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Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis. [ABSTRACT FROM AUTHOR]

sAPP, the secretory domain of the β-amyloid precursor protein (APP), exerts a growth promoting and motogenic activity on keratinocytes. Here we report on the expression of APP and its homologue, the amyloid precursor like protein 2 (APLP2), during cutaneous wound repair using a full-thickness excisional wound healing model in mice. In unwounded skin APP was predominantly expressed in the basal cell layer. During wound healing increased suprabasal expression of APP was observed in all cell layers of the hyperproliferative epithelium at the wound margin. APP mRNA was increased up to 2.3-fold, whereas the APLP2 mRNA was decreased. Immunocytochemically, all proliferation competent keratinocytes of the normal as well as the wound site epidermis showed increased expression of APP but not of APLP2. Using culture models of keratinocyte differentiation the release of sAPP was found to be significantly higher in proliferating cells, i.e., when cultured at subconfluency or at low [Ca2+], than in quiescent, partially differentiated keratinocytes cultured at confluency or at high [Ca2+]. Our results suggest that sAPP secretion is presumably also increased in proliferation competent keratinocytes of the wound margin and that sAPP due to its growth promoting and motogenic function might participate in the control of epidermal wound repair. [Copyright &y& Elsevier]

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. Activation of the cholinergic anti‐inflammatory 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. [ABSTRACT FROM AUTHOR]

Innervation of the intestinal mucosa by the sympathetic nervous system is well described but the effects of adrenergic receptor stimulation on the intestinal epithelium remain equivocal. We therefore investigated the effect of sympathetic neuronal activation on intestinal cells in mouse models and organoid cultures, to identify the molecular routes involved. Using publicly available single-cell RNA sequencing datasets we show that the α2A isoform is the most abundant adrenergic receptor in small intestinal epithelial cells. Stimulation of this receptor with norepinephrine or a synthetic specific α2A receptor agonist promotes epithelial proliferation and stem cell function, while reducing differentiation in vivo and in intestinal organoids. In an anastomotic healing mouse model, adrenergic receptor α2A stimulation resulted in improved anastomotic healing, while surgical sympathectomy augmented anastomotic leak. Furthermore, stimulation of this receptor led to profound changes in the microbial composition, likely because of altered epithelial antimicrobial peptide secretion. Thus, we established that adrenergic receptor α2A is the molecular delegate of intestinal epithelial sympathetic activity controlling epithelial proliferation, differentiation, and host defense. Therefore, this receptor could serve as a newly identified molecular target to improve mucosal healing in intestinal inflammation and wounding. [ABSTRACT FROM AUTHOR]

Enteric glia have been recently recognized as key components of the colonic tumor microenvironment indicating their potential role in colorectal cancer pathogenesis. Although enteric glia modulate immune responses in other intestinal diseases, their interaction with the colorectal cancer immune cell compartment remains unclear. Through a combination of single-cell and bulk RNA-sequencing, both in murine models and patients, here we find that enteric glia acquire an immunomodulatory phenotype by bi-directional communication with tumor-infiltrating monocytes. The latter direct a reactive enteric glial cell phenotypic and functional switch via glial IL-1R signaling. In turn, tumor glia promote monocyte differentiation towards pro-tumorigenic SPP1 + tumor-associated macrophages by IL-6 release. Enteric glia cell abundancy correlates with worse disease outcomes in preclinical models and colorectal cancer patients. Thereby, our study reveals a neuroimmune interaction between enteric glia and tumor-associated macrophages in the colorectal tumor microenvironment, providing insights into colorectal cancer pathogenesis., (© 2024. The Author(s).)

Background and Purpose: ET-1 signalling modulates intestinal motility and inflammation, but the role of ET-1/ET B receptor signalling is poorly understood. Enteric glia modulate normal motility and inflammation. We investigated whether glial ET B signalling regulates neural-motor pathways of intestinal motility and inflammation., Experimental Approach: We studied ET B signalling using: ET B drugs (ET-1, SaTX, BQ788), activity-dependent stimulation of neurons (high K + -depolarization, EFS), gliotoxins, Tg (Ednrb-EGFP)EP59Gsat/Mmucd mice, cell-specific mRNA in Sox10 CreERT2 ;Rpl22-HAflx or ChAT Cre ;Rpl22-HAflx mice, Sox10 CreERT2 ::GCaMP5g-tdT, Wnt1 Cre2 ::GCaMP5g-tdT mice, muscle tension recordings, fluid-induced peristalsis, ET-1 expression, qPCR, western blots, 3-D LSM-immunofluorescence co-labelling studies in LMMP-CM and a postoperative ileus (POI) model of intestinal inflammation., Key Results: In the muscularis externa ET B receptor is expressed exclusively in glia. ET-1 is expressed in RiboTag (ChAT)-neurons, isolated ganglia and intra-ganglionic varicose-nerve fibres co-labelled with peripherin or SP. ET-1 release provides activity-dependent glial ET B receptor modulation of Ca 2+ waves in neural evoked glial responses. BQ788 reveals amplification of glial and neuronal Ca 2+ responses and excitatory cholinergic contractions, sensitive to L-NAME. Gliotoxins disrupt SaTX-induced glial-Ca 2+ waves and prevent BQ788 amplification of contractions. The ET B receptor is linked to inhibition of contractions and peristalsis. Inflammation causes glial ET B up-regulation, SaTX-hypersensitivity and glial amplification of ET B signalling. In vivo BQ788 (i.p., 1 mg·kg -1 ) attenuates intestinal inflammation in POI., Conclusion and Implications: Enteric glial ET-1/ET B signalling provides dual modulation of neural-motor circuits to inhibit motility. It inhibits excitatory cholinergic and stimulates inhibitory nitrergic motor pathways. Amplification of glial ET B receptors is linked to muscularis externa inflammation and possibly pathogenic mechanisms of POI., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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.

Background: Transcriptional profiling of specific intestinal cell populations under health and disease is generally based on traditional sorting approaches followed by gene expression analysis. Therein, specific cell populations are identified either by expressing reporter genes under a cell type-specific promotor or by specific surface antigens. This method provides adequate results for blood-derived and tissue-resident immune cells. However, in stromal cell analysis, cellular stress due to digestion often results in degraded RNA. Particularly, ramified cells integrated into the tissue, such as enteric neurons and glial cells, suffer from these procedures. These cell types are involved in various intestinal processes, including a prominent immune-regulatory role, which requires suitable approaches to generate cell-specific transcriptional profiles., Methods: Sox10 iCreERT2 and choline acetyltransferase (ChAT Cre ) mice were crossed with mice labeling the ribosomal Rpl22 protein upon Cre activity with a hemagglutinin tag (Rpl22-HA, termed RiboTag). This approach enabled cellular targeting of enteric glia and neurons and the immediate isolation of cell-specific mRNA from tissue lysates without the need for cell sorting., Key Results: We verified the specific expression of Rpl22-HA in enteric glia and neurons and provided gene expression data demonstrating a successful enrichment of either Sox-10 + glial or ChAT + neuronal mRNAs by the RiboTag-mRNA procedure using qPCR and RNA-Seq analysis., Conclusions and Inferences: We present a robust and selective protocol that allows the generation of cell type-specific transcriptional in vivo snapshots of distinct enteric cell populations that will be especially useful for various intestinal disease models involving peripheral neural cells., (© 2021 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Macrophages reside in a dense cellular network in the intestinal muscularis externa, and there is emerging evidence that the functionality of these cells determines the local microenvironment. Inflammatory responses during intestinal diseases change the homeostatic functionality of these cells causing inflammation and intestinal dysmotility. Such disturbances are not only induced by a change in the cellular composition in the intestinal muscularis but also by an altered crosstalk with the peripheral and central nervous system. In this review, we summarize the role of muscularis macrophages in the intestine in homeostasis and inflammation. We compare the functionality, the phenotype, and the origin of muscularis macrophages to their neighboring counterparts within the different layers of the intestine. We outline the cellular crosstalk with the enteric and the peripheral nervous system and summarize the current therapeutic approaches to modulate the functionality of these phagocytes.

Purpose: In recent years, multimodal treatment approaches have led to an increased median survival time of patients with colorectal liver metastases. In particular, this results from new perioperative chemotherapy regimens, which in turn are accompanied by an increased risk of perioperative bleeding and/or liver failure due to the hepatotoxic side effects. Nineteen to 58 % of patients treated with oxaliplatin develop sinusoidal obstruction syndrome (SOS). The influence of preexisting SOS on liver surgery remains controversial., Methods: Animals were operated 4 days after SOS induction with monocrotaline and received either vascular occlusion in the form of Pringle maneuver (PM) or hepatectomy (LR; 70 %) or a combination of both (LR + PM). Postoperative liver function was assessed by determination of liver enzyme levels, bile production, and tissue oxygen saturation., Results: Preexisting SOS impaired morbidity after liver resection, reflected by elevated liver enzyme levels, reduced bile secretion, and low liver tissue oxygenation levels. Mortality was increased by up to 25 %. Additional ischemia in the form of PM showed no further impact in the LR ± PM group compared to LR alone., Conclusion: PM without LR results in high enzyme distribution in the SOS group. SOS significantly affects the outcome after liver resection in our experimental rat model only without PM and showed no protective effect in ischemia in the form of PM.

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4 Cre mice were crossed to ChAT fl/fl mice to achieve specific deletion of ChAT in CD4 + T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor β 2 To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT -/- mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT -/- mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansion., (Copyright © 2016 the American Physiological Society.)

Aims: Postoperative ileus (POI) is a frequent complication after abdominal surgery, resulting from an inflammation of the muscularis externa (ME). So far no valid biomarker for occurrence, duration or intensity of POI exists. Extravasation of monocytes and neutrophils from blood circulating into the postoperative ME is well known as a hallmark of POI. In a previous study we demonstrated that a low abundant subset of TH1 cells, activated by IL-12, can be detected in the peripheral blood of a small subset of patients in response to abdominal surgery. The aim of the present study was to investigate if these specific TH1 cells, IL-12 or circulating leukocyte levels could act as a valid marker for POI occurrence., Main Methods: At different time points, blood samples of patients undergoing abdominal or extraabdominal surgery were collected. Serum levels of IL-12 or TH1 cells as well as neutrophils and monocytes were analyzed. Data were compared between both groups and correlated with clinical signs of POI., Key Findings: Time until first flatus and defecation as well as solid food tolerances are delayed after abdominal compared to extraabdominal surgery. Circulating IL-12 levels and numbers of TH1 cells, neutrophils and monocytes did not differ between both groups., Significance: While previous experiments indicated that specific TH1 cells play a crucial role in POI dissemination, our present data from a larger human cohort demonstrate that they do not seem to be suitable to distinguish between abdominal and extraabdominal surgery. Furthermore neither TH1 cells nor leukocytes or serum IL-12 levels are appropriate biomarkers for POI in a clinical setting., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims: The endocannabinoid system and cannabinoid receptor 2 (CB2 receptor) have been associated with modulation of inflammatory response and myocardial adaptation after ischemic injury. In order to elucidate CB2 receptor-related effects during cellular interactions, we investigated cardiomyocyte survival and macrophage function in vitro., Main Methods: Murine embryonic (eCM) and adult (CM) cardiomyocytes, murine macrophages (MO), and their subtypes M1 (M1-MO) and M2 (M2-MO) were derived from wildtype- (WT) and CB2 receptor-deficient (Cnr2(-/-)) mice. Cells were cultured separately or in co-culture under normoxia or hypoxia (2% O2) and pro-inflammatory stimulation using interferon (IFN)γ. Besides immunohistochemistry, we also measured mRNA expression (Taqman®) and performed FACS-analysis of cardiomyocytes. Macrophage migration was assessed using Boyden chamber assay., Key Findings: We found a significant induction of CB2 receptor mRNA and protein in murine eCM as well as M1- and M2-MO in vitro following cultivation under hypoxia or stimulation with IFNγ. A significantly higher amount of apoptotic Cnr2(-/-)-CMs was found after incubation under hypoxia when compared to WT-CMs. We observed a significantly stronger migration potential in Cnr2(-/-)-M1-MOs towards the supernatant of apoptotic CM, than in corresponding WT-cells. Co-culture revealed a significantly higher loss of eCMs and induction of their apoptosis after cultivation with Cnr2(-/-)-M1-MOs. Production of TNF-α in M1-MOs was dependent on CB2 receptor stimulation by anandamide., Significance: Our data provide novel insights into CB2 receptor-mediated protection of cardiomyocytes during hypoxia and pro-inflammatory stimulation. We show CB2 receptor-dependent effects on migration and function of M1-MOs in interaction with cardiomyocytes, thereby influencing their survival., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background/aims: Ischemia reperfusion (I/R) injury after small bowel transplantation leads to inflammatory reactions and loss of structural integrity with subsequent graft contractile dysfunction in the early postoperative phase. The natural tetrahydropyrimidine ectoine (1-,4-,5-,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid; THP) protects the ileal mucosa and muscularis against effects of I/R injury in an experimental model of isolated graft reperfusion. The effects of THP treatment were evaluated in an established experimental intestinal transplant model., Methods: Isogenic, orthotopic small bowel transplantation was performed in Lewis rats (6 h cold ischemia time). Perioperative THP treatment (intraluminal/intravascular) groups were compared to vehicle-treated animals (after 3 and 24 h) and non-transplanted controls (n = 5/group). Park's score defined the effects of I/R injury. The infiltration of neutrophils, monocytes and macrophages, mRNA expression of IL-6 and TNF-α, serum levels of IL-6 and NO and smooth muscle contractility were evaluated., Results: Improved graft outcome after intraluminal and intravascular THP treatment was defined by considerably ameliorated neutrophil infiltration and less histological signs of I/R injury (p ≤ 0.05). In the presence of THP, mRNA expression of IL-6 and TNF-α and IL-6 and NO serum levels were reduced and smooth muscle function was improved., Conclusion: THP treatment offers protection against the effects of I/R injury in intestinal transplantation in vivo, however, only as supplementary treatment option., (Copyright © 2012 S. Karger AG, Basel.)

Abnormal cleavage of amyloid precursor protein (APP) in the central nervous system has been linked to the development of Alzheimer's disease. Recent work has identified additional roles for APP in peripheral tissue, such as cellular proliferation and motility. APP undergoes proteolytic processing to release a soluble NH(2)-terminal ectodomain fragment (sAPP), an Abeta or p3 peptide, and cytosolic COOH-terminal fragments. We have identified the up-regulation of APP expression in pancreatic cancer cells both in vitro and in vivo. APP undergoes high levels of proteolytic processing in pancreatic cancer cells, and sAPP can be detected in collected medium in vitro. Inhibition of sAPP signaling reduces pancreatic cancer cell number via a reduction in cellular proliferation. We propose that APP may function to promote growth in pancreatic cancer cells via signaling through sAPP and may therefore represent a novel therapeutic target in pancreatic cancer.