Stat6-Dependent Inhibition of Mincle Expression in Mouse and Human Antigen-Presenting Cells by the Th2 Cytokine IL-4

The C-type lectin receptors (CLR) Mincle, Mcl and Dectin-2 bind mycobacterial and fungal cell wall glycolipids and carbohydrates. Recently, we described that expression of these CLR is down-regulated during differentiation of human monocytes to dendritic cells (DC) in the presence of GM-CSF and IL-4. Here, we demonstrate that the Th2 cytokine IL-4 specifically inhibits expression of Mincle, Mcl and Dectin-2in human APC. This inhibitory effect of IL-4 was observed across species, as murine macrophages and DC treated with IL-4 also down-regulated these receptors. IL-4 blocked up-regulation of Mincle and Mcl mRNA expression and cell surface protein by murine macrophages in response to the Mincle ligand Trehalose-6,6-dibehenate (TDB), whereas the TLR4 ligand LPS overcame inhibition by IL-4. Functionally, down-regulation of Mincle expression by IL-4 was accompanied by reduced cytokine production upon stimulation with TDB. These inhibitory effects of IL-4 were dependent on the transcription factor Stat6. Together, our results show that the key Th2 cytokine IL-4 exerts a negative effect on the expression of Mincle and other Dectin-2 cluster CLR in mouse and human macrophages and DC, which may render these sentinel cells less vigilant for sensing mycobacterial and fungal ligands

Decidual and placental NOD1 is associated with inflammation in normal and preeclamptic pregnancies

Introduction: Inflammation is a normal physiological process that increases to harmful levels in preeclampsia. It affects the interaction between maternal immune cells and fetal trophoblasts at both sites of the maternal-fetal interface; decidua and placenta. The pattern recognition receptor nucleotide-binding oligomerization domain-containing protein (NOD)1 is expressed at both sites. This study aimed to characterize the cellular expression and functionality of NOD1 at the maternal-fetal interface of normal and preeclamptic pregnancies. Methods: Women with normal or preeclamptic pregnancies delivered by caesarean section were included. Decidual (n = 90) and placental (n = 91) samples were analyzed for NOD1 expression by immunohistochemistry and an automated image-based quantification method. Decidual and placental explants were incubated with or without the NOD1-agonist iE-DAP and cytokine responses measured by ELISA. Results: NOD1 was markedly expressed by maternal cells in the decidua and by fetal trophoblasts in both decidua and placenta, with trophoblasts showing the highest NOD1 expression. Preeclampsia with normal fetal growth was associated with a trophoblast-dependent increase in decidual NOD1 expression density. Compared to normal pregnancies, preeclampsia demonstrated stronger correlation between decidual and placental NOD1 expression levels. Increased production of interleukin (IL)-6 or IL-8 after in vitro explant stimulation confirmed NOD1 functionality. Discussion: These findings suggest that NOD1 contributes to inflammation at the maternal-fetal interface in normal pregnancies and preeclampsia and indicate a role in direct maternal-fetal communication. The strong expression of NOD1 by all trophoblast types highlights the importance of combined assessment of decidua and placenta for overall understanding of pathophysiological processes at the maternal-fetal interface.publishedVersio

Patient derived colonoids as drug testing platforms - Critical importance of oxygen concentration

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology

Divergent Regulation of Decidual Oxidative-Stress Response by NRF2 and KEAP1 in Preeclampsia with and without Fetal Growth Restriction

Utero-placental development in pregnancy depends on direct maternal–fetal interaction in the uterine wall decidua. Abnormal uterine vascular remodeling preceding placental oxidative stress and placental dysfunction are associated with preeclampsia and fetal growth restriction (FGR). Oxidative stress is counteracted by antioxidants and oxidative repair mechanisms regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). We aimed to determine the decidual regulation of the oxidative-stress response by NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) in normal pregnancies and preeclamptic pregnancies with and without FGR. Decidual tissue from 145 pregnancies at delivery was assessed for oxidative stress, non-enzymatic antioxidant capacity, cellular NRF2- and KEAP1-protein expression, and NRF2-regulated transcriptional activation. Preeclampsia combined with FGR was associated with an increased oxidative-stress level and NRF2-regulated gene expression in the decidua, while decidual NRF2- and KEAP1-protein expression was unaffected. Although preeclampsia with normal fetal growth also showed increased decidual oxidative stress, NRF2-regulated gene expression was reduced, and KEAP1-protein expression was increased in areas of high trophoblast density. The trophoblast-dependent KEAP1-protein expression in preeclampsia with normal fetal growth indicates control of decidual oxidative stress by maternal–fetal interaction and underscores the importance of discriminating between preeclampsia with and without FGR.publishedVersio

Arqus Openness Position Paper

The Openness Position Paper published by the Arqus European University Alliance emphasises that Arqus institutions, in line with the policies, roadmaps and strategies of the EU and a wide range of stakeholders, are striving jointly to make further progress towards realising Open Science. The Position Paper identifies and acknowledges aims and values of Open Science and relates them to values, principles, and standards shared by the Arqus Alliance, followed by a vision for a future with Open Science. In the interest of a nuanced picture, the Position Paper discusses not only desired effects, but also possible areas of tension related to Open Science. It presents a wide range of specific aims and recommendations for each of the eleven elements of Open Science defined by the Arqus Openness Task Force: Governance Publications (including Open Access) Data (including research data management, FAIR and Open Data) Infrastructures (including support staff, Open Science software and tools, repositories, Open Labs) Methods (including source code, preregistration, materials, workflows, protocols, lab notes) Awareness and training (including education of early-stage researchers) Evaluation (including Open Metrics, research assessment, Open Peer Review, rewards and incentives) Communication (including multilingualism) Citizen Science Open Education Open Innovation The Position Paper concludes with an annex that highlights the progress already made in the implementation and support of Open Science practices at Arqus institutions.Cofunded by the Erasmus+Programme of the European Unio

D7.4 How to be FAIR with your data. A teaching and training handbook for higher education institutions

This handbook aims to support higher education institutions with the integration of FAIR-related content in their curricula and teaching. It was written and edited by a group of about 40 collaborators in a series of six book sprint events that took place between 1 and 10 June 2021. The document provides practical material, such as competence profiles, learning outcomes and lesson plans, and supporting information. It incorporates community feedback received during the public consultation which ran from 27 July to 12 September 2021

Aktivierung humaner antigenpräsentierender Zellen durch den mykobakteriellen Cord-Faktor und das Glykolipid-Adjuvans Trehalose-6,6’-dibehenate

The mycobacterial cord factor trehalose-6,6’-dimycolate (TDM) is an abundant cell wall glycolipid of Mycobacterium tuberculosis and other mycobacteria. It causes inflammation and adjuvanticity, but it is also a major virulence factor of M. tuberculosis. Its synthetic analogue trehalose-6,6’-dibehenate (TDB) has robust adjuvant activity and induces a Th1/Th17 T cell response in animal models. The TDB-containing liposomal adjuvant formulation Caf01 has entered phase I clinical studies in humans. In mice, the C-type lectin receptors (CLRs) MINCLE and MCL act as sensors for TDB and TDM and activate macrophages and dendritic cells (DC) through the SYK-CARD9 pathway. Yet, little is known about the interaction of human antigen-presenting cells (APC) with TDB and TDM; the question remains open whether the same pathway is engaged. We established an in vitro model to characterize the inflammatory response of primary human monocytes, macrophages and DC to TDB and TDM stimulation. TDB/TDM stimulation induced a robust release of chemokines and cytokines, while the pattern of response was dependent on stimulus and cell type. Whereas monocytes and GMCSF-derived macrophages secreted comparable amounts of chemokines and cytokines when stimulated with TDB and TDM, in MCSF-derived macrophages and DC the response was markedly dependent on the stimulus. Hypothesizing that the receptors for TDB/TDM recognition would be conserved in mice and humans, we analyzed requirements for SYK and CARD9 expression and activity, and demonstrated that they were necessary for glycolipid-induced IL8 secretion. In human monocytes and macrophages mRNA expression of MINCLE and MCL was high. Moreover, we could detect MINCLE and MCL protein with predominantly intracellular localization in resting cells. We found no upregulation of MINCLE mRNA expression induced by MCL, as it has been described in mice; induction by stimulation was weak. This suggests a different expression pattern of the receptors in mouse and man. Regarding the functionality of MINCLE and MCL, IL8 production of GMCSF-derived macrophages in response to glycolipid stimulation was partially reduced after siRNA knockdown. In turn, genetic complementation of human MINCLE, but not MCL, in murine MINCLE-deficient DC was sufficient to restore their responsiveness to TDB/TDM stimulation. Furthermore, human MINCLE showed dose-dependent binding to TDB and TDM. In this comprehensive study, we could demonstrate that in human innate immune cells dependent on the SYK-CARD9 pathway the CLR MINCLE contributes to recognition of TDB/TDM, while the role of MCL remains less clear. These findings will help to better understand the interaction of mycobacteria with immune cells and to interpret the results of the first clinical trials with the prototypic liposomal adjuvant Caf01.Der mykobakterielle Cord-Faktor Trehalose-6,6’-dimycolate (TDM) ist das häufigste Glykolipid in der Zellwand von Mycobacterium tuberculosis und anderen Mykobakterien. TDM ist ein wichtiger mykobakterieller Virulenzfaktor, wirkt jedoch auch als Adjuvans. Ein synthetisches Analogon von TDM, Trehalose-6,6’-dibehenate (TDB), zeigt in Tiermodellen Adjuvanswirkung und vermittelt eine Th1/Th17 T-Zell-abhängige Immunantwort. Das liposomale Adjuvans Caf01 enthält TDB und wird derzeit in klinischen Phase-I-Studien erprobt. In Mäusen fungieren die C-Typ Lektin-Rezeptoren (CLRs) MINCLE und MCL als Rezeptoren für TDB und TDM und können Makrophagen und Dendritische Zellen (DC) abhängig vom SYK-CARD9 Signalweg aktivieren. Über die Interaktion humaner Antigen-präsentierenden Zellen (APC) mit TDB und TDM ist bisher wenig bekannt. Um die Reaktion von humanen Monozyten, Makrophagen und DC auf TDB und TDM zu untersuchen, wurde ein in vitro-Stimulationsmodell etabliert. Stimulation mit TDB/TDM führte zu stimulus- und zelltyp-abhängiger Sekretion von Zytokinen und Chemokinen. In der Annahme, dass die Rezeptoren für TDB/TDM-Erkennung in Maus und Mensch konserviert sind, wurde untersucht, ob Expression und Aktivität von SYK und CARD9 für die Antwort erforderlich sind. In der Tat zeigte sich, dass die Glykolipid-induzierte Sekretion von IL8 abhängig von SYK-CARD9 ist. Die mRNA-Expression von MINCLE und MCL in humanen Monozyten und Makrophagen war hoch. MINCLE und MCL Protein war in ruhenden Zellen überwiegend intrazellulär lokalisiert. Anders als in Mäusen beschrieben, wurde nach Stimulation keine Hochregulation der MINCLE mRNA-Expression, und nur eine geringe Änderung der Proteinexpression beobachtet; dies deutet auf grundsätzliche Expressionsunterschiede in Maus und Mensch hin. Im Hinblick auf die Funktionalität der Rezeptoren, führte der siRNA-Knockdown von MINCLE und MCL in GMCSF-differenzierten Makrophagen zu einer partiellen Reduktion der IL8-Antwort auf Glykolipid-Stimulation. Zusätzlich konnte die retrovirale Transduktion von humanem MINCLE, aber nicht von MCL, in murine MINCLE-defiziente DC die Antwort auf TDB/TDM-Stimulation wieder herstellen. Eine dosisabhängige Bindung von humanem MINCLE an TDB/TDM wurde nachgewiesen. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der humane MINCLE Rezeptor in Abhängigkeit vom SYK-CARD9-Signalweg an der Erkennung von TDB/TDM beteiligt ist. Die Rolle von Mcl für die Glykolipid-Erkennung ist noch nicht vollständig geklärt. Die Ergebnisse tragen dazu bei, die Interakion humaner Immunzellen mit Mykobakterien besser zu verstehen, und helfen, die Ergebnisse der klinischen Studien mit dem prototypischen liposomalen Adjuvans Caf01 zu interpretieren

Contact, Collaboration, Conflict: Signal Integration of Syk-coupled C-type Lectin Receptors

Several spleen tyrosine kinase–coupled C-type lectin receptors (CLRs) have emerged as important pattern recognition receptors for infectious danger. Because encounter with microbial pathogens leads to the simultaneous ligation of several CLRs and TLRs, the signals emanating from different pattern recognition receptors have to be integrated to achieve appropriate biological responses. In this review, we briefly summarize current knowledge about ligand recognition and core signaling by Syk-coupled CLRs. We then address mechanisms of synergistic and antagonistic crosstalk between different CLRs and with TLRs. Emerging evidence suggests that signal integration occurs through 1) direct interaction between receptors, 2) regulation of expression levels and localization, and 3) collaborative or conflicting signaling interference. Accordingly, we aim to provide a conceptual framework for the complex and sometimes unexpected outcome of CLR ligation in bacterial and fungal infection

Demystifying Research Data Management: What do you need to know when you start RDM-ing in Norway?

<p>RDA, NFR, EOSC, BOTT, NVA, Sikt, NeIC, DigDir, UHR, NIRD, UBs, TSD, NESH, REK, KD, ESFRI, QualiFAIR - are you familiar with all the abbreviations? Are you new to research data management (RDM) in Norway or have been in the field for a long time but are still confused as we are?</p><p>Research Data Alliance Norway (NO-RDA) aims to create a structured overview of important actors, infrastructures, networks, services and collaborations in the Norwegian RDM landscape. In this session we will present results from our initial mapping, discuss questions submitted through our survey, and have invited representatives from central actors to present their services and answer questions live.</p><p><strong>Topics</strong></p><ul><li>Important events and policies</li><li>Sorting actors</li><li>Infrastructure</li><li>Data that needs protection</li><li>Where to get help?</li></ul><p><strong>Invited guests</strong></p><ul><li>Nenitha Dagslott, The Research Council of Norway</li><li>Bodil Agasøster, Sikt</li><li>Abdulrahman Azab, NeIC</li><li>Koenraad de Smedt, CLARINO</li><li>Agata Bochynska, QualiFAIR</li></ul&gt

Demystifying Research Data Management: What do you need to know when you start RDM-ing in Norway?

<p>RDA, NFR, EOSC, BOTT, NVA, Sikt, NeIC, DigDir, UHR, NIRD, UBs, TSD, NESH, REK, KD, ESFRI, QualiFAIR - are you familiar with all the abbreviations? Are you new to research data management (RDM) in Norway or have been in the field for a long time but are still confused as we are?</p><p>Research Data Alliance Norway (NO-RDA) aims to create a structured overview of important actors, infrastructures, networks, services and collaborations in the Norwegian RDM landscape. In this session we will present results from our initial mapping, discuss questions submitted through our survey, and have invited representatives from central actors to present their services and answer questions live.</p><p><strong>Topics</strong></p><ul><li>Important events and policies</li><li>Sorting actors</li><li>Infrastructure</li><li>Data that needs protection</li><li>Where to get help?</li></ul><p><strong>Invited guests</strong></p><ul><li>Nenitha Dagslott, The Research Council of Norway</li><li>Bodil Agasøster, Sikt</li><li>Abdulrahman Azab, NeIC</li><li>Koenraad de Smedt, CLARINO</li><li>Agata Bochynska, QualiFAIR</li></ul&gt