Caspase‐8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice

Article2 May 2022Open Access Source DataTransparent process Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice Nathalie Tisch Corresponding Author Nathalie Tisch [email protected] orcid.org/0000-0003-0019-4346 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Carolin Mogler Carolin Mogler orcid.org/0000-0003-3400-7254 Institute of Pathology, TUM School of Medicine, Technical University of Munich, Munich, Germany Contribution: ​Investigation, Writing - review & editing Search for more papers by this author Ana Stojanovic Ana Stojanovic Department of Immunobiochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation, Methodology Search for more papers by this author Robert Luck Robert Luck European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Validation, ​Investigation Search for more papers by this author Emilia A Korhonen Emilia A Korhonen European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Data curation, Methodology Search for more papers by this author Alexander Ellerkmann Alexander Ellerkmann European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Department of General, Visceral and Transplantation Surgery, Heidelberg University, Heidelberg, Germany Contribution: ​Investigation Search for more papers by this author Heike Adler Heike Adler European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Data curation Search for more papers by this author Mahak Singhal Mahak Singhal European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Data curation, Formal analysis, Visualization Search for more papers by this author Géza Schermann Géza Schermann European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation, Visualization, Methodology Search for more papers by this author Lena Erkert Lena Erkert Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: ​Investigation Search for more papers by this author Jay V Patankar Jay V Patankar Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Andromachi Karakatsani Andromachi Karakatsani European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation Search for more papers by this author Anna-Lena Scherr Anna-Lena Scherr National Center for Tumor Diseases, Department of Medical Oncology, Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany Contribution: ​Investigation, Methodology Search for more papers by this author Yaron Fuchs Yaron Fuchs Laboratory of Stem Cell Biology & Regenerative Medicine, Department of Biology, Technion –Israel Institute of Technology, Haifa, Israel Contribution: Resources Search for more papers by this author Adelheid Cerwenka Adelheid Cerwenka European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Department of Immunobiochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Resources, Supervision Search for more papers by this author Stefan Wirtz Stefan Wirtz Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Resources, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Bruno Christian Köhler Bruno Christian Köhler orcid.org/0000-0002-7308-0377 National Center for Tumor Diseases, Department of Medical Oncology, Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany Contribution: Resources, Supervision, Methodology Search for more papers by this author Hellmut G Augustin Hellmut G Augustin orcid.org/0000-0002-7173-4242 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Christoph Becker Christoph Becker Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Conceptualization, Resources, Supervision, Methodology Search for more papers by this author Thomas Schmidt Thomas Schmidt orcid.org/0000-0002-7166-3675 Department of General, Visceral and Transplantation Surgery, Heidelberg University, Heidelberg, Germany Department of General, Visceral, Cancer and Transplantation Surgery, Faculty of Medicine with University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Resources, Supervision Search for more papers by this author Carmen Ruiz de Almodóvar Corresponding Author Carmen Ruiz de Almodóvar [email protected] orcid.org/0000-0001-5975-7815 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Nathalie Tisch Corresponding Author Nathalie Tisch [email protected] orcid.org/0000-0003-0019-4346 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Carolin Mogler Carolin Mogler orcid.org/0000-0003-3400-7254 Institute of Pathology, TUM School of Medicine, Technical University of Munich, Munich, Germany Contribution: ​Investigation, Writing - review & editing Search for more papers by this author Ana Stojanovic Ana Stojanovic Department of Immunobiochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation, Methodology Search for more papers by this author Robert Luck Robert Luck European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Validation, ​Investigation Search for more papers by this author Emilia A Korhonen Emilia A Korhonen European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Data curation, Methodology Search for more papers by this author Alexander Ellerkmann Alexander Ellerkmann European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Department of General, Visceral and Transplantation Surgery, Heidelberg University, Heidelberg, Germany Contribution: ​Investigation Search for more papers by this author Heike Adler Heike Adler European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Data curation Search for more papers by this author Mahak Singhal Mahak Singhal European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Data curation, Formal analysis, Visualization Search for more papers by this author Géza Schermann Géza Schermann European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation, Visualization, Methodology Search for more papers by this author Lena Erkert Lena Erkert Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: ​Investigation Search for more papers by this author Jay V Patankar Jay V Patankar Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Andromachi Karakatsani Andromachi Karakatsani European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: ​Investigation Search for more papers by this author Anna-Lena Scherr Anna-Lena Scherr National Center for Tumor Diseases, Department of Medical Oncology, Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany Contribution: ​Investigation, Methodology Search for more papers by this author Yaron Fuchs Yaron Fuchs Laboratory of Stem Cell Biology & Regenerative Medicine, Department of Biology, Technion –Israel Institute of Technology, Haifa, Israel Contribution: Resources Search for more papers by this author Adelheid Cerwenka Adelheid Cerwenka European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Department of Immunobiochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Resources, Supervision Search for more papers by this author Stefan Wirtz Stefan Wirtz Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Resources, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Bruno Christian Köhler Bruno Christian Köhler orcid.org/0000-0002-7308-0377 National Center for Tumor Diseases, Department of Medical Oncology, Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany Contribution: Resources, Supervision, Methodology Search for more papers by this author Hellmut G Augustin Hellmut G Augustin orcid.org/0000-0002-7173-4242 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Christoph Becker Christoph Becker Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany Contribution: Conceptualization, Resources, Supervision, Methodology Search for more papers by this author Thomas Schmidt Thomas Schmidt orcid.org/0000-0002-7166-3675 Department of General, Visceral and Transplantation Surgery, Heidelberg University, Heidelberg, Germany Department of General, Visceral, Cancer and Transplantation Surgery, Faculty of Medicine with University Hospital Cologne, University of Cologne, Cologne, Germany Contribution: Resources, Supervision Search for more papers by this author Carmen Ruiz de Almodóvar Corresponding Author Carmen Ruiz de Almodóvar [email protected] orcid.org/0000-0001-5975-7815 European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Nathalie Tisch *,1, Carolin Mogler2, Ana Stojanovic3, Robert Luck1,4, Emilia A Korhonen1, Alexander Ellerkmann1,5, Heike Adler1, Mahak Singhal1,4, Géza Schermann1, Lena Erkert6, Jay V Patankar6, Andromachi Karakatsani1, Anna-Lena Scherr7, Yaron Fuchs8, Adelheid Cerwenka1,3, Stefan Wirtz6, Bruno Christian Köhler7, Hellmut G Augustin1,4, Christoph Becker6, Thomas Schmidt5,9 and Carmen Ruiz de Almodóvar *,1,10,11 1European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 2Institute of Pathology, TUM School of Medicine, Technical University of Munich, Munich, Germany 3Department of Immunobiochemistry, Mannheim Institute for Innate Immunoscience (MI3), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 4Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany 5Department of General, Visceral and Transplantation Surgery, Heidelberg University, Heidelberg, Germany 6Department of Medicine 1, Friedrich-Alexander-University, Erlangen, Germany 7National Center for Tumor Diseases, Department of Medical Oncology, Internal Medicine VI, Heidelberg University Hospital, Heidelberg, Germany 8Laboratory of Stem Cell Biology & Regenerative Medicine, Department of Biology, Technion –Israel Institute of Technology, Haifa, Israel 9Department of General, Visceral, Cancer and Transplantation Surgery, Faculty of Medicine with University Hospital Cologne, University of Cologne, Cologne, Germany 10Present address: Institute for Neurovascular Cell Biology, University Hospital Bonn, Bonn, Germany 11Present address: Schlegel Chair for Neurovascular Cell Biology, University of Bonn, Bonn, Germany *Corresponding author. Tel: +41 44 655 73 42; E-mail: [email protected] *Corresponding author. Tel: +49 228 6885303; E-mail: [email protected] EMBO Mol Med (2022)14:e14121https://doi.org/10.15252/emmm.202114121 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 The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function. SYNOPSIS Inflammatory bowel disease (IBD) is a group of disorders that causes severe inflammation of the intestine. This study identified that vascular dysfunction in the small intestine results in classic features of IBD in mice. Adult mice lacking Caspase-8 (Casp8) in endothelial cells (Casp8ECko) die within 3 weeks after tamoxifen treatment with severe intestine inflammation and mucosal hemorrhages, whereas all other organs are not affected. Casp8ECko mice show lymphatic and blood vessel regression in the small intestine that can be prevented by co-deletion of MLKL. Deletion of Casp8 solely in the blood endothelium (not in lymphatics) does not manifest bowel inflammation, suggesting that Casp8 is primarily required as a pro-survival factor in the lymphatic vasculature. Disease development in Casp8ECko mice is dependent on the presence of microbiota. The paper explained Problem The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the blood stream, and as such contributes to gut homeostasis. Vascular dysfunction has been reported in patients with inflammatory bowel disease (IBD). However, the molecular mechanisms that maintain the gut-vascular barrier remain elusive. Furthermore, it remains unknown whether vascular dysfunction is only consequence, or can even be cause of small bowel inflammation. Results Our study addresses the role of Caspase-8 (Casp8), a key pro-survival factor in the necroptosis cell death signaling pathway, in endothelial cells (ECs). Loss of Casp8 function in epithelial cells has been shown to be involved in human IBD patients as well as in mouse models of inflammatory bowel diseases. Our study shows that EC-specific Casp8 knockout mice recapitulate those phenotypes, indicating the additional need of endothelial Casp8 for vascular homeostasis. In particular, lymphatic EC homeostasis largely depends on Casp8 expression. Overall, our data shows that the intestinal vasculature can be a primary driver for small bowel inflammation. Impact Our study reveals that targeting cell death signaling in the intestinal vasculature in inflammatory bowel diseases might prove useful as novel therapeutical strategy to treat IBD patients. Introduction The intestinal epithelium is a single-cell layer lining the small and large intestine that constitutes the body’s largest barrier against the external environment (Martini et al, 2017). The gut microbiota lives in symbiosis with its host and regulates a number of physiological processes, such as integrity and permeability of the gut epithelium, host immunity, and defense against ingested pathogens (Backhed et al, 2005). However, the gut microbiota also poses a potential threat to the host and can lead to inflammation and infection, when immune or epithelial cell homeostasis is compromised (Goto & Kiyono, 2012; Kamada et al, 2013). Therefore, the intestine has acquired a highly specialized immune system. Hereby, Peyer’s patches, organized lymphoid follicles, are important players in immune surveillance (Jung et al, 2010; Morikawa et al, 2016). The gut epithelium is functionally supported by a cooperative dense vascular network that builds a second barrier, termed the gut-vascular barrier (GVB) (Spadoni et al, 2015). The GVB is intermingled by blunt-ended lacteals, the capillaries of the lymphatic system that reside within intestinal villi (Spadoni et al, 2015; Bernier-Latmani & Petrova, 2017), and both systems actively co-regulate each other (Jang et al, 2013). Accumulating evidence indicates that endothelial cells (ECs) forming the inner lining of blood vessels actively contribute to both organ development, maintenance, and repair by the secretion of tissue-specific, so called angiocrine factors that instruct and guide cells in the environment (Rafii et al, 2016; Augustin & Koh, 2017). In line, it is recognized that ECs possess organ-specific morphological and transcriptional properties that are required to develop organotypic properties (Nolan et al, 2013). Similarly, organ-specific lymphatic ECs (LECs) exist (Bernier-Latmani & Petrova, 2017; Oliver et al, 2020). However, the molecular mechanisms that confer these organotypic features and that maintain vascular homeostasis in an organ-specific manner remain elusive. Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the small and large intestine (Kim et al, 2017; Martini et al, 2017). IBD pathogenesis is not just caused by immune cell-mediated mechanisms but is also closely associated with a loss of epithelial barrier integrity that can lead to excessive translocation of commensal microbiota followed by an exaggerated immune response (Martini et al, 2017). Even though vascular impairments in the intestine have been reported in IBD patients (Homan et al, 1976; Dvorak et al, 1980; Wakefield et al, 1991), only little is known about the mechanisms that might lead to gut-specific vascular dysfunction. Whereas it is assumed that these vascular defects arise secondary to increased intestinal inflammation, the possibility that vascular dyshomeostasis can be a primary cause for IBD development has so far not been sufficiently investigated. Caspase-8 (Casp8) is a central regulator of the extrinsic cell death signaling pathway. When fully activated, Casp8 leads to the proteolytic cleavage and activation of the executioner Caspase-3 to induce cellular apoptosis (Lin et al, 1999; Kang et al, 2004). On the other hand, restricted Casp8 activity is required to prevent necroptosis (Oberst et al, 2011; Dillon et al, 2012). Loss of Casp8, or inhibition of its enzymatic activity, results in the formation of the RIPK1-RIPK3 comprising necrosome and the phosphorylation of mixed lineage kinase protein like (MLKL), the ultimate executer of necroptosis (Sun et al, 2012; Zhao et al, 2012). Interestingly, a subpopulation of human patients with a CASP8 deficiency suffering from very early onset IBD has been identified (Lehle et al, 2019). Epithelial cell-specific knockout of Casp8 in adult mice results in mouse lethality due to massive intestinal defects with similar features as seen in human IBD conditions (Gunther et al, 2011; Schwarzer et al, 2020). This is caused by epithelial cell necroptosis (Gunther et al, 2011), indicating that Casp8 is an important pro-survival factor of the gut-epithelial barrier. Epithelial cell death in the absence of Casp8 can be inhibited if cytokine signaling is downregulated (Gunther et al, 2011, 2019), and colonic inflammation in epithelial cell-specific Casp8 knockout mice strongly depends on microbial composition (Stolzer et al, 2020). Whether Casp8 is required in a similar fashion as a pro-survival factor of the GVB, and thus, whether EC dysfunction in the absence of Casp8 in the gut might also contribute to IBD development, remains elusive. In this study, we describe that acute deletion of Casp8 in ECs of adult mice (from hereon Casp8ECko) resulted in a gut-vasculature-specific phenotype and caused lethality of the mice around 3 weeks after the first tamoxifen treatment. Most strikingly, Casp8ECko mice developed severe inflammation and tissue damage. These defects were dependent on EC necroptosis and the presence of microbiota. Importantly, deletion of Casp8 solely in blood ECs in the intestine did not result in disease, thus highlighting the importance of Casp8 expression primarily in the lymphatic vasculature. We therefore identified Casp8 as a novel, organotypic regulator of vascular homeostasis in the small intestine. Furthermore, our data suggests that the intestinal endothelium acts as an important safeguard of overall intestine homeostasis as vascular dysfunction can be a primary driver for intestine inflammation. Results Acute loss of Casp8 in adult ECs is lethal and impairs intestine homeostasis To determine whether Casp8 is required in ECs to maintain vessel homeostasis, we induced the deletion of Casp8 in adult ECs using an EC-specific Casp8 floxed line (Cdh5CreERT2; Casp8fl/fl mice; termed from here on Casp8ECko (Tisch et al, 2019)) (Fig 1A). Around 3 weeks after the first tamoxifen treatment, Casp8ECko mice had significantly reduced weight compared to Casp8fl/fl (from here on Casp8WT mice) (Fig 1B) and ~90% of the Casp8ECko mice died or had to be euthanized due to illness (Fig 1C). Surprisingly, examination of all main organs only revealed hemorrhages in the small intestine (Fig 1D), whereas all others seemed unaffected (Fig EV1A). Figure 1. Acute loss of Casp8 in adult ECs is lethal and impairs intestine homeostasis A. Schematic representation of Casp8 deletion in adult ECs indicating the time points for tamoxifen treatment, analysis of the phenotype (early (day 15) or late (day 18)), and the timeframe of disease progression. B, C. Graphs showing the relative weight (B; n = 15 WT, 12 ECko; curve comparison) and the survival (C; n = 15 WT, 11 ECko; Log-Rank test) of mice upon tamoxifen treatment. D. Representative images of the intestine of Casp8WT and Casp8ECko mice, with mild and severe hemorrhages at a late disease stage. E, F. Representative images of H&E staining (E) and quantification of intestinal pathology (F) of the small intestine at an early disease stage in Casp8WT and Casp8ECko mice (enteropathy score; n = 6 WT, 6 ECko; two-tailed unpaired Student’s t-test). Scale bars: 100 µm; insets 50 µm. G, H. Quantification of intestinal pathology (enteropathy score, G; n = 8 WT, 7 ECko; two-tailed unpaired Student’s t-test) and hemorrhages in the mucosa (H; n = 4 WT, 5 ECko; two-tailed unpaired Student’s t-test) at a late disease stage in Casp8WT and Casp8ECko mice. I. Overview of H&E-stained Swiss roll preparations of Casp8WT and Casp8ECko mice. Scale bars: 2 mm. J. Representative images of H&E staining of the small intestine of Casp8WT (j’) and Casp8ECko mice with mild (j″) and more severely (j‴) affected areas at a late disease stage (white arrows point to hemorrhages, asterisk points to focal erosion of the epithelium). Scale bars: 100 µm. Data information: Data shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Casp8ECko mice do not present defects in other organs A. Representative images of H&E staining of the indicated organs in Casp8WT and Casp8ECko mice at a late disease stage (n = 4–5 WT, 5 ECko). Scale bars 100 µm. B–D. Representative images of H&E staining of the large intestine (colon) (B). Quantification of intestinal pathology (C) and mucosal hemorrhages (D) in Casp8WT and Casp8ECko mice at a late disease stage (enteropathy score; n = 4 WT, 4 ECko; two-tailed unpaired Student’s t-test). Scale bars: 100 µm. Data information: All data is shown as mean ± SEM. ns: not significant. Source data are available online for this figure. Download figure Download PowerPoint To characterize the intestinal defects in Casp8ECko mice in more detail, we scored the grade of inflammation and epithelial cell damage using standard histopathology (Welz et al, 2011) at different disease stages. At 15 days after the first tamoxifen treatment (early disease), Casp8ECko mice did not present obvious histopathological changes (Fig 1E and F). However, starting at 18 days after the first tamoxifen treatment (late disease), Casp8ECko mice developed profound enteritis that most strongly manifested in the terminal ileum (Fig 1G) and mucosal hemorrhages (Fig 1H). Swiss roll preparations of the small intestine of Casp8ECko mice were larger, probably due to cell infiltration and fluid accumulation (Fig 1I). Compared to Casp8WT mice (Fig 1J and j´), Casp8ECko mice presented mild (Fig 1J and j″) to severe (Fig 1J and j‴) tissue lesions that were accompanied by inflammation and focal erosion of the epithelium in hypoxic-necrotic tissue. The large intestine was not affected (Fig EV1B–D). This phenotype was not due to a restricted expression of Cre recombinase in gut ECs as analysis of Cdh5-CreERT2 x RosamTmG reporter mice revealed active Cre recombinase in ECs of all organs (Appendix Fig S1A and B). Furthermore, Casp8 expression was not only reduced in isolated intestine ECs but also in brain, lung, and skin ECs of Casp8ECko mice (Appendix Fig S1C–G). We also ruled out that the Cdh5-CreERT2 was unspecifically active in the intestinal epithelium, as Casp8 mRNA levels in isolated intestinal epithelial cells of Casp8ECko mice were equal to controls (Appendix Fig S1H). In addition, GFP+ cells in Cdh5-CreERT2 x RosamTmG mice only colocalized with blood (CD31+) and lymphatic (CD31+ Lyve-1+) ECs, but never with the intestinal epithelium (Appendix Fig S1I). Together, these findings indicate that deletion of Casp8 in ECs drives a severe intestinal pathology, without affecting other organs. Loss of Casp8 in ECs leads to small bowel inflammation We next characterized intestinal inflammation in Casp8ECko mice in detail using an unbiased proteome profiler array to detect a panel of cytokines, chemokines, and growth factors in ileal tissue lysates at an advanced disease stage. Upregulated cytokines in Casp8ECko mice were linked to inflammatory and bacterial response pathways, as confirmed by qPCR analysis (Figs 2A and EV2A). In line with this inflammatory profile and the presence of hemorrhages, vessel permeability and EC activation markers were strongly expressed in Casp8ECko mice (Fig 2B). Cytokines involved in the complement cascade and mucosal healing were also significantly upregulated in the proteome profiler assay (Figs 2C and D, and EV2B), suggesting that active bacterial defense and compensatory mucosal repair processes to maintain tissue homeostasis were activated. Consistently, intestinal crypts were elongated and presented an increased amount of Ki67+ proliferating epithelial cells (Fig 2E and F), together with increased Ki67 and c-myc expression in ileal lysates (Fig 2G). E-cadherin staining of the epithelial lining showed that the intestinal barrier remained largely intact (Fig 2H and h′), except of local barrier breakdown at the site of severe intestinal lesions (Fig 2H and h″). In line, as shown by FISH-staining of bacterial 16S rRNA (Fig EV2C and D), bacterial translocation wa