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Protein Dysregulation in Immune Cells of Multiple Sclerosis Patients

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Protein Dysregulation in Immune Cells of Multiple Sclerosis Patients Master’s Thesis 2020 60 ECTS Faculty of Chemistry, Biotechnology and Food Sciences Protein Dysregulation in Immune Cells of Multiple Sclerosis Patients Oda Glomstad Kråbøl Biotechnology Faculty of Chemistry, Biotechnology and Food Sciences Protein Dysregulation in Immune Cells of Multiple Sclerosis Patients Oda Glomstad Kråbøl Main supervisor: Tone Berge Co-supervisor: Chiara Cappelletti Internal supervisor: Siv Kjølsrud Bøhn Oslo University Hospital – University of Oslo Multiple Sclerosis Research Group and The Norwegian University of Life Sciences, Faculty of Chemistry, Biotechnology and Food Sciences June 2020 ©Oda Glomstad Kråbøl Protein Dysregulation in Immune Cells of Multiple Sclerosis Patients https://nmbu.brage.unit.no Acknowledgements This thesis was carried out at the MS research group in the Neuroscience Research Unit of Oslo University Hospital (OUS) from August 2019 to June 2020. The thesis is a part of a Master’s Degree in Biotechnology at the Norwegian University of Life Sciences (NMBU), main affiliation being the Faculty of Chemistry, Biotechnology and Food Sciences (KBM). Firstly, I would like to thank Professor Tone Berge for being my supervisor, and for arranging this interesting project. Her expertise and enthusiasm have given me great insights into the challenging yet fascinating multiple sclerosis research field. The guidance I have received throughout this time has been exceptional! I am likewise thankful for my co-supervisor Chiara Cappelletti for devoting her time supervising me in the lab and answering all my questions. She has provided me with protocols as well as theoretical and practical explanations for everything I have needed, which has been invaluable. I would also like to thank the MS research group members for making me feel welcome and included. A special thanks to Anna Eriksson, Ina Brorson and Steffan D. Bos, for sharing their time, knowledge and good advice. Additionally, I would like to thank my supervisor at NMBU, associate professor Siv Kjølsrud Bøhn, for supportive and constructive feedback. Furthermore, I have appreciated the company of my fellow master students Hannah and Stine. Thank you for sharing lunch breaks, experiences and occasional frustrations. Lastly, I am ever grateful for the support from my family and friends during this past year, and especially for Adrian, whose motivational words and encouragement have been essential for finalizing this work. Thank you for always pushing me and inspiring me to do my very best! Oda Glomstad Kråbøl Oslo, June 2020 Abstract Multiple sclerosis (MS) is a complex, autoimmune and inflammatory disease affecting the central nervous system. The pathogenesis of MS is not completely understood, but T cell activation is believed to be an important part of disease etiology. MS susceptibility is provided through a combination of several single contributions involving environmental exposures and multiple genetic risk variants. To date, more than 200 MS associated risk variants have been identified, many of which are linked to immune cell reactions in T cells. In the current project, we aimed at (i) using available proteomic data from CD4+ and CD8+ T cells of genome-wide genotyped MS patients and healthy controls to identify novel MS- linked proteins or pathways. Secondly, (ii) we aimed at collecting new samples from activated CD4+ T cells from the same groups for proteomics profiling by mass spectrometry. (i) A proteomic data set from CD4+ and CD8+ T cells was utilized to correlate the genotype at MS susceptibility variants with the expression of proteins encoded by genes located 100kb upstream and downstream of the MS risk variants. Furthermore, pathway analysis was performed on proteins that were differentially expressed between MS patients and healthy controls in both cell types. (ii) Live CD4+ T cells are stored on liquid nitrogen, and cells from 28 female relapsing-remitting MS patients and 28 healthy individuals were thawed and activated in vitro with a-CD3 and a-CD28 antibodies. The cells were analyzed by flow cytometry for cell surface expression of the T cell activation marker CD69 and with a LIVE/DEAD stain distinguishing live from dead cells. Cells with >50 % CD69 positive cells after activation and with >70 % viability were sent for liquid chromatography-tandem mass spectrometry analysis to analyze the proteome of the samples. (i) Two novel protein quantitative trait loci (pQTL) candidates were identified, i.e. rs1800693 - CD9 and rs137955 - UQCRFS1P1. 26 proteins were differentially expressed in both CD4+ and CD8+ T cells between the groups, and pathway analysis did not identify any specific enriched biological pathways. The identified pQTL candidates might have relevance for MS disease, and can inspire for functional studies to seek a broader understanding of the effect of genetic risk variants, as well as the mechanisms behind MS development. (ii) A total of 20 samples from MS patients and 20 samples from healthy controls fulfilled the inclusion criteria, and were included in the proteomic analysis. Thus, samples from un-activated and cells activated for 24 hours were shipped to the proteomics core facility in Bergen. The samples are lysed an prepared for proteomic profiling, but the results of the analysis is not available yet, due to the Covid-19 situation. Whether the proteomic analysis of activated CD4+ T cells lead to identification of proteins and pathways of importance for MS, remains to be shown. Sammendrag Multippel sklerose er en autoimmun, nevro-inflammatorisk sykdom i sentralnervesystemet med komplekst sykdomsbilde. Årsak til utviklingen av denne kroniske sykdommen er ikke fullstendig kartlagt, men aktiverte T-celler antas å ha en betydningsfull rolle. Mottakelighet for MS er knyttet til kombinasjonen av en rekke genetiske risikovarianter samt eksponering for ulike miljøfaktorer. Over 200 MS- assosierte mottakelighets varianter er i dag indentifisert, hvor mange av disse er koblet opp mot immuncelle responser hos T-celler. Dette prosjektet hadde som hensikt (i) å benytte tilgjengelige proteomikk-data fra CD4+ og CD8+ T-celler fra MS pasienter og friske kontroller i kombinasjon med individenes genotype-data, for å identifisere nye MS- assosierte proteiner eller reaksjonsveier. Videre ønsket vi å (ii) samle nye prøver fra aktiverte CD4+ T-celler fra de samme gruppene til proteomikk-analyser ved massespektrometri. (i) Proteomikk-data fra CD4+ og CD8+ T-celler ble benyttet til korrelasjon av genotypen ved MS mottakelighets-variantene med ekspresjon av proteiner kodet av gener lokalisert i et område på 100kb oppstrøms og nedstrøms for hver MS mottakelighets-variant. Videre ble reaksjonsvei-analyser utført for proteiner som var differensielt uttrykte mellom MS pasienter og friske kontroller i begge celletypene. (ii) Levende CD4+ T celler blir lagret i flytende nitrogen, og celler fra 28 kvinnelige relapserende remitterende MS pasienter og 28 friske kontroller ble tinet og aktivert in vitro ved platebundet a-CD3 og løselig a-CD28 antistoff. Cellene ble analysert ved væskestrømsanalyse ved å detektere T-celle aktiverings-markøren CD69 i kombinasjon med en LIVE/DEAD stain for å skille levende fra døde celler. Celler med > 50 % CD69 positive celler etter aktivering, samt > 70 % levelighet, ble sent til analyser ved væskekromatografi/tandem massespektrometri, for å analysere proteomet i prøvene. (i) To nye protein «quantitative trait loci» (pQTL) kandidater ble identifisert, dvs. rs1800693 - CD9 og rs137955 - UQCRFS1P. 26 proteiner var differensialt uttrykte i både CD4+ og CD8+ T-celler mellom gruppene, men ingen anrikede reaksjonsveier ble identifisert i reaksjonsvei-analysene. De identifiserte pQTL kandidatene kan være relevante for MS og samtidig inspirere til funksjonelle studier som kan belyse effektene av de genetiske risiko-variantene samt de underliggende mekanismene i MS utvikling. (ii) 20 prøver fra MS-pasienter og 20 prøver fra friske kontroller oppfylte inklusjons-kravene og ble inkludert i proteomikk-analysene. Ikke-aktiverte prøver og prøver aktivert i 24 timer sendt til kjerneanlegget for proteomikk i Bergen. Prøvene er lysert og preparert til analyse, men resultatene er ikke tilgjengelige grunnet Covid-19 situasjonen. Det gjenstår å evaluere om proteomikk-resultatene kan bidra til identifisering av proteiner eller reaksjonsveier med betydning for MS. Table of contents 1 - Introduction ...................................................................................................................... - 3 - 1.1 The immune system ...................................................................................................................... - 3 - 1.1.1 T cell differentiation and development ...................................................................... - 5 - 1.1.2 T cell activation .................................................................................................................... - 6 - 1.2 Multiple sclerosis ............................................................................................................................ - 8 - 1.2.1 Multiple sclerosis etiology ............................................................................................... - 8 - 1.2.2 Multiple sclerosis pathogenesis .................................................................................... - 9 - 1.2.3 Multiple sclerosis is an autoimmune disorder ........................................................ - 9 - 1.3 Genetics ..........................................................................................................................................
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