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

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INTRODUCTION Vagal activity enhances macrophage phagocytosis In the gastro-intestinal tract, the vagus nerve regulates motility and digestive functions mostly via the peripheral release of the parasympathetic neurotransmitter acetylcholine (ACh) that activates nicotinic acetylcholine receptors (nAChRs). However, vagal activity has also immune-regulatory properties. While the afferent vagus system is known to regulate the inflammatory response via the hypothalamic pituitary adrenal axis, efferent vagus nerve activity possesses immuno-modulatory potential as well. Borovikova et al 1 have revealed the potency of the vagus nerve to inhibit TNF production by macrophages after systemic endotoxin 2 . Peritoneal and PBMC-derived macrophages express nAChRs, and nAChR activation has been shown to inhibit NF-κB transcriptional activity 3,4 and pro-inflammatory cytokine production 4,5 . In conjunction, electrical vagus nerve stimulation has been shown to ameliorate disease in animal models of inflammatory conditions such as postoperative ileus 4,6 , colitis 7 , peritonitis 8,9 , and hemorrage 10 . An immune regulating role for the cholinergic nervous system may be particularly evident in intestinal tissue, given the dense cholinergic innervation, and the abundant number of resident macrophages that populate the intestinal mucosa and muscularis externa, of which some closely associate with cholinergic fibers 6 . Cholinergic inhibition of pro-inflammatory cytokine production by macrophages has been firmly established 1,3,6 . However, besides an effect on cytokine secretion, the cholinergic nervous system may also affect more professional macrophage functions such as endo- and phagocytosis of bacteria and particles. Especially in the intestinal compartment macrophages may rather function as phagocytes that, along with dendritic cells, form critical effectors in the surveillance of luminal antigens 11,12 . Hence, the question arises whether the anti-inflammatory effect of vagus nerve activity in intestinal inflammation solely rests on reduced macrophage cytokine production, or whether the vagus nerve also regulates other macrophage functions important in host defense. This is supported by the observation that vagus nerve activity affects bacterial clearance and mortality in various mouse models of infectious disease 8,13,14 . Therefore, we studied the effect of nAChR activation on endo- and phagocytosis by macrophages residing in the peritoneal and mucosal compartment. We show here that nAChR α4/β2, rather than α7, activation enhances the phagocytic potential in mouse macrophages. Despite enhanced phagocytosis, the activation of NF-κB activity and pro-inflammatory cytokine production is inhibited. In conjunction, we also demonstrate that in mice, electrical stimulation of the vagus nerve increases the epithelial permeability for luminal bacteria and stimulates phagocytosis by F4/80 + CD11 b+ macrophages residing in the intestinal mucosa. Taken together, our data suggest that vagus nerve activity can enhance macrophage bacterial uptake via activation of the nAChR α4/β2, while reducing inflammatory cell activation. 79
Chapter 5 METHODS Reagents and antibodies. (-)-Nicotine, (+)-Nicotine, Acetylcholine, α-Bugarotoxin, Methylcaconitine, 2,2,6,6-Tetramethylpiperidin-4-yl heptanoate, Dihydro-βerythroidine, 3-Methyl-5-[(2S)-1-methyl-2-pyrrolidinyl]isoxazole, Zymosan A from S. cerevisiae were from Sigma-Aldrich (Zwijndrecht, the Netherlands). Polyclonal antibodies against phosphorylated and unphosporylated Akt and PI3K, p38 and anti- NFkB-p65 were from Cell Signaling. Anti-α4 nAChR was purchased from Santa Cruz Biotechnology; anti-β2 nAChR subunit (MAb clone 270) and anti-Dynamin-2 (clone 3457) were from Abcam (Cambridge, UK). Dil-AcLDL was obtained from (Invitrogen). Rat anti mouse monoclonal CD11b, F4/80 and CD11c were purchased from BDBiosciences (Franklin Lakes, NJ). FITC (Sigma-Aldrich), labelling of 1*10 9 heat-killed E. faecium was performed in 0.1 M NaHCO 3 pH9.0 for 1h at 37°C (in shaking waterbath). Mammalian cell culture. Macrophages from an immortalized spleen macrophage cell line (Mf4/4 15 ) were cultured in RPMI 1640/10% FCS. Primary peritoneal cells (PMF) from BALB/c, C57/Bl6 WT mice, nAChR alpha7-/- or nAChRbeta2-/- mice (kindly provided by Dr. G.LaRosa and Dr. U. Maskos respectively) were collected by lavage 4 days after ip. injection of 1ml of Biogel solution and cultured in OptiMEM (Invitrogen). Phagocytosis assays. For phagocytosis assays, cells were plated to 80 % confluence in 24 well suspension plates and pretreated with cholinergic agonists for 20min. Subsequently, cells were challenged with FITC-labeled Zymosan particles(20particles/ cell) for 10min or other time-points where indicated. Cells were washed by PBS, final washing was in PBS/EDTA/lidocaine and cells were harvested by scraping. After fixation in 2% PFA, bead uptake was determined by flow cytometry (FACS Calibur, Becton Dickinson). Fc Receptor-mediated phagocytosis of RBC. Sheep RBC (300 μl of a 10% suspension; Cappel/ICN) were opsonized with 1:1000 diluted rabbit-anti-sheep RBC IgG (Cappel/ ICN). Opsonized RBCs were allowed to bind to Mf4/4 or peritoneal macrophages at a 1/80 cell to RBC ratio at 37°C. After 15min, slides were exposed to ice-cold water for 15 sec to lyse RBCs that were not internalized, washed in PBS and fixed. Cell stimulations. Peritoneal macrophages were incubated with nicotinic agonists at the concentration indicated for 45 minutes and stimulated with Zymosan(10particles/ cell) for 6hrs. After treatment, the medium was removed; the cells washed three times with ice-cold PBS, and medium and cell lysates were measured by ELISA. 80
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The vagus nerve as a modulator ofin
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The vagus nerve as a modulator of i
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Promotiecommissie Promotores: Prof.
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Table of contents Chapter 1 Introdu
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Introduction and outline The aim of
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Introduction and outline Hence, the
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Introduction and outline 18. van de
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Chapter 2 ABSTRACT The cholinergic
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Chapter 2 neurotransmitter ACh with
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Chapter 2 THE VAGUS NERVE AND CHOLI
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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 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: Chapter 5 ABSTRACT Background and A
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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