Giovanna Farro

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.

Abdominal vagus nerve stimulation as a new therapeutic approach to prevent postoperative ileus

Electrical stimulation of the cervical vagus nerve (VNS) prevents postoperative ileus (POI) in mice. As this approach requires an additional cervical procedure, we explored the possibility of peroperative abdominal VNS in mice and human.

Vagotomy affects the development of oral tolerance and increases susceptibility to develop colitis independently of the alpha-7 nicotinic receptor

Vagotomy (VGX) increases the susceptibility to develop colitis suggesting a crucial role for the cholinergic anti-inflammatory pathway in the regulation of the immune responses. Since oral tolerance and the generation of regulatory T cells (Tregs) are crucial to preserve mucosal immune homeostasis, we studied the effect of vagotomy and the involvement of α7 nicotinic receptors (α7nAChR) at the steady state and during colitis. Therefore, the development of both oral tolerance and colitis (induced by dextran sulfate sodium (DSS) or via T cell transfer) was studied in vagotomized mice and in α7nAChR−/− mice. VGX, but not α7nAChR deficiency, prevented oral tolerance establishment. This effect was associated with reduced Treg conversion in the lamina propria and mesenteric lymphnodes. To the same extent, vagotomized mice, but not α7nAChR−/− mice, developed a more severe DSS colitis compared with control mice treated with DSS, associated with a decreased number of colonic Tregs. However, neither VGX nor absence of α7nAChR in recipient mice affected colitis development in the T cell transfer model. In line, deficiency of α7nAChR exclusively in T cells did not influence the development of colitis induced by T cell transfer. Our results indicate a key role for the vagal intestinal innervation in the development of oral tolerance and colitis, most likely by modulating induction of Tregs independently of α7nAChR.

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

Objective 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. Design IM was performed in mice with defective monocyte migration to tissues (C-C motif chemokine receptor 2, Ccr2−/ − mice) and wild-type (WT) mice to study the role of monocytes and monocyte-derived macrophages (MΦs) during onset and resolution of ME inflammation. Results At early time points, IM-induced GI transit delay and inflammation were equal in WT and Ccr2 − / − mice. However, GI transit recovery after IM was significantly delayed in Ccr2 − / − mice compared with WT mice, associated with increased neutrophil-mediated immunopathology and persistent impaired neuromuscular function. During recovery, monocyte-derived MΦs acquire pro-resolving features that aided in the resolution of inflammation. In line, bone marrow reconstitution and treatment with MΦ colony-stimulating factor 1 enhanced monocyte recruitment and MΦ differentiation and ameliorated GI transit in Ccr2 − / − mice. Conclusion Our study reveals a critical role for monocyte-derived MΦs in restoring intestinal homeostasis after surgical trauma. From a therapeutic point of view, our data indicate that inappropriate targeting of monocytes may increase neutrophil-mediated immunopathology and prolong the clinical outcome of POI, while future therapies should be aimed at enhancing MΦ physiological repair functions.

Smooth muscle and neural dysfunction contribute to different phases of murine postoperative ileus

Postoperative ileus (POI) is characterized by a transient inhibition of gastrointestinal (GI) motility after abdominal surgery mediated by the inflammation of the muscularis externa (ME). The aim of this study was to identify alterations in the enteric nervous system that may contribute to the pathogenesis of POI.

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.

Ghrelin receptor modulates T helper cells during intestinal inflammation

The orexigenic peptide ghrelin has anti‐inflammatory properties in colitis, however, the mechanism of action and the immune cells targeted remain still to be elucidated. Here, we assessed the possible effect of ghrelin on T helper (Th) cells in a T cell transfer model of chronic colitis.

The Spleen Responds to Intestinal Manipulation but Does Not Participate in the Inflammatory Response in a Mouse Model of Postoperative Ileus

Background Postoperative ileus is characterized by a transient impairment of the gastrointestinal motility after abdominal surgery. The intestinal inflammation, triggered by handling of the intestine, is the main factor responsible for the prolonged dysmotility of the gastrointestinal tract. Secondary lymphoid organs of the intestine were identified as essential components in the dissemination of inflammation to the entire gastrointestinal tract also called field effect. The involvement of the spleen, however, remains unclear. Aim In this study, we investigated whether the spleen responds to manipulation of the intestine and participates in the intestinal inflammation underlying postoperative ileus. Methods Mice underwent Laparotomy (L) or Laparotomy followed by Intestinal Manipulation (IM). Twenty-four hours later, intestinal and colonic inflammation was assessed by QPCR and measurement of the intestinal transit was performed. Analysis of homeostatic chemokines in the spleen was performed by QPCR and splenic cell populations analysed by Flow Cytometry. Blockade of the egress of cells from the spleen was performed by administration of the Sphingosine-1-phosphate receptor 1 (S1P1) agonist CYM-5442 10 h after L/IM. Results A significant decrease in splenic weight and cellularity was observed in IM mice 24 h post-surgery, a phenomenon associated with a decreased splenic expression level of the homeostatic chemokine CCL19. Splenic denervation restored the expression of CCL19 and partially prevented the reduction of splenocytes in IM mice. Treatment with CYM-5442 prevented the egress of splenocytes but did not ameliorate the intestinal inflammation underlying postoperative ileus. Conclusions Intestinal manipulation results in two distinct phenomena: local intestinal inflammation and a decrease in splenic cellularity. The splenic response relies on an alteration of cell trafficking in the spleen and is partially regulated by the splenic nerve. The spleen however does not participate in the intestinal inflammation during POI.

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).