Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic ("don't eat me") CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6C$^{hi}$ monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more "pro-phagocytic," suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages

Neuroprotective tissue adaptation induced by IL-12 attenuates CNS inflammation

IL-12 is a well-established driver of type 1 immune responses. Paradoxically, in several autoimmune conditions including neuroinflammation, IL-12 reduces pathology and exhibits regulatory properties. Yet, the mechanism and the involved cellular players behind this immune regulation remain elusive. To identify the IL-12-responsive elements which prevent immunopathology, we generated mouse models lacking a functional IL-12 receptor either in all cells or in specific populations within the immune or central nervous system (CNS) compartments, and induced experimental autoimmune encephalomyelitis (EAE), which models human Multiple Sclerosis (MS). This revealed that the CNS tissue-protective features of IL-12 are mediated by cells of the neuroectoderm, and not immune cells. Importantly, sections of brain from patients with MS show comparable patterns of expression, indicating parallel mechanisms in humans. By combining spectral flow cytometry, bulk and single-nucleus RNA sequencing, we uncovered an IL-12-induced neuroprotective adaption of the neuroectoderm critically involved in maintaining CNS tissue integrity during inflammation

Glucocorticoid Receptor-Deficient Foxp3+ Regulatory T Cells Fail to Control Experimental Inflammatory Bowel Disease

Activation of the immune system increases systemic adrenal-derived glucocorticoid (GC) levels which downregulate the immune response as part of a negative feedback loop. While CD4+ T cells are essential target cells affected by GC, it is not known whether these hormones exert their major effects on CD4+ helper T cells, CD4+Foxp3+ regulatory T cells (Treg cells), or both. Here, we generated mice with a specific deletion of the glucocorticoid receptor (GR) in Foxp3+ Treg cells. Remarkably, while basal Treg cell characteristics and in vitro suppression capacity were unchanged, Treg cells lacking the GR did not prevent the induction of inflammatory bowel disease in an in vivo mouse model. Under inflammatory conditions, GR-deficient Treg cells acquired Th1-like characteristics and expressed IFN-gamma, but not IL-17, and failed to inhibit pro-inflammatory CD4+ T cell expansion in situ. These findings reveal that the GR is critical for Foxp3+ Treg cell function and suggest that endogenous GC prevent Treg cell plasticity toward a Th1-like Treg cell phenotype in experimental colitis. When equally active in humans, a rationale is provided to develop GC-mimicking therapeutic strategies which specifically target Foxp3+ Treg cells for the treatment of inflammatory bowel disease

IL-12 sensing in neurons induces neuroprotective CNS tissue adaptation and attenuates neuroinflammation in mice

Interleukin-12 (IL-12) is a potent driver of type 1 immunity. Paradoxically, in autoimmune conditions, including of the CNS, IL-12 reduces inflammation. The underlying mechanism behind these opposing properties and the involved cellular players remain elusive. Here we map IL-12 receptor (IL-12R) expression to NK and T cells as well as neurons and oligodendrocytes. Conditionally ablating the IL-12R across these cell types in adult mice and assessing their susceptibility to experimental autoimmune encephalomyelitis revealed that the neuroprotective role of IL-12 is mediated by neuroectoderm-derived cells, specifically neurons, and not immune cells. In human brain tissue from donors with multiple sclerosis, we observe an IL-12R distribution comparable to mice, suggesting similar mechanisms in mice and humans. Combining flow cytometry, bulk and single-nucleus RNA sequencing, we reveal an IL-12-induced neuroprotective tissue adaption preventing early neurodegeneration and sustaining trophic factor release during neuroinflammation, thereby maintaining CNS integrity in mice

Evaluating the role of pro-survival BCL-2 family member A1 in T cell immunity

Apoptose ist ein evolutionär konservierter Zelltodprozess, der vor allem in multizellulären Organismen eine bedeutende Rolle spielt. Er kann entweder direkt durch eine Liganden- Rezeptor Interaktionen initiiert werden oder über einen zweiten Signalweg, der innerhalb der Zellen eingeleitet wird (intrinsisch) und von Stimuli wie DNA-Schäden oder Entzug von Wachstumsfaktoren aktiviert wird. Beide Signalwege führen zur Aktivierung von speziellen Proteasen, sogenannten Caspasen, die für eine regulierte Zerlegung der Zelle sorgen. Ordnungsgemäß ablaufende Apoptose ist vor allem für die Entwicklung und Funktion des Immunsystems essentiell, da eine abweichende Funktion des Zelltodes zur Entstehung von Lymphomen, Leukämien und Autoimmunerkrankungen führen kann. Meine Dissertation fokussiert sich auf die Rolle des intrinsischen Signalweges der Apoptose in der Entwicklung und Funktion des Immunsystems. Dieser Signalweg wird von der BCL-2 Familie reguliert, welche sich aus drei Gruppen zusammensetzt: BAX, BAK1 und BOK sind Effektorproteine, die den Zelltod auslösen; die sogenannten BH3-Proteine, bestehend aus BIM, BAD, BID, BMF, HRK, PUMA, und NOXA, sind die pro-apoptotischen Initiatoren und die dritte Gruppe besteht aus den zelltodhemmenden Proteinen BCL-2, BCL-xL, BCL-W, MCL1 und A1. Ich interessiere mich vor allem für die Funktion des Proteins A1 im Immunsystem und insbesondere in T Zellen. A1 ist vorwiegend im hämatopoetischen System exprimiert und kann durch Mitogene stark induziert werden. Aufgrund einer Vervierfachung des A1-Gen Lokus in der Maus konnte die Funktion des Proteins allerdings lange Zeit nicht eindeutig identifiziert werden. Unterschiedliche Studien nutzten RNA-Interferenz zur Proteinreduktion, um die Rolle von A1 im Immunsystem zu untersuchen. Diese ließen eine Funktion von A1 für das Überleben von neutrophilen Granulozyten und Mastzellen sowie bei der Entwicklung von T und B Zellen vermuten. In einer in dieser Arbeit beschriebenen Publikationen zeige ich, dass A1 auch für das Überleben von reifen B Zellen wichtig ist. In einer Kollaboration mit Dr. Marco Herold und Dr. Andreas Strasser vom Walter and Eliza Hall Institut für Medizinische Forschung in Melbourne, Australien, bekam ich letztendlich die Möglichkeit Mäuse zu untersuchen, die komplett A1 defizient sind. Überraschenderweise zeigten die A1 defizienten Mäuse keine offensichtlichen Veränderungen in ihrer Entwicklung und auch der Aufbau sowie die Funktion des Immunsystems erschienen normal. Des Weiteren waren auch aktivierte T Zellen, die äußert hohe A1 Expression in den Kontrollen aufweisen, durch A1 Deletion in Zahl und Funktion nicht beeinträchtigt. Schlussfolgernd kann ich sagen, dass A1 mit zumindest einem anderen zelltodhemmenden Familienmitglied redundant ist. Aufgrund der häufigen Überexpression von A1 in verschiedenen Tumoren sind meine Ergebnisse äußerst interessant, da sie für die Entwicklung von A1 hemmenden Medikamenten und deren sichere Anwendung von Bedeutung sind.Apoptosis is an evolutionary conserved cellular suicide-process that is of importance for all multi-cellular organisms. It can be induced either by ligand binding to specific death receptors referred to as extrinsic apoptosis pathway or initiated by intracellularly sensed types of stress like DNA-damage or deprivation of growth factors referred to as intrinsic apoptosis pathway. Both pathways merge at the level of specialized executioner proteases (caspases) that are responsible for an organized destruction of the cell. Apoptosis is especially needed for a proper development and function of the immune system. Aberrant apoptosis can result in severe malignancies like lymphomas, leukaemias and autoimmune-diseases. The focus of my thesis lies on the intrinsic pathway that is regulated by the BCL-2 protein family, which consists of three main branches: the pro-apoptotic effectors BAX, BAK1 and BOK, the initiators BIM, BAD, BID, BIK, BMF, HRK, PUMA, and NOXA (also known as BH3-only proteins) and the guardians of cellular survival the pro-survival proteins BCL-2, BCLxL, BCL-W, MCL-1 and A1. I was interested in the function of A1 in the immune system and especially in T cells. A1 is expressed mainly in the hematopoietic system and can be rapidly induced by mitogen-mediated activation. However, the physiological role of A1 in the immune system was elusive for a long time due to a quadruplication of the A1 gene in the mouse genome leading to three fully functional A1 paralogues and making genetic targeting very difficult. Different studies using in vivo silencing of A1 with RNA-interference pointed towards a role of the protein in the survival of neutrophils and mast cells but also in the development of T and B cells. I also contributed to a study showing that ablated A1 protein led to impaired survival of mature B cells, which is also described in this thesis. However, as the achieved efficiency of a knockdown could never resemble a full deletion of the gene I was eager to analyze the effects of a fully A1 deficient mouse model which was done in collaboration with Dr. Marco Herold and Dr. Andreas Strasser at the Walter and Eliza Hall Institute of Medical Research. In contrast to the other pro-survival BCL-2 family members, A1 deficient mice did not exhibit any obvious developmental phenotype. Furthermore, we could not detect any defects in the differentiation of the immune cell types analyzed. Unexpectedly, also T cell function upon viral infections remained unaffected although A1 expression is highly upregulated in activated T cells, compared to resting, naïve T cells. Therefore, I conclude that A1 is highly redundant with one or more of the other BCL2 family members. As A1 is often found to be overexpressed in human cancers, my findings are, although unexpected, of special interest for the generation of A1 targeting drugs and their potential use in clinics.by Selma Tuzlak, MScKumulative Dissertation aus drei ArtikelnZusammenfassung in deutscher SpracheMedical University of Innsbruck, Dissertation, 2017OeBB(VLID)197165

Interrogating the relevance of mitochondrial apoptosis for vertebrate development and postnatal tissue homeostasis.

"Programmed cell death or 'apoptosis' is critical for organogenesis during embryonic development and tissue homeostasis in the adult. Its deregulation can contribute to a broad range of human pathologies, including neurodegeneration, cancer, or autoimmunity…" These or similar phrases have become generic opening statements in many reviews and textbooks describing the physiological relevance of apoptotic cell death. However, while the role in disease has been documented beyond doubt, facilitating innovative drug discovery, we wonder whether the former is really true. What goes wrong in vertebrate development or in adult tissue when the main route to apoptotic cell death, controlled by the BCL2 family, is impaired? Such scenarios have been mimicked by deletion of one or more prodeath genes within the BCL2 family, and gene targeting studies in mice exploring the consequences have been manifold. Many of these studies were geared toward understanding the role of BCL2 family proteins and mitochondrial apoptosis in disease, whereas fewer focused in detail on their role during normal development or tissue homeostasis, perhaps also due to an irritating lack of phenotype. Looking at these studies, the relevance of classical programmed cell death by apoptosis for development appears rather limited. Together, these many studies suggest either highly selective and context-dependent contributions of mitochondrial apoptosis or significant redundancy with alternative cell death mechanisms, as summarized and discussed here

Repositioning TH cell polarization from single cytokines to complex help

International audienceWhen helper T (TH) cell polarization was initially described three decades ago, the TH cell universe grew dramatically. New subsets were described based on their expression of few specific cytokines. Beyond TH1 and TH2 cells, this led to the coining of various TH17 and regulatory (Treg) cell subsets as well as TH22, TH25, follicular helper (TFH), TH3, TH5 and TH9 cells. High-dimensional single-cell analysis revealed that a categorization of TH cells into a single-cytokine-based nomenclature fails to capture the complexity and diversity of TH cells. Similar to the simple nomenclature used to describe innate lymphoid cells (ILCs), we propose that TH cell polarization should be categorized in terms of the help they provide to phagocytes (type 1), to B cells, eosinophils and mast cells (type 2) and to non-immune tissue cells, including the stroma and epithelium (type 3). Studying TH cells based on their helper function and the cells they help, rather than phenotypic features such as individual analyzed cytokines or transcription factors, better captures TH cell plasticity and conversion as well as the breadth of immune responses in vivo

Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein-Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways

Repositioning T H cell polarization from single cytokines to complex help

When helper T (TH) cell polarization was initially described three decades ago, the TH cell universe grew dramatically. New subsets were described based on their expression of few specific cytokines. Beyond TH1 and TH2 cells, this led to the coining of various TH17 and regulatory (Treg) cell subsets as well as TH22, TH25, follicular helper (TFH), TH3, TH5 and TH9 cells. High-dimensional single-cell analysis revealed that a categorization of TH cells into a single-cytokine-based nomenclature fails to capture the complexity and diversity of TH cells. Similar to the simple nomenclature used to describe innate lymphoid cells (ILCs), we propose that TH cell polarization should be categorized in terms of the help they provide to phagocytes (type 1), to B cells, eosinophils and mast cells (type 2) and to non-immune tissue cells, including the stroma and epithelium (type 3). Studying TH cells based on their helper function and the cells they help, rather than phenotypic features such as individual analyzed cytokines or transcription factors, better captures TH cell plasticity and conversion as well as the breadth of immune responses in vivo