Application of a RiboTag-based approach to generate and analyze mRNA from enteric neural cells

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.Funding Agencies|Cluster of Excellence - ImmunoSensation2 [EXC2151-190873048]; Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [388159768, WE4205/3-1]</p

Intestinal epithelial barrier maturation by enteric glial cells is GDNF-dependent

Enteric glial cells (EGCs) of the enteric nervous system are critically involved in the maintenance of intestinal epithelial barrier function (IEB). The underlying mechanisms remain undefined. Glial cell line-derived neurotrophic factor (GDNF) contributes to IEB maturation and may therefore be the predominant mediator of this process by EGCs. Using GFAP\8^{cre}\) x Ai14$^{floxed}$ mice to isolate EGCs by Fluorescence-activated cell sorting (FACS), we confirmed that they synthesize GDNF in vivo as well as in primary cultures demonstrating that EGCs are a rich source of GDNF in vivo and in vitro. Co-culture of EGCs with Caco2 cells resulted in IEB maturation which was abrogated when GDNF was either depleted from EGC supernatants, or knocked down in EGCs or when the GDNF receptor RET was blocked. Further, TNFα-induced loss of IEB function in Caco2 cells and in organoids was attenuated by EGC supernatants or by recombinant GDNF. These barrier-protective effects were blunted when using supernatants from GDNF-deficient EGCs or by RET receptor blockade. Together, our data show that EGCs produce GDNF to maintain IEB function in vitro through the RET receptor

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

Frequency and Predictors for Chronic Thromboembolic Pulmonary Hypertension after a first Unprovoked Pulmonary Embolism: results from PADIS studies

International audienceBackground: Chronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening pulmonary embolism's (PE) complication whose incidence and predictors are not precisely determined.Objective: To determine the frequency and predictors for CTEPH after a first unprovoked PE.Patients/methods: In a randomized trial comparing an additional 18-month warfarin versus placebo in patients after a first unprovoked PE initially treated with vitamin K antagonist for 6 months, we applied recommended CTEPH screening strategies through 8-year follow-up to determine cumulative incidence of CTEPH. CTEPH predictors were estimated using Cox models. Pulmonary vascular obstruction (PVO) and systolic pulmonary arterial pressure (sPAP) at PE diagnosis and 6 months were studied by receiver operating curves analysis. All CTEPH cases and whether they were incident or prevalent were adjudicated.Results: During a median follow-up of 8.7 years, 9 CTEPH cases were diagnosed among 371 patients, with a cumulative incidence of 2.8% (95% confidence interval [CI], 0.95-4.64), and of 1.31% (95%CI, 0.01-2.60) after exclusion of 5 cases adjudicated as prevalent. At PE diagnosis, PVO>45% and sPAP>56mmHg were associated with CTEPH with a hazard ratio (HR) of 33.00 (95%CI 1.64-667.00, p=0.02) and 12.50 (95%CI 2.10-74.80, p65 years, lupus anticoagulant antibodies and non-O blood groups were also predictive of CTEPH. PVO>14% and sPAP>34mmHg at 6-month were associated with CTEPH (HRs 63.90 [95%CI, 3.11-1310.00, p<0.01]and 17.2 [95%CI, 2.75-108, p<0.01]).Conclusion: After a first unprovoked PE, CTEPH cumulative incidence was 2.8% during 8-year follow-up. PVO and sPAP at PE diagnosis and at 6 months were the main predictors for CTEPH diagnosis

Frequency and risk factors for Chronic Thromboembolic Pulmonary Hypertension after a first unprovoked Pulmonary Embolism: results from PADIS-studies

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Risk factors for recurrent venous thromboembolism after unprovoked pulmonary embolism: the PADIS-PE randomised trial

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