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

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Vagal anti-inflammatory pathway mediated via JAK2-STAT3 activation each sample. For qualification, resulting PCR products were analyzed on an ethidium bromide-stained 2.5% agarose gel. Immunohistochemistry. For double-labeling of macrophages and cholinergic fibers, rats were anesthetized with pentobarbital sodium (90mg/kg, ip) and transcardially perfused with heparinized saline (20 U/ml), followed by ice-cold 4% phosphatebuffered (pH 7.4) paraformaldehyde. Gastric and intestinal tissue were extracted and postfixed in the same fixative for a minimum of 2 hr. Tissue was cryoprotected overnight in 18% sucrose and 0.05% sodium azide in 0.01M phosphate-buffered saline (PBS). Twenty-micron flat-sections and 25 µm cross-sections of corpus and mid ileum were cryostat cut and processed in PBS. Sections were pretreated with 0.5% sodium borohydride in PBS and subsequently blocked in donkey normal serum. Monoclonal mouse anti-rat ED2 (Serotec, Raleigh, NC), and polyclonal goat anti-Vesicular Acetylcholine Transporter (VAChT; Chemicon, Temecula, CA) were diluted in 0.1% gelatin and 0.05% sodium azide in PBS with 0.5% Triton X-100 (PBST) and incubated for 20 hr at room temperature or for 48 hr at 4 o C. Secondary antibodies used were Cy-3-conjugated donkey anti-mouse (Jackson ImmunoResearch, West Grove, PA) for ED2 and Cy-2-conjugated donkey anti-goat (Jackson ImmunoResearch, West Grove, PA) for VAChT in PBST. Sections were mounted in 100% glycerol with the addition of 5% n-propyl gallate as an antifade agent. In vivo labeling of mouse phagocytes was performed by i.p. injection of 20 µg Alexa 546 -labeled Dextran particles (Mw 10,000; Molecular Probes, Sunnydale, CA) 24 h before surgery. One hour following surgery, anaesthetized mice were perfused with 10 mL of ice-cold 0.9% NaCl containing 1mM Na 3 VO 4 , followed by 20 mL of ice-cold 4 % formaldehyde solution, pH 7.4. Intestinal tissue was isolated, fixed overnight in 4% formaldehyde, dehydrated and embedded in paraffin. Six mm sections were cut and immunostained for PY 705 -Stat-3 using polyclonal rabbit antibodies (Cell Signaling Technologies) and biotin-labeled anti-rabbit antibodies according to the manufacturer’s instructions. Biotin was visualized using 3-amino-9-ethyl carbazole (Sigma, St Louis, MO) as chromogen, followed by counterstaining using haematoxylin. Alternatively, Alexa 488 -streptavidin (Molecular probes) with DaPi nuclear counterstain was used for analysis by confocal microscopy. RESULTS Nicotine activates STAT3 in macrophages. To study the cellular response of macrophages to nicotinic receptor activation, we isolated peritoneal macrophages from mice and investigated the effect of nicotine on LPS-induced cytokine production. Nicotine reduced the LPS-induced release of TNF, MIP-2 and IL-6 but not IL-10 in a dose-dependent way (fig.1a), consistent with published reports on 45
Chapter 3 Figure 1. Nicotine attenuates peritoneal macrophage activation and induces phosphorylation of STAT3 and SOCS3 expression. (a) ELISA of TNF, MIP-2, IL-6 and IL-10 in the supernatants of peritoneal macrophages stimulated with 100 ng/ml of LPS in vitro in the presence of nicotine (dose, horizontal axes). Data represent mean +/-s.e.m. of four independent experiments in triplicate. (b) Immunoblots for phosphotyrosine-STAT3 (PY-STAT3), STAT3 and SOCS3 in cell lysates of peritoneal macrophages stimulated with 1 ng/ml of LPS (right) or no LPS (left) in the presence of nicotine (concentration, above lanes). Blot is representative of five independent experiments. (c) Immunoblot of phosphorylated STAT3 (PY-STAT3) and STAT3 in cell lysates of peritoneal macrophages stimulated with 100 nM nicotine (time, above lanes). Blot is one representative of three independent experiments. (d) Immunoblot of phosphorylated STAT3 (PY-STAT3), STAT3 and SOCS3 in cell lysates of peritoneal macrophages pretreated with vehicle, actinomycin-D (Act-D) or cycloheximide (CHX) and incubated with saline (-) or 100 nM nicotine (+). Blot is representative of three independent experiments. (e) Immunoblot of phosphorylated STAT1 (PY-STAT1) and STAT1 in peritoneal macrophages incubated with nicotine (concentration, above lanes) and stimulated with 100 ng/ml of interferon-γ (IFN-γ). Actin, loading control. 46
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: Chapter 3 for preparation of whole
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
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
Page 109 and 110:
Chapter 7 macrofagen aan te tonen (
Page 111 and 112:
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
Page 121:
Curriculum Vitae Curriculum Vitae E
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