated whether activation of nAChR on macrophages would attenuate intestinal inflammation in vivo. We assessed the effect of stimulation of the vagus nerve on the inflammation that follows intestinal manipulation in our mouse model11 , because this immune response is associated with the activation of macrophages12,22 . We electrically stimulated the left cervical vagus nerve during intestinal manipulation surgery and investigated the effects on muscular inflammation and gastric emptying 24 h later (Fig. 5). Consistent with published findings11,23 , intestinal manipulation of mice resulted in a delayed gastric emptying compared with that of mice that underwent only laparotomy, indicative of the development of postoperative ileus11 (gastric retention after 60 min, 14.5% +/-2.7% for laparotomy and 43.0 +/-6.7% for intestinal manipulation). However, stimulation of the vagus nerve prevented the intestinal manipulation−induced gastroparesis 24 h after surgery (gastric retention, 25.2% +/-3.2%;Fig. 5). Notably, stimulation of the vagus nerve in itself may alter gastric emptying during the vagus stimulation protocol24 . However, we found that stimulation of the vagus nerve did not affect basal gastric emptying 24 h after surgery (gastric retention, 15.7% +/-3.6%; Fig 5). The last finding demonstrates that normalization of gastric emptying after stimulation of the vagus nerve was not a direct effect on gastric motility but resulted from reduced inflammation of the manipulated bowel segment11 . We next analyzed muscularis tissue for granulocytic infiltrates by measuring myeloperoxidase activity in muscularis tissue homogenates and quantifying cellular infiltrates (Supplementary figure 1). The intestinal manipulation−induced inflammation of the muscularis externa in mice that received stimulation of the vagus nerve was reduced in a voltage-dependent way compared with that of mice that received intestinal manipulation plus sham stimulation. Prior vagotomy of the proximal end of the stimulated vagus nerve did not affect these results (data not shown), indicating that the anti-inflammatory effect of stimulation of the vagus nerve was not dependent on the activation of central nuclei, which confirms published 50
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: Chapter 3 Figure 2. Inhibition of m
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
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
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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
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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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