Mariola Lysson

Inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents

Background: Abdominal surgery results in a molecular and cellular inflammatory response in the intestine, leading to postoperative ileus. It was hypothesised that resident macrophages within the intestinal muscularis have an important role in this local inflammation. Aims: To investigate whether chemical or genetic depletion of resident muscularis macrophages would lead to a reduction in the local inflammation and smooth-muscle dysfunction. Methods: Two rodent models were used to deplete and inactivate macrophages: (1) a rat model in which resident macrophages were depleted by chlodronate liposomes; (2) a model of mice with osteopetrosis mice, completely lacking the resident muscularis macrophages, used as an additional genetic approach. Animals with normal or altered intestinal macrophages underwent surgical intestinal manipulation. The inflammatory response was investigated by quantitative reverse transcriptase-polymerase chain reaction for mRNA of MIP-1α, interleukin (IL)1β, IL6, intracellular adhesion molecule 1 (ICAM-1) and monocyte chemotractant protein 1 (MCP)-1 in the isolated small bowel muscularis. In addition, muscularis whole mounts were used for histochemical and immunohistochemical analysis to quantify leucocyte infiltration and detect cytokine expression. Subsequently, in vitro muscle contractility and in vivo gastrointestinal transit were measured. Results: Both models resulted in markedly decreased expression of MIP-1α, IL1β, IL6, ICAM-1 and MCP-1 after manipulation compared with controls. In addition to this decrease in inflammatory mediators, recruitment of leucocytes into the muscularis was also diminished. Macrophage-altered animals had near normal in vitro jejunal circular muscle function and gastrointestinal transit despite surgical manipulation. Conclusions: Resident intestinal muscularis macrophages are initially involved in inflammatory responses resulting in postoperative ileus. Depletion and inactivation of the muscularis macrophage network prevents postoperative ileus.

Postoperative Ileus Involves Interleukin-1 Receptor Signaling in Enteric Glia

BACKGROUND & AIMS Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. METHODS We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. RESULTS Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. CONCLUSIONS IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

Mechanical strain and TLR4 synergistically induce cell-specific inflammatory gene expression in intestinal smooth muscle cells and peritoneal macrophages

Mechanical trauma of the gut is an unavoidable event in abdominal surgery. Former studies demonstrated that intestinal manipulation induces a strong inflammation within the tunica muscularis. We hypothesized that mechanical strain initiates or aggravates proinflammatory responses in intestinal smooth muscle cells (iSMC) or macrophages. First, an appropriate isolation and culture method for neonatal rat iSMC was established. Purified iSMC and primary peritoneal macrophages (pMacs) were subjected to static or cyclic strain, and gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6, and IL-1β was analyzed by quantitative PCR. Supernatants from stretched iSMC were transferred to untreated pMacs or contrariwise, and medium transfer-triggered inflammatory gene expression was measured in unstretched cells. Finally, we investigated the synergistic effect of static strain on LPS-induced proinflammatory gene expression. Although cyclic strain failed, static strain significantly induced iNOS, COX-2, and IL-1β mRNA in iSMC. pMacs showed an increase in all inflammatory genes investigated as well as macrophage inflammatory protein (MIP)-1α and MIP-2 mRNA after static strain. Both cell entities liberated unknown mediators in response to stretch that mutually stimulated iNOS gene expression. Finally, mechanostimulation amplified LPS-induced iNOS and IL-1β gene expression in iSMC as well as COX-2 and IL-6 mRNA in pMacs. In conclusion, static strain initiates proinflammatory gene expression in iSMC and pMacs and triggers a bidirectional paracrine communication between both cultured cell entities via the liberation of unknown mediators. Furthermore, static strain synergistically operates with Toll-like receptor 4 ligation in a cell-specific manner. Hence, this study demonstrates that mechanical strain functions as an immunomodulatory stimulus in abdominal cells.

Cathepsin B release from rodent intestine mucosa due to mechanical injury results in extracellular matrix damage in early post-traumatic phases.

Abstract An in vivo model was used to investigate the role of cathepsins in mouse intestine after mechanical manipulation. Inspection of different intestine segments by immunofluorescence microscopy provided evidence for a local release of cathepsin B from cells of individual gut sections shortly after traumatic injury. Densitometry of immunoblots ruled out alterations in cathepsin B expression levels. Because similar results were obtained with both mouse and rat intestine trauma models, we were interested in identifying potential targets of released cathepsin B in early post-traumatic phases. Immunoblotting revealed initial declines followed by an increase in protein levels of claudin-1 and E-cadherin, indicating that tight junctions and cell-cell adhesions were only transiently compromised by surgical trauma. Apical aminopeptidase N and dipeptidyl peptidase IV were only slightly affected, whereas basolateral low-density lipoprotein receptors were strongly up-regulated in response to trauma. As potential targets of cathepsin B released from injured cells, we identified collagen IV and laminin of the basement membrane that was damaged during initial post-traumatic stages. Because increased collagen IV expression was observed in the intestine of cathepsin B-deficient animals, we propose a direct role of cathepsin B in that it contributes to acute post-traumatic extracellular matrix damage and may thereby facilitate onset of post-operative ileus.

A role for 12/15-lipoxygenase-derived proresolving mediators in postoperative ileus: protectin DX-regulated neutrophil extravasation

Resolution of inflammation is an active counter‐regulatory mechanism involving polyunsaturated fatty acid‐derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15‐lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1+/Ly6C+ infiltrating monocytes rather than Ly6G+ neutrophils. Mass spectrometry analyses demonstrated that a 12/15‐lipoxygenase increase was accompanied by production of docosahexaenoic acid‐derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17‐hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood‐derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G+/Ly6C+ neutrophil extravasation after protectin DX treatment, whereas Ly6G‐/Ly6C+ monocyte numbers were not affected. 12/15‐lipoxygenase‐deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid‐rich lipid emulsion reduced postoperative leukocyte infiltration in wild‐type mice but failed in 12/15‐lipoxygenase‐deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15‐lipoxygenase‐deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15‐lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid‐derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15‐lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.

The novel CGRP receptor antagonist BIBN4096BS alleviates a postoperative intestinal inflammation and prevents postoperative ileus.

Abdominal surgery results in neuronal mediator release and subsequent acute intestinal hypomotility. This phase is followed by a longer lasting inflammatory phase resulting in postoperative ileus (POI). Calcitonin gene‐related peptide (CGRP) has been shown to induce motility disturbances and in addition may be a candidate mediator to elicit neurogenic inflammation. We hypothesized that CGRP contributes to intestinal inflammation and POI.

The novel orally active guanylhydrazone CPSI-2364 prevents postoperative ileus in mice independently of anti-inflammatory vagus nerve signaling

PurposePostoperative ileus (POI) is an iatrogenic complication of abdominal surgery, mediated by a severe inflammation of the muscularis externa (ME). Previously, we demonstrated that intravenous application of the tetravalent guanylhydrazone semapimod (CNI-1493) prevents POI, but the underlying mode of action could not definitively be confirmed. Herein, we investigated the effect of a novel orally active salt of semapimod (CPSI-2364) on POI in rodents and distinguished between its inhibitory peripheral and stimulatory central nervous effects on anti-inflammatory vagus nerve signaling.MethodsDistribution of radiolabeled orally administered CPSI-2364 was analyzed by whole body autoradiography and liquid scintillation counting. POI was induced by intestinal manipulation with or without preoperative vagotomy. CPSI-2364 was administered preoperatively via gavage in a dose- and time-dependent manner. ME specimens were assessed for p38-MAP kinase activity by immunoblotting, neutrophil extravasation, and nitric oxide production. Furthermore, in vivo gastrointestinal (GIT) and colonic transit were measured.ResultsAutoradiography demonstrated a near-exclusive detection of CPSI-2364 within the gastrointestinal wall and contents. Preoperative CPSI-2364 application significantly reduced postoperative neutrophil counts, nitric oxide release, GIT deceleration, and delay of colonic transit time, while intraoperatively administered CPSI-2364 failed to improve POI. CPSI-2364 also prevents postoperative neutrophil increase and GIT deceleration in vagotomized mice.ConclusionsOrally administered CPSI-2364 shows a near-exclusive dispersal in the gastrointestinal tract and effectively reduces POI independently of central vagus nerve stimulation. Its efficacy after single oral dosage affirms CPSI-2364 treatment as a promising strategy for prophylaxis of POI.

Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus

Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery.

Mo1158 IL-1 Receptor Signaling Mediates Postoperative Ileus in a Mouse Model of Intestinal Manipulation

neuromuscular function by increasing basal tone and response to cholinergic stimulus. These effects are not reversible after short-term observation. It is known that temperature affects both the ionic conductances and the rate constants modulating the action potential propagation in giant squid axon. We hypothesize that temperature gradients, by changing the ion dynamics, could influence the electrochemical behaviour of intestinal muscle, with a memory effect which should require long time to be deleted. Further studies are needed to establish the exact involved mechanisms in order to better understand clinical consequences of hypothermia during abdominal surgery.

Eine perioperative parenterale Nahrungsergänzung mehrfach-ungesättigter Fettsäuren (PUFA) vermindert die postoperative Darmatonie

Introduction: Abdominal surgery results in the inflammation of the tunica muscularis (ME), subsequently leading to postoperative ileus (POI) [1]. Previously, we demonstrated that inhibition of macrophage function and cytokine production prevents rodents from POI [2]. Omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are known to exert anti- or proinflammatory effects, respectively. The aim of this study was to analyze the effect of omega-3 and omega-6 PUFA on POI in rodents. Methods: Male rats and mice underwent intestinal manipulation (IM) after 5 days preoperative enteral or parenteral application of vehicle, omega-3 (Omegaven®) or omega-6 (Lipovenos®-MCT) PUFA-enriched emulsions (1 ml/100 g bodyweight, once daily). Erythrocyte membranes were analyzed by gas chromatography for fatty acid composition. Inflammatory activity was determined by detection of neutrophils 24 h after IM. Production of nitric oxide (NO) from ME specimen was determined by Griess reaction. Contractility of jejunal ME strips was measured in vitro in an organ bath setup. Gastrointestinal and colonic transits were determined in vivo. Results: Omegaven treatment significantly increased omega-3 to omega-6 PUFA rates in erythrocytes (1 : 9), compared to untreated controls (1 : 15). Furthermore, Omegaven reduced neutrophil levels (–35 %) and NO production (–38 %) after IM and improved contractility and gastrointestinal and colonic transit time. Interestingly, enteral Omegaven treatment showed increased NO production and tended toward increased neutrophils levels. In all experiments, Lipovenos-MCT treatment solely showed a trend to reduced inflammation and improved motility. Conclusion: Perioperative parenteral omega-3 PUFA enriched nutrition reduces intestinal inflammation and prevents postoperative ileus.