MAGI2 Gene Region and Celiac Disease

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.This work was partially funded by the Basque Department of Education grant IT1281-19 and ISCIII Research Project PI16/00258, cofunded by the European Union ERDF, A way to make Europe to JB. AC-R is supported by an Ikerbasque Fellowship and funded by a research project grant 2017111082 from the Basque Goverment. IS was funded by a research project grant 2015111068 from the Basque Department of Health. AJ-M and AO-G are predoctoral fellows funded by FPI grants from the Basque Department of Education, Universities and Research and IR-G and MS are predoctoral fellows funded by the University of Basque Country

Identification and functional characterization of LNCRNAS Associated with TYPE 1 Diabetes.

216 p.Estudios de asociación de genoma completo han detectado polimorfismos asociados con el riesgo a padecer diabetes de tipo 1 (DM1) en ARN largos no codificantes (lncRNAs). Sin embargo, todavía se desconocen los mecanismos moleculares por los que la mayoría de estos lncRNAs contribuyen al desarrollo de la DM1.Los estudios recogidos en esta tesis doctoral confirman la presencia de lncRNAs implicados en el desarrollo de DM1. Así mismo, se ha realizado la caracterización funcional de 2 de estos lncRNAs; llamados Lnc13 y ARGI. Gracias a estos estudios se demuestra la importancia de estas moléculas no sólo en el funcionamiento celular (en esta tesis centrada en el estudio de las células ß pancreáticas), sino también en el desarrollo de diversas enfermedades como son la diabetes

A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells

IntroductionMost of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. MethodsHere, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. ResultsWe have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. ConclusionsThese findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.This work was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (PID2019-104475GA-I00 to I.S, and PGC2018-097573-A-I00 to AC-R) and the European Foundation for the Study of Diabetes (EFSD) - EFSD/JDRF/Lilly Programme on Type 1 Diabetes Research to IS. FO (MS19/00109) is recipient of the Miguel Servet scheme, and AL (FI19/00045) was supported by the Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia, Innovación y Universidades, Gobierno de España (ES). HR-M (PRE2019-089350) is supported by predoctoral grant from the Ministerio de Ciencia, Innovacion y Universidades, Gobierno de España (ES) IG-M, MS-C, JM-S and AO-G were supported by Predoctoral Fellowship Grants from the UPV/EHU (Universidad del Pais Vasco/EuskalHerrikoUnibertsitatea) and the Basque Department of Education. MC is supported by the Fonds National de la RechercheScientifique (FNRS), the Francophone Foundation for Diabetes Research (sponsored by the French Diabetes Federation, Abbott, Eli Lilly, Merck Sharp & Dohme, and Novo Nordisk) and FF and MC by the EFSD/BoehringerIngelheim European Research Programme on Multi-System Challenges in Diabetes. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication

Identification and characterization of novel non-coding regulators of innate immune responses in human cells

The onset of immune response against microbial stimuli activates induction of many anti- inflammatory genes and ISGs for effective clearance of the pathogen. This response includes transcriptional activation of several non-coding transcripts such as miRNAs and long non-coding RNAs (lncRNAs). LncRNAs constitutes the largest class of non-coding genome and are arbitrarily described as transcripts greater than 200 base pairs. Similar to protein coding mRNAs, lncRNAs are RNA polymerase II transcripts and undergo mRNA processing such as capping, splicing and polyadenylation. In recent years, high throughput sequencing has enabled an in-depth exploration of the human genome and subsequent discovery of lncRNAs. Several studies have highlighted the crucial role of lncRNAs in many biological processes including as regulators of gene expression as well as molecular effectors of host-pathogen driven immune responses. To date, majority of lncRNAs have been studied in murine models with limited understanding in human cells. In order to elucidate the role of lncRNAs in human immune cell regulation, the goal of this thesis is to identify and characterize novel lncRNAs critical to host-pathogen innate immune responses. RNA sequencing in LPS, IAV and HSV stimulated cells revealed lncRNA LUCAT1 as most differentially regulated lncRNA. CRISPR-cas9 and shRNA mediated depletion of LUCAT1 showed enhanced IFN-I genes signature, which was suppressed upon overexpression of LUCAT1. Additionally, LPS stimulated hDCs showed enrichment of LUCAT1 in the nucleus and its association with the chromatin markers. Further, LUCAT1 depletion contributed to enhanced occupancy of transcriptional coactivators at the promoters of IFN-I genes. Global identification of RNA associated proteins revealed LUCAT1 association with STAT1 in the nucleus thus emphasizing its role in transcriptional regulation of Type I IFN genes in inflammatory responses. This thesis furthers the understanding about the molecular factors affecting immune regulation and describes the novel role of LUCAT1 as an attenuator of immune cell response to pathogens

Regulation of IRF7 by m6A-mediated mechanisms: a link between viral infections and celiac disease

137 p (eusk.) 130 p. (eng.)La enfermedad celíaca es una enfermedad inflamatoria crónica desarrollada en individuos con susceptibilidad genética por una respuesta inmune inadecuada al gluten de la dieta. Sin embargo, la respuesta inmunitaria inducida por el gluten derivada de la susceptibilidad genética explica menos del 50% de la heredabilidad, lo que sugiere que además del gluten podría haber factores ambientales adicionales que contribuyan al inicio de la respuesta inmunitaria. En base a esto, se han propuesto las infecciones virales como posibles agentes desencadenantes en la enfermedad celiaca.Con el fin de investigar la implicación de las infecciones virales en el desarrollo de la enfermedad celiaca, el proyecto actual se ha centrado en la búsqueda de determinantes funcionales relacionados con la respuesta inmunitaria innata contra las infecciones por virus de ARN. Más concretamente, en la presente tesis doctoral se ha analizado una nueva capa de regulación génica de IRF7 tras la combinación de infecciones virales entéricas y el consumo de gluten, y se ha estudiado la implicación de la metilación del ARN m6A en la regulación de este transcrito antiviral y su actividad aguas abajo en la vía de señalización del IFN-I

Long non-coding RNAs involved in myeloid cell differentiation and macrophage activation

The human genome encodes for ~ 20.000 long non-coding RNAs (lncRNAs), yet their molecular functions, especially in the immune system, remain largely unknown. In this study, two main aspects were addressed: the participation of lncRNAs in the differentiation of myeloid immune cells and their involvement in pro-inflammatory activation of human macrophages. In order to address the first aspect, RNA sequencing results from distinct immune cell subsets were analysed. These data showed that lncRNAs define immune-cell identity equally well as protein-coding genes, such as surface receptors considered as precise markers of leukocyte subsets. In the present work, non-coding RNA LINC00211 was identified as a specific myeloid cell lineage marker. Functional characterisation demonstrated that this lncRNA regulates the expression of several genes, including CHI3L1 and S100A9, which participate in myeloid cell differentiation. Furthermore, LINC00211 was regulated by PU.1, a transcription factor with fundamental roles in immune cell lineage commitment. Additionally, LINC00211 could be characterised as a biomarker of pulmonary inflammation, since high expression was observed in bronchoalveolar lavage fluid from infected individuals and in lung extracts from IPF patients, correlating with the degree of neutrophil infiltration. In order to investigate the involvement of lncRNAs in pro-inflammatory activation of human macrophages, RNA sequencing experiments were performed and unveiled several differentially expressed lncRNAs in resting and immune-activated human macrophages. Furthermore, a multidimensional approach was established to categorize human lncRNAs according to their subcellular localization and co-sedimentation with cellular protein complexes in macrophages. The resulting data revealed that lncRNAs constitute a highly heterogeneous class of RNA co-sedimenting with various cellular machineries, including ribosomes. Using these data, lncRNA MaIL1 was identified as a highly immune-responsive, cytosolic and non-ribosome associated intergenic lncRNA (lincRNA). Functional analysis associated MaIL1 with type I interferon production after Toll-like Receptor (TLR) activation. RNA antisense purification and mass spectrometry (RAP-MS) showed that MaIL1 interacts with Optineurin, a protein known to be required for signal transduction within the TBK1-IRF3 axis, thus facilitating type I interferon production. More specifically, MaIL1 regulates Optineurin ubiquitination, a modification essential for Optineurin function. When MaIL1 was knocked down, IRF3 phosphorylation and subsequently type I interferon production was impaired. Moreover, MaIL1 was found to be essential for defence against Legionella pneumophila, a Gram-negative bacterium that predominantly replicates inside alveolar macrophages and causes pneumonia. In addition, MaIL1 levels were increased during pulmonary infections and correlated linearly with IFNβ mRNA levels in human bronchoalveolar lavage fluid. Thus, the present work identifies MaIL1 as a critical regulator of TLR-induced IFN responses to infection. In summary, both studies revealed detailed information about the function of lncRNAs in myeloid immune cells and provide a rich resource and blueprint for future investigations of lncRNA functions in the immune system

Exploring the world of non-coding genes in stem cells and autoimmunity.

Numerous studies have contributed to our current understanding of autoimmune diseases (AIDs), however, pathogenesis of many AIDs can still not be fully explained. Both genetic factors and environmental factors are involved in the onset of autoimmunity. Which mechanisms explain the contribution of these genetic and environmental factors to disease pathogenesis, and how the different factors interplay remain unanswered key questions. The studies presented in this thesis aimed at identifying and unravelling some of the enigmatic mechanisms in rheumatoid arthritis (RA) and systemic sclerosis (SSc). LUMC / Geneeskund

Post-transcriptional Regulation through Long Noncoding RNAs (lncRNAs)

This book is a collection of eight articles, of which seven are reviews and one is a research paper, that together form a Special Issue that describes the roles that long noncoding RNAs (lncRNA) play in gene regulation at a post-transcriptional level

Role of microbiota and related metabolites in gastrointestinal tract barrier function in NAFLD

The Gastrointestinal (GI) tract is composed of four main barriers: microbiological, chemical, physical and immunological. These barriers play important roles in maintaining GI tract homeostasis. In the crosstalk between these barriers, microbiota and related metabolites have been shown to influence GI tract barrier integrity, and alterations of the gut microbiome might lead to an increase in intestinal permeability. As a consequence, translocation of bacteria and their products into the circulatory system increases, reaching proximal and distal tissues, such as the liver. One of the most prevalent chronic liver diseases, Nonalcoholic Fatty Liver Disease (NAFLD), has been associated with an altered gut microbiota and barrier integrity. However, the causal link between them has not been fully elucidated yet. In this review, we aim to highlight relevant bacterial taxa and their related metabolites affecting the GI tract barriers in the context of NAFLD, discussing their implications in gut homeostasis and in disease