Andrea Nemethova

A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen

The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca2+ imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca2+ response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

New therapeutic strategies for postoperative ileus

Patients undergoing an abdominal surgical procedure develop a transient episode of impaired gastrointestinal motility or postoperative ileus. Importantly, postoperative ileus is a major determinant of recovery after intestinal surgery and leads to increased morbidity and prolonged hospitalization, which is a great economic burden to health-care systems. Although a variety of strategies reduce postoperative ileus, including multimodal postoperative rehabilitation (fast-track care) and minimally invasive surgery, none of these methods have been completely successful in shortening the duration of postoperative ileus. The aetiology of postoperative ileus is multifactorial, but insights into the pathogenesis of postoperative ileus have identified intestinal inflammation, triggered by surgical handling, as the main mechanism. The importance of this inflammatory response in postoperative ileus is underscored by the beneficial effect of pharmacological interventions that block the influx of leukocytes. New insights into the pathophysiology of postoperative ileus and the involvement of the innate and the adaptive (T-helper type 1 cell-mediated immune response) immune system offer interesting and important new approaches to prevent postoperative ileus. In this Review, we discuss the latest insights into the mechanisms behind postoperative ileus and highlight new strategies to intervene in the postoperative inflammatory cascade.

Mast cells play no role in the pathogenesis of postoperative ileus induced by intestinal manipulation

Introduction Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling. Design The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn. Results KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Conclusions Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.

Inhibition of spleen tyrosine kinase as treatment of postoperative ileus

Objective Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. Design In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. Results In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4±0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2±0.7; GSK143 (1 mg/kg): 7.6±0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0–90 μM) and trinitrophenyl (0–4 μg/ml) induced a concentration-dependent release of β-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03–10 μM) concentration dependently blocked substance P and trinitrophenyl induced β-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. Conclusions The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.

Nicotine attenuates activation of tissue resident macrophages in the mouse stomach through the β2 nicotinic acetylcholine receptor.

Background The cholinergic anti-inflammatory pathway is an endogenous mechanism by which the autonomic nervous system attenuates macrophage activation via nicotinic acetylcholine receptors (nAChR). This concept has however not been demonstrated at a cellular level in intact tissue. To this end, we have studied the effect of nicotine on the activation of resident macrophages in a mouse stomach preparation by means of calcium imaging. Methods Calcium transients ([Ca2+]i) in resident macrophages were recorded in a mouse stomach preparation containing myenteric plexus and muscle layers by Fluo-4. Activation of macrophages was achieved by focal puff administration of ATP. The effects of nicotine on activation of macrophages were evaluated and the nAChR involved was pharmacologically characterized. The proximity of cholinergic nerves to macrophages was quantified by confocal microscopy. Expression of β2 and α7 nAChR was evaluated by β2 immunohistochemistry and fluorophore-tagged α-bungarotoxin. Results In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900nm. The ATP induced [Ca2+]i increase was significantly inhibited in 65% or 55% of macrophages by 100µM or 10µM nicotine, respectively. This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express β2 but not α7 nAChR at protein level, while those in the intestine express both receptor subunits. Conclusion This study is the first in situ demonstration of an inhibition of macrophage activation by nicotine suggesting functional signaling between cholinergic neurons and macrophages in the stomach. The data suggest that the β2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.

Absence of intestinal inflammation and postoperative ileus in a mouse model of laparoscopic surgery

Postoperative ileus (POI) is characterized by impaired gastrointestinal motility resulting from intestinal handling‐associated inflammation. The introduction of laparoscopic surgery has dramatically reduced the duration of POI. However, it remains unclear to what extent this results in a reduction of intestinal inflammation. The aim of the present study is to compare the degree of intestinal inflammation and gastrointestinal transit following laparoscopic surgery and open abdominal surgery.

Tu1730 Novel Transgenic Mast Cell-Deficient Mouse Model Reveals No Role for Mast Cells in the Pathogenesis of Postoperative Ileus

Background & Aims: The neuropeptide NT and its receptor NTR1 mediate intestinal inflammation and their expression is increased in IBD. Colitis and associated hypoxia through the activation of the transcription factor hypoxia-inducible factor HIF-1modulates the expression of miR-210. NT promotes the transcriptional activity of HIF-1α, an event associated with increased intestinal angiogenesis (DDW2013: #1738), and induces the expression of miR210 (Gastroenterology, 2011; 141:1749). Here we examined the implication of HIF-1α in miR-210 regulation by NT, and assessed the role of miR-210 in the development of colitis and intestinal angiogenesis. Methods: NCM460 cells overexpressing NTR1 (NCM460-NTR1) were exposed to NT (0.1 μM, 6 h), +/the HIF-1α inhibitor PX-478 (40 μM, 18 h); miR-210 expression was evaluated by qPCR. Acute colonic inflammation was induced by intracolonic administration of TNBS (5 mg/kg, 48 h) in wild type (WT) and NTR1 knockout (KO) mice. Colonic miR-210 was inhibited by intracolonic administration of locked nucleic acid anti-miR-210 (LNA-anti-miR-210). The degree of inflammation was evaluated on distal colon segments stained with H&E and angiogenesis was evaluated by von-Willebrand (vWB) staining. Results: NT treatment of NCM460-NTR1 cells induced miR-210 expression (p<0.006) that was attenuated (by ~65%, p<0.005) by treatment with the HIF-1α inhibitor, PX-478. TNBS treatment increased colonic miR-210 expression in WT mice, an effect suppressed in NTR1 KO mice (p=0.02, n=6/group). Moreover, miR-210 levels were elevated in tissue samples from patients with Ulcerative Colitis (UC) compared to controls (p=0.003, n=11/group). Intracolonic administration of LNA-anti-miR-210 in mice reduced endogenous colonic miR-210 levels by ~55% (p=0.01), without affecting expression of other miRs. LNAanti-miR-210 pre-treated colons (n=8/group) exhibited reduced levels of IL-6, Cxcl1, and TNFα (p<0.05 for all), diminished mucosal integrity score (p=0.0001), neutrophil infiltration (p=0.0004), total colitis score (p=0.0001), and vWB staining (p=0.008) compared to colons treated with TNBS alone. Conclusions: Our results demonstrate the importance of NT/miR210 axis in the pathophysiology of colitis and IBD. The successful intracolonic microRNA silencing indicates the utility of microRNA inhibitors as a novel therapeutic approach towards IBD. Supported by NIH grant DK60729 (CP), the Crohns and Colitis Foundation of America (KB) and the Blinder Research Foundation for Crohns Disease (IKML).

Tu1629 Spleen Tyrosine Kinase Inhibition Reduces Intestinal Inflammation and Prevents Postoperative Ileus

Background: Recent advances underline intestinal inflammation, induced by handling of the gut during surgery, as crucial mechanism in the pathophysiology of postoperative ileus (POI). Macrophages, in the muscularis layer, and mast cells are the key players in the induction of this inflammatory process. Spleen tyrosine kinase (Syk) is an important kinase involved in macrophage activation as well as in mast cell degranulation and therefore inhibition of Syk pathway may represent an interesting therapeutic approach for POI. In the current study, we have evaluated the effect of the Syk-inhibitor GSK143 as potential treatment to shorten POI. Methods: The effect of an oral single dose (3mg/kg, 1.5 hours before surgery) was evaluated in a mouse model of POI, by analyzing gastrointestinal transit and intestinal muscularis inflammation. The in vitro effect of GSK143 (1 and 3 μM) was evaluated on cultured peritoneal mast cells (pMCs) and bone marrow derived macrophages (BMDMs) stimulated with immune complexes with IgE-anti-TNP (40 ng/mL) and LPS (100 ng/ml) respectively. Results: Administration of GSK143 (3 mg/kg) resulted in a serum level of 0.38±0.24 μM 1.5 hours after oral delivery. Treatment with a single dose of GSK143 (3 mg/kg) significantly improved gastrointestinal transit after surgical intestinal manipulated mice compared to placebo (Geometric Center (GC): Placebo, 4.2 ±0.4 vs GSK143, GC; 6.9±0.6). In addition, GSK143 was able to reduce the number of recruited myeloperoxidase (MPO) positive cells (placebo, 187±30 vs GSK143, 44±8, per 0.5mm2), neutrophils (placebo, 4.6x10-5±0.2 x10-5 vs GSK143, 1.7x10-5 ±0.7 x10-5) and monocytes (placebo, 1.6x106±0.4 x10-6 vs GSK143, 0.8x10-6 ±0.25 x10-6) in the muscularis externa compared to placebo. To define the possible target cells affected, GSK143 was tested in vitro on pMCs and BMDMs. Thirty minutes after GSK143 (1 and 3 μM) or vehicle treatment, pMCs degranulation, quantified by assessment of β-hexosaminidase, was induced by immune complexes with IgE-anti-TNP. Interestingly, GSK143 significantly reduced pMCs degranulation compared to vehicle in a dose dependent manner (GSK143 1μM, 62±7.5% and GSK143 3μM, 76±3.3% inhibition vs vehicle). In line, pre-treatment of BMDMs with GSK143 (1 and 3 μM) prior to LPS challenge significantly lowered the expression of IL-6 (21±4% to 31± 3 % inhibition vs vehicle ), TNFα (39±0.8% to 54±0.7% inhibition vs vehicle ), IL1 β (31±1.3% to 39±1.06% inhibition vs vehicle) and CCL2 (33±5.3% to 43±7% inhibition vs vehicle). Conclusion: Pre-treatment with the Syk-inhibitor GSK143 attenuates intestinal inflammation and consequently restores gastrointestinal transit in a model of POI. In vitro data suggests that both macrophages and mast cells are targeted by GSK143. These findings strongly suggest that Syk inhibition may be a new therapeutical tool to shorten POI.