X-chromosome and kidney function:evidence from a multi-trait genetic analysis of 908,697 individuals reveals sex-specific and sex-differential findings in genes regulated by androgen response elements

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research.</p

Target genes, variants, tissues and transcriptional pathways influencing human serum urate levels.

Elevated serum urate levels cause gout and correlate with cardiometabolic diseases via poorly understood mechanisms. We performed a trans-ancestry genome-wide association study of serum urate in 457,690 individuals, identifying 183 loci (147 previously unknown) that improve the prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardiometabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urate-associated loci and colocalization with gene expression in 47 tissues implicated the kidney and liver as the main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in the liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A transactivated the promoter of ABCG2, encoding a major urate transporter, in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardiometabolic traits.The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Variant annotation was supported by software resources provided via the Caché Campus program of the InterSystems GmbH to Alexander Teumer

Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells

Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of &gamma; radiation. GPCs were treated with 2.5 Gy of &gamma; radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape

Detrimental effects of exuberant and restrained immune responses against the central nervous system in the context of multiple sclerosis and glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and most aggressive astrocytic tumor of the central nervous system (CNS) in adults. The standard treatment consisting of surgery, followed by a combinatorial radio- and chemotherapy, is only palliative and prolongs patient median survival to 12 to 15 months. The tumor subpopulation of stem cell-like glioma-initiating cells (GICs) shows resistance against radiation as well as chemotherapy, and has been suggested to be responsible for relapses of more aggressive tumors after therapy. The efficacy of immunotherapies, which exploit the immune system to specifically recognize and eliminate malignant cells, is limited due to strong immunosuppressive activities of the GICs and the generation of a specialized protective microenvironment. The molecular mechanisms underlying the therapy resistance of GICs are largely unknown. rnThe first aim of this study was to identify immune evasion mechanisms in GICs triggered by radiation. A model was used in which patient-derived GICs were treated in vitro with fractionated ionizing radiation (2.5 Gy in 7 consecutive passages) to select for a more radio-resistant phenotype. In the model cell line 1080, this selection process resulted in increased proliferative but diminished migratory capacities in comparison to untreated control GICs. Furthermore, radio-selected GICs downregulated various proteins involved in antigen processing and presentation, resulting in decreased expression of MHC class I molecules on the cellular surface and diminished recognition potential by cytotoxic CD8+ T cells. Thus, sub-lethal fractionated radiation can promote immune evasion and hamper the success of adjuvant immunotherapy. Among several immune-associated proteins, interferon-induced transmembrane protein 3 (IFITM3) was found to be upregulated in radio-selected GICs. While high expression of IFITM3 was associated with a worse overall survival of GBM patients (TCGA database) and increased proliferation and migration of differentiated glioma cell lines, a strong contribution of IFITM3 to proliferation in vitro as well as tumor growth and invasiveness in a xenograft model could not be observed. rnMultiple sclerosis (MS) is the most common autoimmune disease of the CNS in young adults of the Western World, which leads to progressive disability in genetically susceptible individuals, possibly triggered by environmental factors. It is assumed that self-reactive, myelin-specific T helper cell 1 (Th1) and Th17 cells, which have escaped the control mechanisms of the immune system, are critical in the pathogenesis of the human disease and its animal model experimental autoimmune encephalomyelitis (EAE). It was observed that in vitro differentiated interleukin 17 (IL-17) producing Th17 cells co-expressed the Th1-phenotypic cytokine Interferon-gamma (IFN-γ) in combination with the two respective lineage-associated transcription factors RORγt and T-bet after re-isolation from the CNS of diseased mice. Pathogenic molecular mechanisms that render a CD4+ T cell encephalitogenic have scarcely been investigated up to date. rnIn the second part of the thesis, whole transcriptional changes occurring in in vitro differentiated Th17 cells in the course of EAE were analyzed. Evaluation of signaling networks revealed an overrepresentation of genes involved in communication between the innate and adaptive immune system and metabolic alterations including cholesterol biosynthesis. The transcription factors Cebpa, Fos, Klf4, Nfatc1 and Spi1, associated with thymocyte development and naïve T cells were upregulated in encephalitogenic CNS-isolated CD4+ T cells, proposing a contribution to T cell plasticity. Correlation of the murine T-cell gene expression dataset to putative MS risk genes, which were selected based on their proximity (± 500 kb; ensembl database, release 75) to the MS risk single nucleotide polymorphisms (SNPs) proposed by the most recent multiple sclerosis GWAS in 2011, revealed that 67.3% of the MS risk genes were differentially expressed in EAE. Expression patterns of Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7, and Thada were confirmed in independent experiments, suggesting a contribution to T cell pathogenicity. Functional analysis of Nfatc1 revealed that Nfatc1-deficient CD4+ T cells were restrained in their ability to induce clinical signs of EAE. Nfatc1-deficiency allowed proper T cell activation, but diminished their potential to fully differentiate into Th17 cells and to express high amounts of lineage cytokines. As the inducible Nfatc1/αA transcript is distinct from the other family members, it could represent an interesting target for therapeutic intervention in MS.rnGlioblastoma multiforme (GBM) ist der häufigste und aggressivste astrozytäre Tumor des Zentralnervensystems bei Erwachsenen. Die Standardtherapie (operativer Entfernung des Tumors mit anschließender Radio- und Chemotherapie) wirkt nur palliativ und verlängert das Patientenleben im Mittel um 12-15 Monate. Die Tumor-Subpopulation der stammzellähnlichen Gliom-initiierenden Zellen (englisch: GIC) ist resistent gegenüber den Therapien, und wird für das Entstehen von aggressiveren Sekundärtumoren verantwortlich gemacht. Die Wirksamkeit von Immuntherapien, die das Immunsystem ausnutzen, spezifisch maligante Zellen zu erkennen und zu eliminieren, ist begrenzt aufgrund starker immunsuppressiver Aktivitäten der GICs. rnDas erste Ziel dieser Studie war die Identifizierung von Mechanismen der Immunevasion bei GICs, die durch Bestrahlung ausgelöst werden. Ein Model wurde genutzt, bei dem GICs aus Biopsien in vitro mit fraktionierter ionisierender Strahlung behandelt wurden (2.5 Gy in 7 aufeinanderfolgenden Zellpassagen) um einen strahlungsresistenteren Phänotypen zu selektieren. In der Modell-GIC-Zelllinie 1080 führte dies zu einer erhöhten Zellteilungsrate aber geringerer Migration im Vergleich zu unbehandelten Kontroll-GICs. Des Weiteren haben die strahlungsselektierten GICs diverse Proteine, die in der Antigenprozessierung und –präsentation involviert sind, herunter reguliert, was eine Verringerung von MHC Klasse I Moleküle auf der Zelloberfläche zur Folge hat. Das zeigt, dass eine subletale fraktionierte Bestrahlung eine Immunevasion fördern und somit den Erfolg von kombinierter Immuntherapie hintern kann. Unter den verschiedenen immunassoziierten Proteinen wurde eine erhöhte Expression des Interferon-induzierten Transmembranproteins 3 (IFITM3) in strahlungsselektierten GICs festgestellt. Während eine Assoziation von erhöhter IFITM3 Expression und einem schlechteren Gesamtüberleben festgestellt (TCGA Datenbank), und auch eine erhöhte Proliferations- und Migrationsrate bei differenzierten Gliom-Zelllinien gezeigt wurde, konnte ein signifikanter Beitrag von IFITM3 in der Proliferation von GICs als auch im Tumorwachstum und der Invasivität von GICs in einem Xenograft-Model nicht beobachtet werden. rnMultiple Sklerose (MS) ist die häufigste Autoimmunerkrankung des ZNS in jungen Erwachsenen der westlichen Welt, die zur progressiven Behinderung in genetisch empfänglichen Individuen führt, die vermutlich durch Umweltfaktoren ausgelöst wird. Selbst-reaktive, Myelin-spezifische T Helfer 1 (Th1) Zellen und Th17 Zellen, die den Kontrollmechanismen des Immunsystems entkommen sind, sind entscheidend für die Pathogenese der human Erkrankung als auch des Tiermodells der experimentellen autoimmunen Enzephalomyelitis (EAE) sind. Es wurde gezeigt, dass in vitro differenzierte Interleukin 17 (IL-17) -produzierende Th17 Zellen, nach Isolierung aus dem ZNS erkrankter Mäuse, zusätzlich das Th1-typische Zytokin Interferon-gamma (IFN-γ) in Kombination mit den entsprechenden Sublinien-assoziierten Transkriptionsfaktoren RORγt und T-bet exprimieren. Pathogene molekulare Mechanismen die der Enzephalitogenität von CD4+ T Zellen unterliegen, sind bis heute nur unzureichend untersucht. rnIm zweiten Teil der Arbeit wurden Veränderungen des gesamten Transkriptoms von in vitro differenzierten Th17 Zellen im Verlauf einer EAE analysiert. Die Evaluation von Signalnetzwerken ergab eine Überrepräsentation von Genen die in der Kommunikation zwischen dem angeborenen und adaptiven Immunsystem und in metabolischen Veränderungen, u.a. der Cholesterin-Biosynthese mitwirken. Die Transkriptionsfaktoren Cebpa, Fos, Klf4, Nfatc1 und Spi1, die mit der T Zell Entwicklung und naiven T Zellen assoziiert werden, waren in encephalitogenen ZNS-isolierten CD4+ T Zellen erhöht exprimiert, was ihren Beitrag zur T Zell Plastizität suggeriert. Eine Korrelation der murinen T Zell-basierten Transkriptomanalyse mit MS-Suszeptibilitätsgenen, die aufgrund ihrer nahen Lokalisation (± 500 kb, ensembl Datenbank, release 75) zu den MS-Risiko Nucleotid-Polymorphismen (SNPs), die in der Gesamtgenom-Assoziationsstudie (genome-wide association studies, GWAS) 2011 identifiziert wurden, selektiert wurden, ergab, dass 67.3% der MS Risikogene in der EAE differentiell exprimiert wurden. Die Expressionsmuster von Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7, und Thada wurden in unabhängigen Experimenten bestätigt, was eine Beteiligung an T Zell Pathogenität suggeriert. Eine erste funktionale Analyse von Nfatc1 ergab, dass Nfatc1-defiziente CD4+ T Zellen keine klinischen Symptome der EAE auslösen können. Nfatc1-defiziente T Zellen können zwar aktiviert werden, ihre Differenzierung in Th17 Zellen mit entsprechender Zytokinexpression ist verringert. Das induzierbare Nfatc1/αA Transcript unterscheidet sich von den anderen NFAT-Familienmitgliedern und könnte ein interessantes Target für eine therapeutische Intervention bei MS darstellen

Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells

Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of γ radiation. GPCs were treated with 2.5 Gy of γ radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape

Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism’s lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process

Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System.

SummaryDuring early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism’s lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process

X-chromosome and kidney function: evidence from a multi-trait genetic analysis of 908,697 individuals reveals sex-specific and sex-differential findings in genes regulated by androgen response elements.

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research