A novel P2X2‐dependent purinergic mechanism of enteric gliosis in intestinal inflammation
2020; Springer Nature; Volume: 13; Issue: 1 Linguagem: Inglês
10.15252/emmm.202012724
ISSN1757-4684
AutoresReiner Schneider, Patrick Leven, Tim R. Glowka, Ivan Kuzmanov, Mariola Lysson, Bianca Schneiker, Anna Miesen, Younis Baqi, Claudia Spanier, Iveta Grants, Elvio Mazzotta, Egina C. Villalobos-Hernández, Jörg C. Kalff, Christa E. Müller, Fedias L. Christofi, Sven Wehner,
Tópico(s)Esophageal and GI Pathology
ResumoArticle17 December 2020Open Access Source DataTransparent process A novel P2X2-dependent purinergic mechanism of enteric gliosis in intestinal inflammation Reiner Schneider Reiner Schneider orcid.org/0000-0002-7194-2703 Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Patrick Leven Patrick Leven Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Tim Glowka Tim Glowka Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Ivan Kuzmanov Ivan Kuzmanov Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Mariola Lysson Mariola Lysson Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Bianca Schneiker Bianca Schneiker Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Anna Miesen Anna Miesen Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Younis Baqi Younis Baqi Faculty of Science, Department of Chemistry, Sultan Qaboos University, Muscat, Oman Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Claudia Spanier Claudia Spanier Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Iveta Grants Iveta Grants Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Elvio Mazzotta Elvio Mazzotta Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Egina Villalobos-Hernandez Egina Villalobos-Hernandez Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Jörg C Kalff Jörg C Kalff Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Christa E Müller Christa E Müller Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Fedias L Christofi Fedias L Christofi Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Sven Wehner Corresponding Author Sven Wehner [email protected] orcid.org/0000-0002-8632-7631 Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Reiner Schneider Reiner Schneider orcid.org/0000-0002-7194-2703 Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Patrick Leven Patrick Leven Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Tim Glowka Tim Glowka Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Ivan Kuzmanov Ivan Kuzmanov Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Mariola Lysson Mariola Lysson Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Bianca Schneiker Bianca Schneiker Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Anna Miesen Anna Miesen Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Younis Baqi Younis Baqi Faculty of Science, Department of Chemistry, Sultan Qaboos University, Muscat, Oman Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Claudia Spanier Claudia Spanier Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Iveta Grants Iveta Grants Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Elvio Mazzotta Elvio Mazzotta Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Egina Villalobos-Hernandez Egina Villalobos-Hernandez Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Jörg C Kalff Jörg C Kalff Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Christa E Müller Christa E Müller Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany Search for more papers by this author Fedias L Christofi Fedias L Christofi Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA Search for more papers by this author Sven Wehner Corresponding Author Sven Wehner [email protected] orcid.org/0000-0002-8632-7631 Department of Surgery, University of Bonn, Bonn, Germany Search for more papers by this author Author Information Reiner Schneider1, Patrick Leven1, Tim Glowka1, Ivan Kuzmanov1, Mariola Lysson1, Bianca Schneiker1, Anna Miesen1, Younis Baqi2,3, Claudia Spanier3, Iveta Grants4, Elvio Mazzotta4, Egina Villalobos-Hernandez4, Jörg C Kalff1, Christa E Müller3, Fedias L Christofi4 and Sven Wehner *,1 1Department of Surgery, University of Bonn, Bonn, Germany 2Faculty of Science, Department of Chemistry, Sultan Qaboos University, Muscat, Oman 3Pharmaceutical Institute, Pharmaceutical & Medical Chemistry, University of Bonn, Bonn, Germany 4Department of Anesthesiology, Wexner Medical Center, The Ohio State University, Columbus, OH, USA *Corresponding author. Tel: +49 228 287 11007; E-mail: [email protected] EMBO Mol Med (2021)13:e12724https://doi.org/10.15252/emmm.202012724 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Enteric gliosis was shown to be part of an intestinal immune response upon abdominal surgery. ATP activates enteric glial cells via selective purinergic receptor signalling in mice and humans. Inhibition of this pathogenic pathway by the newly identified P2X2 antagonist ambroxol blocks ATP-induced enteric gliosis and protects against postoperative ileus. Reactive enteric glia actively contribute to intestinal neuroinflammation and disruption of motility in intestinal disorders and GI diseases. ATP induces a gliosis phenotype in enteric glia as occurs in postoperative ileus. ATP triggers P2X2-signaling to promote enteric gliosis and inflammation contributing to disruption of motility in the mouse and human gut. P2X2 antagonism with a newly identified P2X2 antagonist drug ambroxol reduces gliosis and improves clinical symptoms of postoperative bowel inflammation. The paper explained Problem In various inflammation-induced intestinal disorders, it has been shown that reactive enteric glia play a role in disease progression by contributing to inflammatory processes. However, less is known about the underlying pathogenic mechanism of EGC activation. Results Herein, we show that enteric gliosis occurs upon abdominal surgery and leads to postoperative ileus (POI), an inflammation-based intestinal motility disorder. Activation of EGC in this process depends on ATP and selective purinergic signaling in EGCs. Within a comprehensive set of in vivo, ex vivo, and in vitro analyses in mice and human specimens, we found that ATP is released during abdominal surgery and activates purinergic P2X2 signaling that triggers gliosis in human and murine EGC. We further identified a novel P2X2 antagonist and P2X2 antagonism with ambroxol proved to ameliorate gliosis, reduce inflammatory responses, and improve clinical symptoms of POI. Impact We conclude that enteric gliosis and P2X-purinergic receptors might be promising drug targets for therapeutic approaches in immune-driven intestinal diseases. Introduction Enteric glial cells (EGCs) are a unique population of cells in the enteric nervous system (Furness, 2012) playing a pivotal role in the maintenance of gut homeostasis (Sharkey, 2015). They shape the immune environment through interactions with resident immune cells and other cell types (Brierley & Linden, 2014; Yoo & Mazmanian, 2017). In line with this, EGCs secrete neuroprotective (Abdo et al, 2010) and immune-modulatory factors (Yoo & Mazmanian, 2017) and targeted ablation of glia (Rao et al, 2017) or inhibition (McClain et al, 2014) of glial signaling through connexin-43 hemichannel communication between glia can disrupt motility. However, the neuroinflammatory effect of glial ablation is still unclear, as in some cases a fatal bowel inflammation was documented (Bush et al, 1998; Cornet et al, 2001; Aubé et al, 2006) while in a recent study, utilizing a new genetic mouse model, no immune-modulatory effect was observed (Rao et al, 2017). In contrast to their immune-modulatory role, several in vivo and in vitro studies by us and others provide evidence that murine or human EGCs can turn into reactive glia in an immune-stimulated environment, e.g., under LPS presence (Rosenbaum et al, 2016; Liñán-Rico et al, 2016), after viral protein HIV-1 Tat (Esposito et al, 2017) or IL-1 stimulation upon which EGCs release inflammatory mediators like cytokines, nitric oxide or reactive oxygen species (Stoffels et al, 2014; Brown et al, 2016; Liñán-Rico et al, 2016; Rosenbaum et al, 2016). EGCs were also shown to interact with bacteria, and they can discriminate between beneficial and harmful bacteria (Turco et al, 2014). Immune responses are often a consequence of tissue damage which leads to the release of intracellular molecules that act as danger-associated molecular patterns (DAMP) and trigger innate immune processes (Yoo & Mazmanian, 2017). One prominent DAMP is ATP that is produced and utilized by all cell types (Idzko et al, 2014). In the healthy gut, ATP is involved in intestinal homeostasis, gastrointestinal motility, blood flow and synaptic transmission (Christofi, 2008). However, increased extracellular ATP concentrations resulting from tissue damage and trauma, excessive mechanical stimulation, shear stress in diseased blood vessels, cancer, inflammatory cells or a variety of acute or chronic diseases represent a pathogenic pro-inflammatory mechanism contributing to symptomatology (Idzko et al, 2014; Di Virgilio et al, 2018). ATP signaling is complex and is mediated by purinergic receptors to which ATP either binds directly or as an enzymatically metabolized form, e.g., ADP or adenosine (Galligan, 2008). Purinergic receptors are classified broadly into ionotropic P2X, metabotropic P2Y and P1 receptor families. ATP, or other nucleotides can variably activate P2X and P2Y while adenosine activates metabotropic P1 receptors (Galligan, 2008). Recent studies demonstrated the expression of purinergic receptors on EGCs and their role in the regulation of gastrointestinal motility (McClain et al, 2014), neuron-to-glia communication (Gulbransen & Sharkey, 2009) and neuronal survival (Brown et al, 2016). We have identified P1, P2X and P2Y purinergic receptors in primary human EGCs in primary culture networks and the molecular identity of the reactive hEGC phenotype was revealed by LPS induction (Liñán-Rico et al, 2016). Recent progress in the field suggests that EGC may represent “a new frontier in neurogastroenterology and motility” (Ochoa-Cortes et al, 2016). Overall, EGCs modulate motility, maintain gut homeostasis, and contribute to neuroinflammation in intestinal diseases and motility disorders (Gulbransen & Christofi, 2018). The latter includes postoperative gastrointestinal dysfunction and postoperative ileus (POI), a common clinical complication observed upon abdominal surgery that is characterized by a transient impairment of gastrointestinal (GI) function after surgery. POI is associated with increased morbidity in patients, and despite implementation of enhanced recovery protocols for elective colorectal surgery (Hedrick et al, 2018), no good treatment option exists. POI remains a huge health care problem costing billions of dollars in extended hospitalizations (Iyer et al, 2009). POI is well known to originate from postoperative neuronal dysregulation and is based on an inflammation of the muscularis externa (ME) (Wehner et al, 2007). Recently, we demonstrated that this postoperative inflammation involves EGC reactivity (Stoffels et al, 2014), but the molecular identity of the induction and trigger mechanisms of EGC activation are not fully understood. Herein, we tested the hypothesis that surgical manipulation and trauma triggers ATP release that drives enteric gliosis and intestinal inflammation leading to impairment of motility in POI. We accessed the relevance of reactive EGC in human bowel specimens and the well characterized mouse model of acute posttraumatic bowel inflammation resulting in POI. By transferring the discovered mechanistic insights to a clinically relevant treatment option of selective purinergic receptor antagonism with ambroxol, a newly identified P2X2 antagonist “drug”, we confirmed the potential therapeutic importance of ATP-activated EGCs for inflammation-induced POI that may be relevant to other motility disorders. Results Enteric glial cells respond to injury and inflammation and contribute to damage and regenerative processes (Grubišić & Gulbransen, 2017). Our investigation uncovered a purinergic pathway in reactive murine and human EGCs involved in the response to surgical trauma and inflammation. ATP induction of a reactive EGC phenotype is dependent on a p38 MAPK signaling pathway To evaluate enteric glia reactivity, we applied ATP, a trigger of purinergic signaling and an inflammatory mediator, to primary msEGC in culture. Our msEGC cultures were highly enriched in GFAP-expressing cells (mean, 86 ± 2%, Fig EV1A) that also showed Sox10 and S100β immunoreactivity (Fig EV1B) representing the main EGC phenotype seen in vivo (Boesmans et al, 2015) and enriched glial marker expression (Appendix Fig S1A). Click here to expand this figure. Figure EV1. ExATP induces gliosis in enteric glia cells Histological analysis of EGC culture purity by quantification of EGCs and fibroblasts in vitro. Representative immunofluorescence image shows GFAP (violet)-positive EGCs and α smooth muscle actin (αSMA, green)-positive fibroblasts with DAPI as counterstain. Scale bar 50 µm. Representative immunofluorescence image of s100β (violet)- and Sox10 (green)-positive EGCs with DAPI as counterstain. Scale bar 10 µm. PCA plot of gene expression by ATP-treated and untreated EGCs. Blue circles represent ATP-treated EGC cultures, and white circles are matching controls; n = 5–6, respectively. Heat map of ATP-target genes, showing a collection of known target genes of ATP signaling (n = 5–6, msEGCs). Representative Western blots of phospho-p38-MAPK (pp38) and p38-MAPK (p38) in 1 h ATP-treated EGCs. Actin was used as loading control (n = 3, msEGCs). Representative images of GFAP (violet)- and phospho-p38-MAPK (pp38, green)-positive msEGCs with or without ATP treatment (100 µM) for 1 h. White arrows show pp38-positive (ATP-treated) or negative (untreated) EGCs. Scale bar is 10 µm. Effect of p38 inhibition on ATP-induced IL-6 release. Cells were treated with the p38-MAPK inhibitor SB203580 (1, 5, 10 µM) alone or together with ATP (100 µM) for 24 h. ELISA measurement of IL-6 in msEGCs supernatants (n = 7–22, msEGCs). Effect of p38 inhibition on ATP induced mRNA expression of gliosis markers in msEGCs. Cells were treated with SB203580 (10 µM) alone or together with ATP (100 µM) for 6 h (n = 4, msEGCs). Data information: In (A), data are represented as percentage + SEM normalized to the total cell numbers, n = 8, msEGCs. In (G and H), data are represented as fold induction + SEM. Statistics were done in (G and H) by applying unpaired Student's t-test and one-way ANOVA with a subsequent Bonferroni test. * indicates significance to control, and # indicates significance to the ATP treatment with */#P < 0.05, ##P < 0.01, and ***/###P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint RNA-Seq analysis of the glial transcriptome identified the unique gene dysregulation profile induced by ATP in msEGCs. We found profound changes in msEGC gene expression with 2,027 up-regulated and 2,218 down-regulated genes after ATP stimulation (fold change ≥ 1.5; P-value: < 0.05, Fig 1A and principal component analysis shown in Fig EV1C). Therefore, ATP caused up-regulation in 10% and down-regulation in 11% of total glial transcriptome. Induction of genes, known to be expressed in direct response to ATP, including members of the regulator of calcineurin (RCAN) (Canellada et al, 2008) and FOS (Pacheco-Pantoja et al, 2016) gene families were confirmed by both RNA-Seq and qPCR (Figs 1H and EV1D). Gene ontology (GO) enrichment analyses demonstrated a general glial activation in ATP-treated msEGCs showing enriched genes for “ATP binding” and “glial proliferation” (Fig 1B and Appendix Fig S1B). Importantly, challenge with ATP induced genes involved in the regulation of cell motility, cytokine response genes (Fig 1C and D and Dataset EV1) and the mitogen-activated protein kinase (MAPK) pathways (Fig 1E and Dataset EV1) underlining the transition of msEGCs to an activated immune phenotype, also referred to as “gliosis”. The term gliosis is commonly used to describe reactive astrocytes, the CNS counterparts to EGCs. Transcriptionally, gliosis is characterized by the up-regulation of a particular gene set, including, inflammatory response genes. To analyze the reactivity of the EGCs, we created a new GO term for gliosis based on all recent reports discussing CNS gliosis induced by inflammatory stimuli in vivo and in vitro (Zamanian et al, 2012; Hara et al, 2017; Liddelow et al, 2017; Fujita et al, 2018; Mathys et al, 2019; Rakers et al, 2019; Schirmer et al, 2019). Notably, we found that many gliosis-related genes are also regulated in ATP-activated msEGCs (Fig 1F, Appendix Fig S1C and Dataset EV1). Quantitative PCR confirmed the up-regulation of key markers of gliosis including GFAP and NESTIN (Fig 1G) as well as inflammatory mediators like CXCL2 and IL-6 (Fig 1I and J). The latter has been shown to be an important EGC-derived cytokine released upon IL-1β stimulation during surgical trauma (Stoffels et al, 2014). Our data confirmed a robust dose-dependent and statistically significant increase in the levels of IL-6, in both mRNA and protein (Fig 1J and K) upon ATP stimulation, indicating a prominent role of IL-6 in activated EGCs, subsequently making it a reliable marker in enteric gliosis and a central part of our further investigations. To efficiently analyze and describe the glia transformation to a reactive phenotype, we chose six targets; NESTIN and GFAP, two structural glia genes; IL-6 and CXCL2, two inflammatory mediators and FOSb and RCAN, two transcriptional targets of ATP signaling, as a reliable gliosis marker panel developed from our in silico-based method to further evaluate purinergic enteric gliosis in subsequent studies. Figure 1. ATP induces a gliosis in msEGCs A. Volcano plot showing significantly regulated genes between control and ATP-treated msEGCs. B. Visual representation of GO terms associated with enriched genes in ATP-treated msEGCs compared to control. C–F. Heat maps of indicated GO terms in ATP-treated msEGCs compared to control. G–I. qPCR analysis of indicated gliosis genes in ATP-treated EGCs. J. qPCR analysis of IL-6 in msEGCs that were treated for 6 h with ATP. K. IL-6 protein levels in supernatants from msEGCs collected after 24 h treatment with ATP. Data information: In (A), data are shown as fold change > 1.5, P-value < 0.05; (n = 5 for untreated and n = 6 for ATP-treated EGCs). In (G–K), data are shown as fold change + SEM. (G–I) n = 6–9, msEGCs. (J) n = 4–9, msEGCs. (K) n = 6–18, msEGCs. In (A–I): ATP concentration was 100 µM. In (J, K), ATP concentration was 1, 10, or 100 µM. Statistics were performed by applying unpaired Student's t-test (G–K) and/or one-way ANOVA with a subsequent Bonferroni test (J and K). In (A) a limma-trend pipeline model and in (B) the Fishers exact test were performed. * indicates significance to control, and # indicates significance to ATP treatment with *P < 0.05, **/##P < 0.01 and ***P < 0.001. Download figure Download PowerPoint Given that ATP treatment led to an activation of MAPK pathways (Fig 1E), we investigated the involvement of p38-MAPK, an important molecular switch of inflammatory pathways and astrogliosis in the central nervous system (Roy Choudhury et al, 2014). ATP was shown to elevate phospho-p38-MAPK protein (Fig EV1E) which is strongly localized in the nucleus of GFAP-positive msEGCs, absent in untreated msEGCs (Fig EV1F). Furthermore, ATP-induced IL-6 protein release was dose-dependently suppressed using the p38-MAPK-inhibitor SB203580 (Fig EV1G); qPCR confirmed the transcriptional reduction of IL-6 and other gliosis markers like GFAP, CXCL2, and RCAN (Fig EV1H). Altogether, our data demonstrate that EGC gliosis can be triggered by ATP and induction of enteric gliosis depends on activation of the p38-MAPK signaling pathway. P2X receptors mediate the ATP-triggered IL-6 release from msEGC ATP can be enzymatically dephosphorylated and is, together with its metabolites ADP and adenosine, able to signal via multiple purinergic receptors. Those receptors, broadly divided into the P2X, P2Y and P1 classes (Galligan, 2008), make ATP's signaling repertoire rather complex. Many of these receptor subtypes have been identified in enteric glia, although their role in normal or disease states remains unclear (Ochoa-Cortes et al, 2016; Grubišić & Gulbransen, 2017; Gulbransen & Christofi, 2018). As a starting point to pinpoint the purinergic receptor subtype(s) involved in enteric gliosis, we performed pharmacological screening with various agonists and antagonists of the purinergic signaling system. In our analysis, adenosine failed to stimulate IL-6 release from msEGCs, suggesting that the P1 class is not involved in the ATP-induced phenotype (Fig EV2A). Next, we tested the non-selective P2-class antagonist suramin that showed a blockade of ATP-dependent IL-6 release in a concentration-dependent manner (Fig 2A). Similar results were observed with PPADS, another P2 antagonist (Fig EV2B). Additionally, the degradation resistant ATP isoform and P2 agonist ATPγS, dose-dependently increased the IL-6 release with comparable or even stronger efficacy than ATP itself (Fig 2B) and induced the expression of established gliosis marker genes (data not shown). These findings indicated that ATP, but not ADP, AMP, adenosine or inosine are likely involved in ATP-induced EGC gliosis, thereby limiting the involved receptors to members of the P2 class. Click here to expand this figure. Figure EV2. ATP-induced gliosis is mediated by p38-MAPK and P2X2-purinergic signaling A. IL-6 release measurement by ELISA of IL-6 in msEGCs. Cells were treated adenosine (1 and 100 µM) or with ATP (100 µM) for 24 h; n = 14–16, msEGCs. B. Protein release measurement by ELISA of IL-6 in msEGCs. Cells were treated with P2 antagonist PPADS (5, 30 µM) alone or together with ATP (10 or 100 µM) for 24 h; n = 11–12, msEGCs. C. Protein release measurement by ELISA of IL-6 in msEGCs. Cells were treated with P2X2 antagonist PSB-1011 (0.2, 2, 20 µM) or PSB-0711 (0.2, 2, 20 µM) alone or together with ATP (10 µM) for 24 h; n = 9–13, msEGCs. D. Schematic overview of the isolation of msEGCs from small bowel muscularis externa of GFAPcre-Ai14fl/wt mice: FACS-sorted tdTomato+ msEGCs were either analyzed directly (ME-tissue) or in cultured msEGCs before tdTomato-FACS-sorting and further analysis; n = 3–6. E. Gene expression analysis by qPCR of GFAP and Sox10 in msEGC cultures (n = 10) and mouse ME tissue (n = 10). F, G. Representative images of co-localization of GFAP (green) and tdTomato+ msEGC (red) in the ME and in cultured EGCs. Scale bars 50 µm. H–K. qPCR analysis of P2-purinergic receptors in msEGCs isolated from ME (H, J; n = 3) or from cultured cells (I, K; n = 6), respectively. L, M. Representative Western blots of P2X2 in msEGCs transfected with siRNA-control or siRNA-P2X2 for 72 h together with an optical density measurement, see in M. Actin was used as loading control and normalization (n = 6, msEGCs). Data information: In (A–C and E), data are represented as fold induction + SEM. In (H–K), data are represented as mean + SEM normalized to GAPDH expression. In (M), data are represented as optical density + SEM normalized to actin expression. Statistics were done by applying unpaired Student's t-test in (A-C, M and E) or both by unpaired Student's t-test and one-way ANOVA with a subsequent Bonferroni test in (B and C). * indicates significance to control, and # indicates significance to the ATP treatment with #P < 0.05, **/##P < 0.01, and ***/###P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. ATP-induced gliosis is mediated by p38-MAPK and selective purinergic signaling Effect of P2 receptor antagonism on ATP-induced IL-6 release. Cells were treated with P2 antagonist suramin (1, 10, and 100 µM) alone or together with ATP (100 µM) for 24 h. ATP-induced IL-6 release in msEGCs measured by ELISA. Cells were treated with the indicated concentrations of ATP and ATPɣS for 24 h. Effects of P2X antagonists on ATP-induced IL-6 release. Cells were treated for 24 h alone or together with ATP (100 µM) in absence or presence of P2X2, P2X4, and P2X7 antagonists PSB-1011, 5-BD-BD, and A740003, respectively. P2X2 antagonism of ATP induced mRNA expression of IL-6, GFAP, and RCAN by qPCR in msEGCs. Cells were treated with the P2X2 antagonist PSB-1011 (20 µM) alone or together with ATP (10 µM) for 6 h. Representative confocal images of P2X2 (green)- and GFAP (violet)-positive msEGCs in vivo and in vitro. White arrows mark double-positive (white) cells. Scale bar 50 µm. P2X2-siRNA reduces P2X2-mRNA and dampens the gliosis gene expression after ATPɣS (100 µM) treatment for 6 h. P2X2-siRNA reduces IL-6 release after ATPɣS treatment (10, 100 µM) for 6 h. Data information: In (A–D and F), data are shown as fold induction + SEM and (G) as IL-6 pg/ml + SEM, (A): n = 10–15, msEGCs; (B): n = 3–15, msEGCs; (C): n = 8–17, msEGCs; (D): n = 4, msEGCs; (E): n = 3–5, msEGCs; (F): n = 3–5, msEGCs; (G): n = 3–5, msEGCs. Statistics in (A–D, F, G) were performed by applying unpaired Student's t-test and/or one-way ANOVA with a subsequent Bonferroni test. * indicates significance to control, and # indicates significance to ATP treatment with */#P < 0.05, **/##P < 0.01, and ***/###P < 0.001. Download figure Download PowerPoint Next, a P2 receptor mRNA expression profile in msEGC was determined in cells isolated from GFAPcre x Ai14fl/wt mice, expressing tdTomato in all GFAP+ cells. Cells were either directly sorted upon ME digestion or sorted upon an intermediate cell culture period (Fig EV2D, F and G). Highly increased gene expression of GFAP and Sox10 in tdTomato+ compared to tdTomato− cells confirmed a successful enrichment of msEGC in both procedures (Fig EV2E). Comprehensive gene expression analyses of purinergic receptors in isolated EGC showed a distinct and comparable ex vivo and in vitro gene expression profile with three P2X receptor genes reaching the highest levels, exceeding not only P1 (Appendix Fig S2A and B), but also P2Y expression levels by several times. Accordingly, we directed our focus toward these P2X receptors expressed in enteric glia in the order P2X7 > P2X4 > P2X2 (Fig EV2H–K). P2X2 receptors mediate the ATP-triggered EGC gliosis P2X7 has been shown to be prominently involved in inflammatory processes. However, neither blockade of P2X7 receptors (Fig 2C) nor its activation with selective
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