AIM2 inflammasome-derived IL-1 beta 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)-1 alpha and IL-1 beta and focused on the mechanism of IL-1 beta production. IL-1 alpha and IL-1 beta deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1 alpha originated from radio-resistant cells while IL-1 beta 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-1 beta production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1 beta production. Our study provides new insights into distinct roles of IL-1 alpha and IL-1 beta signalling during POI. While IL-1 alpha release is most likely an immediate passive response to the surgical trauma, IL-1 beta 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

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 β2-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 motil-ity 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 denerva-tion 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 im-portant mediator in noninfectious postoperative inflammation. Upon denervation, MMs anti-in-flammatory genes were reduced, and the muscularis externa profile is shaped toward a proinflam-matory 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

Non-invasive transcutaneous auricular vagus nerve stimulation prevents postoperative ileus and endotoxemia in mice

Background: The cholinergic anti-inflammatory pathway comprises the perception of peripheral inflammation by afferent sensory neurons and reflex activation of efferent vagus nerve activity to regulate inflammation. Activation of this pathway was shown to reduce the inflammatory response and improve outcome of postoperative ileus (POI) and sepsis in rodents. Herein, we tested if a non-invasive auricular electrical transcutaneous vagus nerve stimulation (tVNS) affects inflammation in models of POI or endotoxemia. Methods: Mice underwent tVNS or sham stimulation before and after induction of either POI by intestinal manipulation (IM) or endotoxemia by lipopolysaccharide administration. Some animals underwent a preoperative right cervical vagotomy. Neuronal activation of the solitary tract nucleus (NTS) and the dorsal motor nucleus of the vagus nerve (DMV) were analyzed by immunohistological detection of c-fos+ cells. Gene and protein expression of IL-6, MCP-1, IL-1β as well as leukocyte infiltration and gastrointestinal transit were analyzed at different time points after IM. IL-6, TNFα, and IL-1β serum levels were analyzed 3 hours after lipopolysaccharide administration. Results: tVNS activated the NTS and DMV and reduced intestinal cytokine expression, reduced leukocyte recruitment to the manipulated intestine segment, and improved gastrointestinal transit after IM. Endotoxemia-induced IL-6 and TNF-α release was also reduced by tVNS. The protective effects of tVNS on POI and endotoxemia were abrogated by vagotomy. Conclusion: tVNS prevents intestinal and systemic inflammation. Activation of the DMV indicates an afferent to efferent central circuitry of the tVNS stimulation and the beneficial effects of tVNS depend on an intact vagus nerve. tVNS may become a non-invasive approach for treatment of POI

Sympathetic Innervation Modulates Mucosal Immune Homeostasis and Epithelial Host Defense

Intestinal mucosal cells, such as resident macrophages and epithelial cells, express adrenergic receptors and are receptive to norepinephrine, the primary neurotransmitter of the sympathetic nervous system (SNS). It has been suggested that the SNS affects intestinal immune activity in conditions, such as inflammatory bowel disease; however, the underlying mechanisms remain ambiguous. Here, we investigated the effect of SNS on mucosal immune and epithelial cell functions. We employed 6-OHDA-induced sympathetic denervation (cSTX) to characterize muscularis-free mucosal transcriptomes by RNA-seq and qPCR, and quantified mucosal immune cells by flow cytometry. The role of norepinephrine and cytokines on epithelial functions was studied using small intestinal organoids. cSTX increased the presence of activated CD68+CD86+ macrophages and monocytes in the mucosa. In addition, through transcriptional profiling, the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ were induced, while Arg-1 and CD163 expression was reduced. Further, cSTX increased intestinal permeability in vivo and induced genes involved in barrier integrity and antimicrobial defense. In intestinal organoids, similar alterations were observed after treatment with proinflammatory cytokines, but not norepinephrine. We conclude that a loss in sympathetic input induces a proinflammatory mucosal state, leading to reduced epithelial barrier functioning and enhanced antimicrobial defense. This implies that the SNS might be required to maintain intestinal immune functions during homeostasis

Sympathetic Innervation Modulates Mucosal Immune Homeostasis and Epithelial Host Defense

Intestinal mucosal cells, such as resident macrophages and epithelial cells, express adrenergic receptors and are receptive to norepinephrine, the primary neurotransmitter of the sympathetic nervous system (SNS). It has been suggested that the SNS affects intestinal immune activity in conditions, such as inflammatory bowel disease; however, the underlying mechanisms remain ambiguous. Here, we investigated the effect of SNS on mucosal immune and epithelial cell functions. We employed 6-OHDA-induced sympathetic denervation (cSTX) to characterize muscularis-free mucosal transcriptomes by RNA-seq and qPCR, and quantified mucosal immune cells by flow cytometry. The role of norepinephrine and cytokines on epithelial functions was studied using small intestinal organoids. cSTX increased the presence of activated CD68+CD86+ macrophages and monocytes in the mucosa. In addition, through transcriptional profiling, the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ were induced, while Arg-1 and CD163 expression was reduced. Further, cSTX increased intestinal permeability in vivo and induced genes involved in barrier integrity and antimicrobial defense. In intestinal organoids, similar alterations were observed after treatment with proinflammatory cytokines, but not norepinephrine. We conclude that a loss in sympathetic input induces a proinflammatory mucosal state, leading to reduced epithelial barrier functioning and enhanced antimicrobial defense. This implies that the SNS might be required to maintain intestinal immune functions during homeostasis

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

<div><p>Aim</p><p>To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus.</p><p>Methods</p><p>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 <i>E</i>. <i>coli</i> and <i>B</i>. <i>vulgatus</i> was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay.</p><p>Results</p><p>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 <i>E</i>. <i>coli</i> 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.</p><p>Conclusion</p><p>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.</p></div

Mucosal IL-1R1 signaling is involved in POI.

<p>(A-C) WT mice underwent IM. Gene expression of (A) IL-1R1; (B) IL-1α and (C) IL-1β was analyzed in mucosal tissue of jejunal and ileal bowel segments after indicated time. (D) Basal gene expression of antimicrobial proteins was quantified in IL-1R1 deficient compared to control mice of jejunal and ileal mucosa. n = 5 for all groups. Statistical analysis was done by 1-way ANOVA, followed by Bonferroni post hoc test or t-test, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 vs. indicated groups.</p

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

Abstract 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

Primers used for qPCR.

<p>Primers used for qPCR.</p