CCR2-dependent monocyte-derived macrophages resolve inflammation and restore gut motility in postoperative ileus

Postoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinal muscularis externa (ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of infiltrating monocytes in a murine model of intestinal manipulation (IM)-induced POI in order to clarify whether monocytes mediate tissue damage and intestinal dysfunction or they are rather involved in the recovery of gastrointestinal (GI) motility.status: publishe

Pharmacological and electrical stimulation of the enteric nervous system as a new therapeutic approach for postoperative ileus.

Each abdominal surgical intervention leads to impaired motility of the entire gastrointestinal (GI) tract lasting several days with symptoms like nausea, vomiting, intolerance to food and absence of defecation, which is referred to as postoperative ileus (POI). This condition is a major source of patient morbidity and prolonged hospital stay and represents a significant economic burden to health care systems. Previous research has showed that POI results from activation of resident macrophages in the muscularis of the intestine induced by intestinal manipulation during abdominal surgery. This event leads to an inflammatory cascade which greatly impacts the muscular function. A novel approach which could prevent the initiation of this inflammatory cascade (i.e. inhibition of macrophage activation) could thus be able to reduce the incidence of POI. In a murine model of POI, we and others previously showed that electrical stimulation of the cervical vagus nerve (VNS) has anti-inflammatory properties and reduces POI. Cervical VNS namely dampens the activation of resident macrophages via the binding of acetylcholine with α7 nicotinerge acetylcholine receptor leading to a reduced influx of inflammatory cells, a decreased pro-inflammatory gene expression in the muscularis externa and a fastened recovery of the GI tract. These data suggest that cervical VNS could be a novel therapeutic approach to shorten POI. Currently, cervical VNS is safely used in patients with epilepsy, rheumatoid arthritis and Crohn’s disease. In brief, a coiled electrode is surgically positioned around the cervical vagus nerve and connected to a pacemaker for daily and chronic stimulation. In our preclinical model however, one single period of electrical stimulation of the cervical vagus nerve already reduces GI inflammation and improves POI. Hence, adding an additional surgical procedure to isolate the cervical vagus nerve is not acceptable. The branches of the abdominal vagus nerve are however easily accessible at the hiatal diaphragm during abdominal surgery, and thus can be electrically stimulated without the need of an extra surgical procedure. In this thesis, the possibility of abdominal VNS was explored in detail in mouse and pig as alternative technique to stimulate the vagus nerve and thus treat POI and to subsequently translate this novel approach to clinical care. First, we compared the composition of cervical and abdominal vagus nerve (Chapter 1). We observed that the cervical vagus contained a significant higher percentage of myelinated fibers compared to the abdominal vagus nerve. In addition, the cervical vagus nerve was composed of 3 different myelinated fiber types (small, medium and large), while the abdominal vagus nerve only consisted of small myelinated fibers. This insights into the composition and type of vagal nerve fibers are important, since they could determine the optimal stimulation parameters. In Chapter 2, we therefore determined in a model of POI whether abdominal VNS has the same anti-inflammatory properties as cervical VNS using the same stimulation parameters for both approaches. This study showed that similar to cervical VNS, abdominal VNS improved intestinal transit and reduced manipulation-induced inflammation. In Chapter 3, we investigated which was the best approach to electrically stimulate the vagus nerve. To this end, we tested several electrodes in pigs and determined which electrode caused the greatest vagal activation using different stimulation parameters (Chapter 2-3). This study showed that abdominal VNS is best achievable with pacing wires within a 15 minute period and does not cause cardiovascular effects. Based on these findings, we conducted a pilot study in patients undergoing abdominal surgery. These patients were treated peroperatively with low or high frequency stimulation of the abdominal vagus nerve to determine which frequency had the greatest anti-inflammatory effect in human. We showed that high, but not low frequency stimulation of the abdominal nervus vagus led to anti-inflammatory effects in the blood as compared to sham-stimulated patients. Furthermore, this technique was safe in humans, i.e. VNS did not cause cardiovascular side effects or leakage of the anastomosis (Chapter 2). Recently, we and others also reported that the vagus nerve does not directly interact with the resident macrophages, but rather indirectly modulates them via enteric neurons. Therefore, we determined whether enteric neurons possess anti-inflammatory properties and contribute to the tolerogenic phenotype of muscularis macrophages. Using immunohistochemical stainings, we showed that muscularis macrophages are in close proximity to cholinergic enteric neurons both in murine and human ileum. Moreover, supernatant of enteric neuron cultures imprinted bone marrow derived macrophages with a tolerogenic (M2-like) phenotype similar to that of muscularis macrophages. Next, using live Ca2+ imaging of Cx3cr1GFP/wt muscularis macrophages in an isolated muscularis preparation, we showed that electrical or pharmacological (using the 5-HT4 receptor agonist prucalopride) stimulation of enteric neurons reduced ATP-induced activation of muscularis macrophages. To further explore the anti-inflammatory properties of enteric neurons, we evaluated the effect of VNS (acting via activation of enteric neurons) and treatment with prucalopride in a murine model of POI. Both treatments significantly reduced the surgery-induced inflammatory response and improved intestinal transit, a mechanism mediated by α7 and β2 nicotinic receptors located on the muscularis macrophages. Finally, we confirmed our findings in patients undergoing abdominal surgery. Preoperative administration of prucalopride decreased intestinal inflammation in the muscularis collected during surgery and significantly improved clinical recovery. Taken together, our data demonstrate that enteric neurons release mediators that install the tolerogenic phenotype and dampen the activation of muscularis macrophages. This anti-inflammatory property of enteric neurons should be further explored as new treatment for POI.nrpages: I-156status: publishe

Intestinal neuro-immune interactions: focus on macrophages, mast cells and innate lymphoid cells

Intestinal homeostasis relies on the reciprocal crosstalk betweenenteric neurons and immune cells, which together form neuro-immune units that occupy distinct anatomical niches within thegut.Here wewill review the recent advancesin our understandingof neuro-immune crosstalk within the gut, with focus onmacrophages, mast cells and innate lymphoid cells. In particular,we will discuss the role of neuron-immune cell crosstalk inhomeostasis, and how aberrant communication may underliedisease in the gastro-intestinal tract.status: publishe

The pig as preclinical model for laparoscopic vagus nerve stimulation

Cervical vagus nerve stimulation (VNS) prevents manipulation-induced intestinal inflammation and improves intestinal transit in a mouse model of postoperative ileus (POI). Cervical VNS, however, is accompanied by cardiovascular and respiratory side effects. In view of potential clinical application, we therefore evaluated the safety and feasibility of abdominal VNS via laparoscopic approach in a porcine model.status: publishe

The Versatile Role of the Vagus Nerve in the Gastrointestinal Tract

status: publishe

Intestinal macrophages and their interaction with the enteric nervous system in health and inflammatory bowel disease

Over the past decades, there has been an increasing understanding of cellular and molecular mechanisms that mediate modulation of the immune system by the autonomic nervous system. The discovery that vagal nerve stimulation (VNS) attenuates endotoxin-induced experimental sepsis paved the way for further studies investigating neuro-immune interaction. In particular, great attention is now given to intestinal macrophages: several studies report the existence of both intrinsic and extrinsic neural mechanisms by which intestinal immune homoeostasis can be regulated in different layers of the intestine, mainly by affecting macrophage activation through neurotransmitter release. Given the important role of inflammation in numerous disease processes, such as inflammatory bowel disease (IBD), cholinergic anti-inflammatory mechanisms are under intense investigation both from a basic and clinical science perspective in immune-mediated diseases such as IBD. This review discusses recent insights on the cross-talk between enteric neurons and the immune system, especially focusing on macrophages, and provides an overview of basic and translational aspects of the cholinergic anti-inflammatory response as therapeutic alternative to reinstall immune homoeostasis in intestinal chronic inflammation.status: publishe

Comparison between the cervical and abdominal vagus nerves in mice, pigs, and humans

Background Vagus nerve (VN) stimulation is currently evaluated as a novel approach to treat immune-mediated disorders. The optimal stimulation parameters, however, largely depend on the VN composition potentially impacting on its clinical translation. Hence, we evaluated whether morphological differences exist between the cervical and abdominal VNs across different species. Materials and methods The cervical and abdominal VNs of mouse, pig, and humans were stained for major basic protein and neurofilament F to identify the percentage and size of myelinated and non-myelinated fibers. Results The percentage of myelinated fibers was comparable between species, but was higher in the cervical VN compared with the abdominal VN. The cervical VN contained 54 +/- 4%, 47 +/- 7%, and 54 +/- 7% myelinated fibers in mouse, pig, and humans, respectively. The myelinated fibers consisted of small-diameter (mouse: 71%, pig: 80%, and humans: 63%), medium-diameter (mouse: 21%, pig: 18%, and humans: 33%), and large-diameter fibers (mouse: 7%, pig: 2%, and humans: 4%). The abdominal VN predominantly contained unmyelinated fibers (mouse: 93%, pig: 90%, and humans: 94%). The myelinated fibers mainly consisted of small-diameter fibers (mouse: 99%, pig: 85%, and humans: 74%) and fewer medium-diameter (mouse: 1%, pig: 13%, and humans: 23%) and large-diameter fibers (mouse: 0%, pig: 2%, and humans: 3%). Conclusion The VN composition was largely similar with respect to myelinated and unmyelinated fibers in the species studied. Human and porcine VNs had a comparable diameter and similar amounts of fibrous tissue and contained multiple fascicles, implying that the porcine VN may be suitable to optimize stimulation parameters for clinical trials

Vagus Nerve Stimulation dampens intestinal inflammation in a murine model of experimental Food Allergy.

Background: The vagus nerve has emerged as an important modulator of the intesti‐nal immune system. Its anti‐inflammatory properties have been previously shown in innate and Th1/Th17 predominant inflammatory models. To what extent the vagus nerve is of importance in Th2 inflammatory responses like food allergy is still unclear. In this study, we therefore aimed to investigate the effect of vagotomy (VGX) and vagus nerve stimulation (VNS), on the development and severity of experimental food allergy.Methods: Balb/C mice were first sensitized with ovalbumin (OVA) in the presence of alum. Prior to oral challenges with OVA, mice were subjected to VGX or VNS. Disease severity was determined by assessing severity and onset of diarrhoea, OVA‐specific antibody production, mast cell number and activity, inflammatory gene expression in duodenal tissue and lamina propria immune cells by flow cytometry analysis.Results: When compared to control mice, VGX did not significantly affect the devel‐opment and severity of the disease in our model of food allergy. VNS, on the other hand, resulted in a significant amelioration of the different inflammatory parameters assessed. This effect was independent of α7nAChR and is possibly mediated through the dampening of mast cells and increased phagocytosis of OVA by CX3CR1hi macrophages.Conclusions: These results underscore the anti‐inflammatory properties of the vagus nerve and the potential of neuro‐immune interactions in the intestine. Further insight into the underlying mechanisms could ultimately lead to novel therapeutic ap‐proaches in the treatment of not only food allergy but also other immune‐mediated diseases.status: publishe

Functional characterization of oxazolone-induced colitis and survival improvement by vagus nerve stimulation.

BACKGROUND:Oxazolone-induced colitis has been frequently used in literature as a model of IBD, but insights into the underlying immune response and pathological features are surprisingly still very limited. Vagus nerve stimulation (VNS) has proven to be effective in innate and Th1/Th17 predominant inflammatory models, including pre-clinical models of colitis, however to what extent VNS is also effective in ameliorating Th2-driven colitis remains to be studied. In the present study, we therefore further characterized the immune response in oxazolone-induced colitis and investigated the potential therapeutic effect of VNS. METHODS:Colitis was induced in Balb/c mice by cutaneous sensitization with 3% oxazolone followed by intracolonic administration of 1% oxazolone 7 days later. To evaluate the effect of VNS on the development of Th2-driven colitis, VNS and sham-treated mice were challenged with 1% oxazolone. RESULTS:Intracolonic oxazolone administration resulted in a severe destruction of the colonic mucosa and a rapid drop in body temperature leading to a 65% mortality rate at day 5. Severe infiltration of neutrophils and monocytes was detected 6h after oxazolone administration which was associated with a Th2-type inflammatory response. VNS significantly improved survival rate which correlated with decreased levels of HMGB1 and reduced colonic (il6 and cxcl1 mRNA) and serum cytokine levels (IL-6, TNFα and CXCL1) compared to sham treated mice. CONCLUSIONS:Oxazolone-induced colitis rather represents a model of sepsis and, at best, may resemble a severe type of ulcerative colitis, associated with early and severe mucosal damage and a high mortality rate. VNS reduces colonic inflammation and improves survival in this model, supporting the anti-inflammatory properties of VNS, even in an aggressive model as oxazolone-induced colitis