Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease
2022; Springer Nature; Volume: 14; Issue: 9 Linguagem: Inglês
10.15252/emmm.202215687
ISSN1757-4684
AutoresMarilena Letizia, Yin‐Hu Wang, Ulrike Kaufmann, Lorenz Gerbeth, A Sand, Max Brunkhorst, Patrick Weidner, J Ziegler, Chotima Böttcher, Stephan Schlickeiser, C. Perez Fernandez, Megumi Yamashita, Kenneth A. Stauderman, Katherine Sun, Désirée Kunkel, Murali Prakriya, Ashley D. Sanders, Britta Siegmund, Stefan Feske, Carl Weidinger,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoArticle2 August 2022Open Access Source DataTransparent process Store-operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease Marilena Letizia Marilena Letizia orcid.org/0000-0002-8039-9415 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Yin-Hu Wang Yin-Hu Wang orcid.org/0000-0002-3637-8852 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Ulrike Kaufmann Ulrike Kaufmann orcid.org/0000-0003-4317-5794 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Project administration Search for more papers by this author Lorenz Gerbeth Lorenz Gerbeth orcid.org/0000-0001-6638-1959 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Visualization Search for more papers by this author Annegret Sand Annegret Sand Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Max Brunkhorst Max Brunkhorst Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Patrick Weidner Patrick Weidner orcid.org/0000-0003-2448-5981 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany Single Cell Approaches for Personalized Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Methodology Search for more papers by this author Jörn Felix Ziegler Jörn Felix Ziegler orcid.org/0000-0001-7445-541X Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Chotima Böttcher Chotima Böttcher Experimental and Clinical Research Center, Berlin, A Cooperation of Charité and MDC, Berlin, Germany Contribution: Formal analysis, Supervision, Methodology Search for more papers by this author Stephan Schlickeiser Stephan Schlickeiser Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Flow & Mass Cytometry Core Facility, Berlin, Germany Contribution: Formal analysis, Supervision Search for more papers by this author Camila Fernández Camila Fernández Experimental and Clinical Research Center, Berlin, A Cooperation of Charité and MDC, Berlin, Germany Contribution: Formal analysis Search for more papers by this author Megumi Yamashita Megumi Yamashita Department of Pharmacology, Northwestern University, Chicago, IL, USA Contribution: Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Kenneth Stauderman Kenneth Stauderman orcid.org/0000-0002-8090-4549 CalciMedica Inc., La Jolla, CA, USA Contribution: Formal analysis, Investigation Search for more papers by this author Katherine Sun Katherine Sun Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Formal analysis Search for more papers by this author Désirée Kunkel Désirée Kunkel Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Flow & Mass Cytometry Core Facility, Berlin, Germany Contribution: Conceptualization, Methodology Search for more papers by this author Murali Prakriya Murali Prakriya Department of Pharmacology, Northwestern University, Chicago, IL, USA Contribution: Conceptualization, Supervision, Investigation Search for more papers by this author IBDome Researchers IBDome Researchers TRR 241 Research Initiative, Berlin-Erlangen, Germany Search for more papers by this author Ashley Sanders Ashley Sanders orcid.org/0000-0003-3945-0677 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany Single Cell Approaches for Personalized Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Supervision, Methodology Search for more papers by this author Britta Siegmund Britta Siegmund Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Supervision Search for more papers by this author Stefan Feske Corresponding Author Stefan Feske [email protected] orcid.org/0000-0001-5431-8178 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Carl Weidinger Corresponding Author Carl Weidinger [email protected] orcid.org/0000-0002-9948-0088 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Clinician Scientist Program, Berlin Institute of Health, Berlin, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Marilena Letizia Marilena Letizia orcid.org/0000-0002-8039-9415 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Yin-Hu Wang Yin-Hu Wang orcid.org/0000-0002-3637-8852 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Ulrike Kaufmann Ulrike Kaufmann orcid.org/0000-0003-4317-5794 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Project administration Search for more papers by this author Lorenz Gerbeth Lorenz Gerbeth orcid.org/0000-0001-6638-1959 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Visualization Search for more papers by this author Annegret Sand Annegret Sand Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Max Brunkhorst Max Brunkhorst Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Formal analysis, Validation, Investigation Search for more papers by this author Patrick Weidner Patrick Weidner orcid.org/0000-0003-2448-5981 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany Single Cell Approaches for Personalized Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Visualization, Methodology Search for more papers by this author Jörn Felix Ziegler Jörn Felix Ziegler orcid.org/0000-0001-7445-541X Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Formal analysis, Investigation Search for more papers by this author Chotima Böttcher Chotima Böttcher Experimental and Clinical Research Center, Berlin, A Cooperation of Charité and MDC, Berlin, Germany Contribution: Formal analysis, Supervision, Methodology Search for more papers by this author Stephan Schlickeiser Stephan Schlickeiser Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Flow & Mass Cytometry Core Facility, Berlin, Germany Contribution: Formal analysis, Supervision Search for more papers by this author Camila Fernández Camila Fernández Experimental and Clinical Research Center, Berlin, A Cooperation of Charité and MDC, Berlin, Germany Contribution: Formal analysis Search for more papers by this author Megumi Yamashita Megumi Yamashita Department of Pharmacology, Northwestern University, Chicago, IL, USA Contribution: Formal analysis, Investigation, Visualization, Methodology Search for more papers by this author Kenneth Stauderman Kenneth Stauderman orcid.org/0000-0002-8090-4549 CalciMedica Inc., La Jolla, CA, USA Contribution: Formal analysis, Investigation Search for more papers by this author Katherine Sun Katherine Sun Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Formal analysis Search for more papers by this author Désirée Kunkel Désirée Kunkel Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Flow & Mass Cytometry Core Facility, Berlin, Germany Contribution: Conceptualization, Methodology Search for more papers by this author Murali Prakriya Murali Prakriya Department of Pharmacology, Northwestern University, Chicago, IL, USA Contribution: Conceptualization, Supervision, Investigation Search for more papers by this author IBDome Researchers IBDome Researchers TRR 241 Research Initiative, Berlin-Erlangen, Germany Search for more papers by this author Ashley Sanders Ashley Sanders orcid.org/0000-0003-3945-0677 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany Single Cell Approaches for Personalized Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany Contribution: Formal analysis, Supervision, Methodology Search for more papers by this author Britta Siegmund Britta Siegmund Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Contribution: Supervision Search for more papers by this author Stefan Feske Corresponding Author Stefan Feske [email protected] orcid.org/0000-0001-5431-8178 Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Contribution: Conceptualization, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Carl Weidinger Corresponding Author Carl Weidinger [email protected] orcid.org/0000-0002-9948-0088 Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA Clinician Scientist Program, Berlin Institute of Health, Berlin, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Marilena Letizia1,2,†, Yin-Hu Wang3,†, Ulrike Kaufmann3,†, Lorenz Gerbeth1,2, Annegret Sand1,2, Max Brunkhorst1,2, Patrick Weidner1,4,5, Jörn Felix Ziegler1,2, Chotima Böttcher6, Stephan Schlickeiser1,7, Camila Fernández6, Megumi Yamashita8, Kenneth Stauderman9, Katherine Sun3, Désirée Kunkel1,7, Murali Prakriya8, 10, Ashley Sanders1,4,5, Britta Siegmund1,2, Stefan Feske *,3,† and Carl Weidinger *,1,2,3,11,† 1Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany 2Department of Gastroenterology, Infectious Diseases and Rheumatology, Campus Benjamin Franklin, Berlin, Germany 3Department of Pathology, New York University Grossman School of Medicine, New York, NY, USA 4Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany 5Single Cell Approaches for Personalized Medicine, Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany 6Experimental and Clinical Research Center, Berlin, A Cooperation of Charité and MDC, Berlin, Germany 7Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Flow & Mass Cytometry Core Facility, Berlin, Germany 8Department of Pharmacology, Northwestern University, Chicago, IL, USA 9CalciMedica Inc., La Jolla, CA, USA 10TRR 241 Research Initiative, Berlin-Erlangen, Germany 11Clinician Scientist Program, Berlin Institute of Health, Berlin, Germany † These authors contributed equally to this work *Corresponding author. Tel: +1 212 263 9066; E-mail: [email protected] author. Tel: +49 30 450 614 413; E-mail: [email protected] EMBO Mol Med (2022)14:e15687https://doi.org/10.15252/emmm.202215687 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 Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses. Using mass cytometry (CyTOF) to analyze the immune cell composition in the lamina propria (LP) of patients with ulcerative colitis (UC) and Crohn's disease (CD), we observed an enrichment of CD4+ effector T cells producing IL-17A and TNF, CD8+ T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store-operated Ca2+ entry (SOCE), which results from the opening of Ca2+ release-activated Ca2+ (CRAC) channels formed by ORAI and STIM proteins. We observed that the pharmacologic inhibition of SOCE attenuated the production of proinflammatory cytokines including IL-2, IL-4, IL-6, IL-17A, TNF, and IFNγ by human colonic T cells and ILCs, reduced the production of IL-6 by B cells and the production of IFNγ by myeloid cells, but had no effect on the viability, differentiation, and function of intestinal epithelial cells. T cell-specific deletion of CRAC channel genes in mice showed that Orai1, Stim1, and Stim2-deficient T cells have quantitatively distinct defects in SOCE, which correlate with gradually more pronounced impairment of cytokine production by Th1 and Th17 cells and the severity of IBD. Moreover, the pharmacologic inhibition of SOCE with a selective CRAC channel inhibitor attenuated IBD severity and colitogenic T cell function in mice. Our data indicate that SOCE inhibition may be a suitable new approach for the treatment of IBD. Synopsis The immune cell composition, signaling cascades, and cytokine networks controlling inflammation in therapy-refractory inflammatory bowel diseases (IBD) remain incompletely understood. The colon lamina propria (LP) of ulcerative colitis (UC) and Crohn's disease (CD) patients is enriched with CD4+ and CD8+ T cells, IL-17-producing innate immune cells (ILC) and Treg cells. Pharmacological inhibition of store-operated Ca2+ Entry (SOCE) inhibits the production of proinflammatory cytokines and certain activation markers by human LP T cells, B cells, ILCs and myeloid cells. Inhibition of SOCE does not impair the differentiation and function of human or mouse intestinal epithelial cells in colonic organoid cultures. Pharmacologic inhibition of SOCE or T cell-specific deletion of the SOCE genes Orai1 and Stim1 in T cells ameliorates intestinal inflammation in mouse models of colitis. SOCE is an important regulator of intestinal immune cell function and a potential drug target for the treatment of IBD. Introduction Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal (GI) tract that manifests predominantly as two related disease entities, ulcerative colitis (UC) and Crohn's disease (CD). Both forms of IBD are associated with diarrhea, abdominal pain, fatigue as well as the development of colorectal cancer in patients with longstanding colitis and the development of fistulae and stricturing disease in patients with CD (Uhlig & Powrie, 2018). While UC predominantly affects the colon and is characterized by a superficial inflammation of the LP, CD can involve the entire GI tract and cause trans-mural inflammation. New single-cell technologies such as single-cell RNA-sequencing and mass cytometry have greatly advanced our pathophysiological understanding of IBD (Corridoni et al, 2020; Mitsialis et al, 2020) and have helped better delineate the composition of intestinal immune cells in IBD patients that drive inflammation. The LP of UC patients is characterized by a significant enrichment of TNF producing CD8+ effector T cells (Corridoni et al, 2020), IL-17 producing effector memory CD4+ T cells, and an expansion of T regulatory (Treg) cells producing inflammatory cytokines (Mitsialis et al, 2020). HLA-DR+CD56+ granulocytes as well as TNF and IFNγ-producing B cells are increased in the mucosa of CD patients (Mitsialis et al, 2020). Despite these findings, the immune cell composition, signaling cascades, and the cytokine networks controlling inflammation in therapy-refractory IBD remain incompletely understood. Recent advances that have revolutionized IBD treatment include blocking antibodies against pro-inflammatory cytokines such as TNF (e.g. infliximab or adalimumab), IL-12, and IL-23 (ustekinumab) and against integrins such as vedolizumab (Neurath, 2019). Nevertheless, intestinal and colonic resections are still frequently required in patients with therapy-refractory IBD, and novel treatment modalities are urgently needed to improve IBD outcomes. SOCE is the predominant Ca2+ influx pathway in most immune cells and is required for the activation, differentiation, and function of murine and human lymphocytes including B, NK, and T cells. Stimulation of the T cell receptor (TCR) leads to production of the second messenger inositol-1,4,5 triphosphate (IP3), which triggers a transient release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores through IP3 receptors into the cytoplasm (Taylor et al, 2009). The concomitant reduction of Ca2+ concentrations in the ER is sensed by stromal interaction molecules (STIM) 1 and STIM2 in the ER membrane, which are subsequently activated and translocated to the plasma membrane (Liou et al, 2005). Activated STIM1 and STIM2 bind to ORAI1 and its homologs ORAI2 and ORAI3, which form the pore of the Ca2+ release-activated Ca2+ (CRAC) channel. SOCE through CRAC channels causes a sustained elevation of intracellular Ca2+ levels (Prakriya et al, 2006; Vaeth et al, 2020), which is required for the activation of numerous Ca2+-dependent enzymes including calcineurin, calmodulin kinases, or Erk1/2 and transcription factors such as NF-κΒ, CREB, and NFAT (Berry et al, 2018; Vaeth & Feske, 2018b; Wang et al, 2020). SOCE is essential for the transcription of cytokines including IL-2, IFNγ, and TNF by T cells and other immune cells. In addition, SOCE regulates several metabolic pathways such as glycolysis and mitochondrial respiration, thereby controlling lymphocyte proliferation and effector functions (Vaeth et al, 2017; Kaufmann et al, 2019; Wang et al, 2020). The pathophysiological importance of SOCE for immune function is emphasized by patients with loss-of-function mutations in STIM1 or ORAI1 genes, who suffer from combined immunodeficiency with recurrent infections (McCarl et al, 2009; Picard et al, 2009; Fuchs et al, 2012; Lacruz & Feske, 2015; Kahlfuss et al, 2020). Previous studies indicate that SOCE is increased in T cells isolated from inflamed compared to non-inflamed mucosa of IBD patients (Schwarz et al, 2004). Another study has attributed this increase to an elevated expression of STIM1 in CD45+ lamina propria mononuclear cells (LPMCs) from inflamed intestinal tissue (Liang et al, 2022). Together, these findings suggest that SOCE acts as an important signaling axis that promotes intestinal inflammation. Accordingly, genetic or pharmacological inhibition of SOCE suppresses pro-inflammatory T cell functions and intestinal inflammation in animal models of colitis (McCarl et al, 2010; Vaeth et al, 2017). Given the critical role of SOCE in immune cells, we hypothesized that pharmacologic blockade of SOCE might attenuate the pro-inflammatory function of lymphoid and myeloid immune cells from patients with therapy-refractory IBD and therefore represent a new strategy for immune modulation. Using a murine T cell transfer model of colitis, we show that deletion of the CRAC channel genes Stim1, Stim2, or Orai1 in T cells prevents IBD and that the severity of intestinal inflammation correlates with the level of SOCE. By applying mass cytometry (CyTOF), we characterized immune cell subsets in the colonic LP of therapy refractory IBD patients and investigated the effects of the SOCE inhibitors BTP2 and CM4620 on intestinal immune cell populations from IBD patients. SOCE inhibition efficiently suppressed various pro-inflammatory functions of human T cells in a dose-dependent manner and inhibited the function of innate lymphoid cells (ILC) and, to a lesser degree, that of myeloid cells and B cells. Importantly, treatment with BTP2 had no detectable effects on epithelial barrier functions of primary human and murine intestinal epithelial cells (IEC) in vitro. Treatment of mice in which IBD had been induced by adoptive T cell transfer with the selective SOCE inhibitor CM4620 attenuated the clinical course of colitis, which was associated with reduced neutrophil infiltration of the LP and decreased IFNγ and TNFα production by CD4+ T cells, suggesting that SOCE inhibition may be a new treatment option for IBD. Results SOCE in T cells is required for the induction of colitis in mice We had previously shown that murine T cells lacking functional CRAC channels are unable to induce colitis upon adoptive transfer into lymphopenic mice (McCarl et al, 2010; Vaeth et al, 2017). Naive CD4+ T cells isolated from Stim1fl/fl Cd4Cre mice lacked STIM1 expression and had strongly suppressed SOCE. Likewise, CD4+ T cells from Orai1R93W knock-in mice that express a channel-dead version of ORAI1 lacked SOCE almost completely, likely because of a dominant negative effect of the mutant ORAI1 protein on other ORAI paralogues (ORAI2 and ORAI3) with which ORAI1 can form heteromeric channels (Thompson et al, 2009; McCarl et al, 2010; Vaeth et al, 2017). To better understand the role of CRAC channel components in colitogenic T cells and the quantitative requirements of SOCE in T cell-mediated intestinal inflammation, we here used mice with T cell-specific deletion of Orai1, Stim1, and Stim2. Naïve CD4+ T cells from Orai1fl/flCd4Cre, Stim1fl/flCd4Cre, and Stim2fl/flCd4Cre mice were polarized into Th1, Th17, and induced Treg (iTreg) cells in vitro. Whereas deletion of Stim1 strongly suppressed SOCE, lack of Orai1 reduced the amplitude of SOCE by approximately half compared to wildtype T cells (Fig 1A). Deletion of Stim2 resulted in only moderately reduced SOCE. It is noteworthy that deletion of Orai and Stim genes affected SOCE in Th1, Th17, and iTreg cells to a similar degree. Moreover, Th1, Th17, and Treg cells from wildtype mice expressed comparable levels of Orai and Stim genes (Appendix Fig S1). We used T cells from this allelic series of mice with gradual defects of SOCE to investigate the dose–response relationship of SOCE and colitogenic T cell function in vivo. We isolated naive CD4+ T cells from Orai1fl/flCd4Cre, Stim1fl/flCd4Cre, and Stim2fl/flCd4Cre and wildtype mice and injected them into lymphopenic Rag1−/− host mice (Fig 1B). Whereas the transfer of Stim2-deficient CD4+ T cells induced weight loss comparable to wildtype T cells, the injection of Orai1-deficient and Stim1-deficient CD4+ T cells resulted in significantly attenuated or no weight loss, respectively. The histological analysis of intestinal inflammation showed severe colitis in host mice that had received wildtype or Stim2-deficient CD4+ T cells, whereas inflammation was partially reduced or absent following transfer of Orai1-deficient and Stim1-deficient CD4+ T cells, respectively (Fig 1C). Flow cytometric analysis of CD4+ T cells obtained from mesenteric lymph nodes (mLN) showed similar frequencies of cells producing IFNγ, TNF, and IL-17A in mice that had received T cells from Stim2fl/flCd4Cre and wildtype mice (Fig 1D and E). By contrast, IFNγ, TNF, and IL-17A production by adoptively transferred CD4+ T cells from Orai1 and Stim1-deficient mice was significantly reduced in vivo compared to wildtype T cells. The frequencies of Treg cells were significantly reduced in the absence of Orai1 or Stim1, but not in the absence of Stim2 (Fig 1D and E). Together, these findings suggest that moderate inhibition of SOCE in Stim2-deficient CD4+ T cells has no effect on colitogenic T cell function, whereas more pronounced inhibition of SOCE in Orai1 or Stim1-deficient CD4+ T cells gradually suppresses inflammatory cytokine production and their ability to i
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