Project description:BACKGROUND Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS With ~4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glia reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity. CONCLUSIONS Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

Project description:BackgroundNiemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. The pathological mechanisms, underlying NPC1 are not yet completely understood. Especially the contribution of glial cells and gliosis to the progression of NPC1, are controversially discussed. As an analysis of affected cells is unfeasible in NPC1-patients, we recently developed an in vitro model system, based on cells derived from NPC1-patient specific iPSCs. Here, we asked if this model system recapitulates gliosis, observed in non-human model systems and NPC1 patient post mortem biopsies. We determined the amount of reactive astrocytes and the regulation of the intermediate filaments GFAP and vimentin, all indicating gliosis. Furthermore, we were interested in the assembly and phosphorylation of these intermediate filaments and finally the impact of the activation of protein kinase C (PKC), which is described to ameliorate the pathogenic phenotype of NPC1-deficient fibroblasts, including hypo-phosphorylation of vimentin and cholesterol accumulation.MethodsWe analysed glial cells derived from NPC1 patient specific induced pluripotent stem cells, carrying different NPC1 mutations. The amount of reactive astrocytes was determined by means of immuncytochemical stainings and FACS-analysis. Semi-quantitative western blot was used to determine the amount of phosphorylated GFAP and vimentin. Cholesterol accumulation was analysed by Filipin staining and quantified by Amplex Red Assay. U18666A was used to induce NPC1 phenotype in unaffected cells of the control cell line. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC.ResultsImmunocytochemical detection of GFAP, vimentin and Ki67 revealed that NPC1 mutant glial cells undergo gliosis. We found hypo-phosphorylation of the intermediate filaments GFAP and vimentin and alterations in the assembly of these intermediate filaments in NPC1 mutant cells. The application of U18666A induced not only NPC1 phenotypical accumulation of cholesterol, but characteristics of gliosis in glial cells derived from unaffected control cells. The application of phorbol 12-myristate 13-acetate, an activator of protein kinase C resulted in a significantly reduced number of reactive astrocytes and further characteristics of gliosis in NPC1-deficient cells. Furthermore, it triggered a restoration of cholesterol amounts to level of control cells.ConclusionOur data demonstrate that glial cells derived from NPC1-patient specific iPSCs undergo gliosis. The application of U18666A induced comparable characteristics in un-affected control cells, suggesting that gliosis is triggered by hampered function of NPC1 protein. The activation of protein kinase C induced an amelioration of gliosis, as well as a reduction of cholesterol amount. These results provide further support for the line of evidence that gliosis might not be only a secondary reaction to the loss of neurons, but might be a direct consequence of a reduced PKC activity due to the phenotypical cholesterol accumulation observed in NPC1. In addition, our data support the involvement of PKCs in NPC1 disease pathogenesis and suggest that PKCs may be targeted in future efforts to develop therapeutics for NPC1 disease.

Project description:IntroductionEnteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed.MethodsWe examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice.ResultsWe identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation.DiscussionTogether, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

Project description:Impaired gut motility may contribute, at least in part, to the development of systemic hyperammonemia and systemic neurological disorders in inherited metabolic disorders, or in severe liver and renal disease. It is not known whether enteric neurotransmission regulates intestinal luminal and hence systemic ammonia levels by induced changes in motility. Here, we propose and test the hypothesis that ammonia acts through specific enteric circuits to influence gut motility. We tested our hypothesis by recording the effects of ammonia on neuromuscular transmission in tissue samples from mice, pigs, and humans and investigated specific mechanisms using novel mutant mice, selective drugs, cellular imaging, and enzyme-linked immunosorbent assays. Exogenous ammonia increased neurogenic contractions and decreased neurogenic relaxations in segments of mouse, pig, and human intestine. Enteric glial cells responded to ammonia with intracellular Ca2+ responses. Inhibition of glutamine synthetase and the deletion of glial connexin-43 channels in hGFAP::CreERT2+/-/connexin43f/f mice potentiated the effects of ammonia on neuromuscular transmission. The effects of ammonia on neuromuscular transmission were blocked by GABAA receptor antagonists, and ammonia drove substantive GABA release as did the selective pharmacological activation of enteric glia in GFAP::hM3Dq transgenic mice. We propose a novel mechanism whereby local ammonia is operational through GABAergic glial signaling to influence enteric neuromuscular circuits that regulate intestinal motility. Therapeutic manipulation of these mechanisms may benefit a number of neurological, hepatic, and renal disorders manifesting hyperammonemia.NEW & NOTEWORTHY We propose that local circuits in the enteric nervous system sense and regulate intestinal ammonia. We show that ammonia modifies enteric neuromuscular transmission to increase motility in human, pig, and mouse intestine model systems. The mechanisms underlying the effects of ammonia on enteric neurotransmission include GABAergic pathways that are regulated by enteric glial cells. Our new data suggest that myenteric glial cells sense local ammonia and directly modify neurotransmission by releasing GABA.

Project description:Cardiometabolic risk may differ based on a stress-induced alpha(α)-adrenergic response versus a predominant beta(β)-adrenergic response. Whether these responses might serve as significant markers of distinct cardiometabolic risk profiles based on hemodynamic reactivity remain unknown. We (1) characterized predominant α-and β-adrenergic hemodynamic response patterns to acute mental stress; and (2) determined the cardiometabolic risk profile within predominant α-or β-adrenergic responders, irrespective of age, sex, or ethnicity. We included 117 South African teachers (aged 20-65 years) and administered an acute mental stress task (Color-Word-Conflict test) for one-minute. Participants' hemodynamic response profiles were characterized as predominant α-adrenergic (decreases in cardiac output (CO) and Windkessel arterial compliance (Cwk) (lowest quartile)) (n = 48) and β-adrenergic (increases in CO, Cwk (highest quartile)) responses (n = 69) via Finometer beat-to-beat hemodynamic monitoring. Ambulatory-BP was measured and the number of 24 H-ischemic events determined by ECG. Cardiometabolic markers were analyzed using fasting blood samples, and abnormal glucose tolerance (Abnl-GT), combining prediabetes and diabetes, was defined as glycated hemoglobin (HbA1c) ≥ 5.7% and/or fasting glucose > 100 mg/dL and/or diabetes medication usage. Predominant α-adrenergic responders presented with an overall poorer cardiometabolic profile, with higher levels of HbA1c, insulin, greater insulin resistance and higher total cholesterol and lower HDL-cholesterol. Adjusted analyses indicated that a predominant α-adrenergic profile had higher odds of central obesity (P = 0.031), low HDL-cholesterol (P = 0.042), 24-H-hypertension (P < 0.001), cardiac stress (P = 0.025), ischemic events (P = 0.048) and medium-to-high 10-year stroke probability (P < 0.001), compared to β-adrenergic responders. In the β-adrenergic responders, higher odds for ischemic events, stroke probability and Abnl-GT were found (all P ≤ 0.022), compared to α-adrenergic responders. Independent of age, sex or ethnicity, the risk profile identified in predominant α-adrenergic responders mainly involved the effects of a high-pressure system, cardiac stress, and ischemia. Whereas in predominant β-adrenergic responders, the risk profile pointed to a more metabolic and hyperperfusion injury-related cardiometabolic risk.

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β-adrenergic signaling triggers enteric glial reactivity and acute enteric gliosis during surgery

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