Microbiota-dependent presence of murine enteric glial cells requires myeloid differentiation primary response protein 88 signaling.

Enteric glial cells (EGCs) were shown to maintain the barrier integrity and immune homeostasis of the bowel. Postnatally, EGCs develop from progenitor cells located in the myenteric plexus and are continuously replenished through adulthood. Both, murine EGC development and replenishment were shown to depend on the microbiome. The underlying mechanisms are still unknown, and we hypothesized that the myeloid differentiation primary response protein 88 (Myd88) or toll-like receptor signaling pathways may be involved. Adult and neonatal C57BL/6 wild-type (wt) and Myd88-/- mice were housed under specific pathogen-free (SPF) or germ-free (GF) conditions. GF mice were further conventionalized by gavaging stools from, and cohousing with, SPF mice having intact microbiomes. The small bowels were harvested at various time points, and immunohistochemistry and qPCR analysis of EGC markers in the muscularis externa and mucosa were performed. In wt mice, after conventionalization, the glial cell-specific markers, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein ß (S100ß), were upregulated in the mucosa and muscularis externa. In Myd88-/- mice, this upregulation did not occur. Importantly, GFAP (only in the mucosa) and S100ß (in both the mucosa and muscularis externa) were significantly reduced in conventionalized Myd88-/- mice compared with the conventionalized wt mice. In neonatal mice, the gene expressions of GFAP and S100ß increased between the day of birth (P0) and postnatal day 15 (P15) in the mucosa and muscularis externa of both wt and Myd88-/- mice. Notably, in the mucosa but not the muscularis externa, at P15, the gene expressions of GFAP and S100ß were significantly reduced in Myd88-/-. Our data demonstrated that postnatal development and replenishment of EGCs require intestinal microbiota and depend on Myd88. The specific upstream mechanisms may involve toll-like-receptor recognition of the microbiota and will be the subject of further research.

Treatment with the 5-Lipoxygenase Antagonist Zileuton Protects Mice from Postoperative Ileus.

INTRODUCTION: Previous work of our group showed that lipoxygenase (LOX) pathways become activated upon surgical manipulation of the bowel wall and revealed a beneficial immune modulating role of the LOX-derived anti-inflammatory mediator protectin DX in postoperative ileus (POI). While we found a particular role of 12/15-LOX in the anti-inflammatory LOX action during POI, the role of 5-LOX, which produces the pro-inflammatory leukotriene B4 (LTB4), remained unknown. The purpose of this study was to investigate the role of 5-LOX within the pathogenesis of POI in a mouse model. METHODS: POI was induced by intestinal manipulation (IM) of the small bowel in C57BL/6, 5-LOX-/-, and CX3CR1GFP/+. Mice were either treated with a vehicle or with the synthetic 5-LOX antagonist zileuton or were left untreated. Cellular localization of 5-LOX and LTB4 release were visualized by immunofluorescence or ELISA, respectively. POI severity was quantified by gastrointestinal transit (GIT) and leukocyte extravasation into the muscularis externa (ME) by immunohistochemistry. RESULTS: 5-LOX expression was detected 24 h after IM within infiltrating leukocytes in the ME. LTB4 levels increased during POI in wild type but not in 5-LOX-/- after IM. POI was ameliorated in 5-LOX-/- as shown by decreased leukocyte numbers and normalized GIT. Zileuton normalized the postoperative GIT and reduced the numbers of infiltrating leukocytes into the ME. DISCUSSION/CONCLUSION: Our data demonstrate that 5-LOX and its metabolite LTB4 play a crucial role in POI. Genetic deficiency of 5-LOX and pharmacological antagonism by zileuton protected mice from POI. 5-LOX antagonism might be a promising target for prevention of POI in surgical patients.

Sympathetic Denervation Alters the Inflammatory Response of Resident Muscularis Macrophages upon Surgical Trauma and Ameliorates Postoperative Ileus in Mice.

Interactions between the peripheral nervous system and resident macrophages (MMs) modulate intestinal homeostatic functions. Activation of ß2-adrenergic receptors on MMs has been shown to reduce bacterial challenges. These MMs are also crucial for the development of bowel inflammation in postoperative ileus (POI), an iatrogenic, noninfectious inflammation-based motility disorder. However, the role of the sympathetic nervous system (SNS) in the immune modulation of these MMs during POI or other noninfectious diseases is largely unknown. By employing 6-OHDA-induced denervation, we investigated the changes in the muscularis externa by RNA-seq, quantitative PCR, and flow cytometry. Further, we performed transcriptional phenotyping of sorted CX3CR1+ MMs and ex vivo LPS/M-CSF stimulation on these MMs. By combining denervation with a mouse POI model, we explored distinct changes on CX3CR1+ MMs as well as in the muscularis externa and their functional outcome during POI. Our results identify SNS as an important mediator in noninfectious postoperative inflammation. Upon denervation, MMs anti-inflammatory genes were reduced, and the muscularis externa profile is shaped toward a proinflammatory status. Further, denervation reduced MMs anti-inflammatory genes also in the early phase of POI. Finally, reduced leukocyte infiltration into the muscularis led to a quicker recovery of bowel motility in the late phase of POI.

A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation.

Enteric glial cells (EGC) modulate motility, maintain gut homeostasis, and contribute to neuroinflammation in intestinal diseases and motility disorders. Damage induces a reactive glial phenotype known as "gliosis", but the molecular identity of the inducing mechanism and triggers of "enteric gliosis" are poorly understood. We tested the hypothesis that surgical trauma during intestinal surgery triggers ATP release that drives enteric gliosis and inflammation leading to impaired motility in postoperative ileus (POI). ATP activation of a p38-dependent MAPK pathway triggers cytokine release and a gliosis phenotype in murine (and human) EGCs. Receptor antagonism and genetic depletion studies revealed P2X2 as the relevant ATP receptor and pharmacological screenings identified ambroxol as a novel P2X2 antagonist. Ambroxol prevented ATP-induced enteric gliosis, inflammation, and protected against dysmotility, while abrogating enteric gliosis in human intestine exposed to surgical trauma. We identified a novel pathogenic P2X2-dependent pathway of ATP-induced enteric gliosis, inflammation and dysmotility in humans and mice. Interventions that block enteric glial P2X2 receptors during trauma may represent a novel therapy in treating POI and immune-driven intestinal motility disorders.

Author Correction: AIM2 inflammasome-derived IL-1ß induces postoperative ileus in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Population of Radio-Resistant Macrophages in the Deep Myenteric Plexus Contributes to Postoperative Ileus Via Toll-Like Receptor 3 Signaling.

Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.

AIM2 inflammasome-derived IL-1ß induces postoperative ileus in mice.

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL) -1α and IL-1ß and focused on the mechanism of IL-1ß production. IL-1α and IL-1ß deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1α originated from radio-resistant cells while IL-1ß was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1ß production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1ß production. Our study provides new insights into distinct roles of IL-1α and IL-1ß signalling during POI. While IL-1α release is most likely an immediate passive response to the surgical trauma, IL-1ß production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients.

A comparative study about the immunomodulatory effects of tramadol and metamizole in a murine model of postoperative ileus.

Postoperative ileus (POI) is a common complication after abdominal surgery characterized by motility disturbances leading to increased morbidity and mortality in surgical patients. Intestinal manipulation of the murine small bowel is an established animal model resulting in an increased postsurgical inflammation within the intestinal muscular externa and a delayed gastrointestinal transit. Some analgesics have been shown to affect inflammation. In this study, we compared the immunomodulatory effects of two different analgesics. Mice were treated with tramadol, metamizole or saline as a control in our established POI model. The postoperative inflammatory response was assessed by gene expression of pro-inflammatory cytokines at different time points and immunocytes extravasation into the muscularis externa. Functional motility analyses were performed by a gastrointestinal transit measurement. Metamizole application reduced the pro-inflammatory response after surgery and improved gastrointestinal motility, while tramadol showed no alteration in cytokine gene expression, influx of immunocytes and gastrointestinal transit compared with the controls. In conclusion. we suggest tramadol as analgesia in immunological studies on POI in mice as it does not affect the underlying inflammation of POI.

Leukocyte-Derived Interleukin-10 Aggravates Postoperative Ileus.

Objective: Postoperative ileus (POI) is an inflammation-mediated complication of abdominal surgery, characterized by intestinal dysmotility and leukocyte infiltration into the muscularis externa (ME). Previous studies indicated that interleukin (IL)-10 is crucial for the resolution of a variety of inflammation-driven diseases. Herein, we investigated how IL-10 affects the postoperative ME inflammation and found an unforeseen role of IL-10 in POI. Design: POI was induced by a standardized intestinal manipulation (IM) in C57BL/6 and multiple transgenic mouse strain including C-C motif chemokine receptor 2-/-, IL-10-/-, and LysMcre/IL-10fl/fl mice. Leukocyte infiltration, gene and protein expression of cytokines, chemokines, and macrophage differentiation markers as well as intestinal motility were analyzed. IL-10 serum levels in surgical patients were determined by ELISA. Results: IL-10 serum levels were increased in patient after abdominal surgery. In mice, a complete or leucocyte-restricted IL-10 deficiency ameliorated POI and reduced the postoperative ME neutrophil infiltration. Infiltrating monocytes were identified as main IL-10 producers and undergo IL-10-dependent M2 polarization. Interestingly, M2 polarization is not crucial to POI development as abrogation of monocyte infiltration did not prevent POI due to a compensation of the IL-10 loss by resident macrophages and neutrophils. Organ culture studies demonstrated that IL-10 deficiency impeded neutrophil migration toward the surgically traumatized ME. This mechanism is mediated by reduction of neutrophil attracting chemokines. Conclusion: Monocyte-derived macrophages are the major IL-10 source during POI. An IL-10 deficiency decreases the postoperative expression of neutrophil-recruiting chemokines, consequently reduces the neutrophil extravasation into the postsurgical bowel wall, and finally protects mice from 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-1ß 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-1ß 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.

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.

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-1ß 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-1ß 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-1ß 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-1ß 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-1ß 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-1ß 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.

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.

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-1alpha, interleukin (IL)1beta, 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-1alpha, IL1beta, 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.