Monotsüütide, monotsüütidest pä rinevate makrofaagide ja dendriitrakkude epigeneetilised profiilid

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Immuunsüsteemi peamiseks ülesandeks on kaitsta organismi nakkushaiguste eest vältides samal ajal oma kudede kahjustamist. See tagatakse immuunrakkude mitmetasandilise reguleeritud koostöö tulemusena. Käesolev uurimus keskendub dendriitrakkudele, mis on immuunsüsteemi peamised antigeeni esitlevad rakud. Dendriitrakkudel on oluline roll nii immuunvastuse esilekutsumises kui ka kahjulike reaktsioonide mahasurumises. Nad töötlevad võõrast materjali ning presenteerivad seda oma raku pinnal teistele immunrakkudele, juhtides seeläbi teiste immuunrakkude tegevust. Koostöös monotsüütide, makrofaagide ja teiste immuunrakkudega, tagavad dendriitrakud organismi immuuntasakaalu. Katseklaasis, aga ka põletikukoldes, saadakse dendriitrakke veres tsirkuleerivatest monotsüütidest. Monotsüütidest pärinevaid dendriitrakke uuritakse jätkuvalt väga põhjalikult ning nad on praegusel hetkel ühed kõige lootustandvamad rakud vähi ja mitmete autoimmuunsete haiguste immuunteraapias. Selle töö eesmärgiks oli uurida geeniregulatsiooni muutusi, mis toimuvad monotsüütides nende diferentseerumisel dendriitrakkudeks ning tuvastada erinevusi monotsüütides noortel ning vanadel inimestel. Uurisime geenide avaldumise regulatsiooni epigeneetilisi muutuste abil monotsüütide diferentseerumisel. Epigeneetika on teadus, mis proovib selgitada, kuidas väga piiratud arve geene saab aluse panna erinevatele rakutüüpidele mõjutamata sealjuures DNA järjestust. Monotsüüdi diferentseerumisel makrofaagiks ja dendriitrakuks leiavad aset mitmed epigeneetilised muutused. Kirjeldasime monotsüütide, monotsüütidest pärinevatel makrofaagide ja dendriitrakkude geeniekspressioone, histoonide modifikatsioone ja mikroRNA profiile. Näitasime, et geenide aktiivsus ja histooni modifikatsioonide muster on väga hästi korreleeritav. Kirjeldasime dendriitrakkude ja makrofaagide mikroRNA ekspressiooni profiile, millest leidsime mitmeid ülesreguleeritud mikroRNAsid, sealhulgas ka varem kirjeldamata miR-511. Lisaks diferentseerumisele, kirjeldasime ka mitmeid erinevalt metüleeritud alasid vananemisel noorte ja vanade indiviidide monotsüütides, mis võivad osutuda kasulikuks mõistmaks paremini epigeneetika rolli vananemisel ning pakkumaks tulevikus uusi lähenemisi põletikuliste protsesside kontrolliks.The purpose of the immune system is to protect the body against diseases meanwhile avoiding damaging of tissues. This is provided by tightly regulated multilevel cooperation of the immune cells. This thesis focuses on dendritic cells; main antigen presenting cells in immune system. Dendritic cells are important both in inducing immune responses and in suppressing harmful reactions. They process foreign material and present it on their cell surface to other immune cells, thereby mediate the action of other immune cells. In cooperation with monocytes, macrophages and other immune cells, dendritic cells help to maintain the immune homeostasis in organisms. In vitro, but also in case of an infection, dendritic cells are rapidly generated in large numbers from monocytes. These monocyte-derived dendritic cells are under going extensive study and are currently the most promising cell type to be used in immunotherapy for cancer and various autoimmune diseases. The aim of this thesis is to study the changes in gene regulation in monocyte differentiation to dendritic cells and to identify differences in monocytes in young and elderly individuals. We studied gene expression regulation through epigenetic changes in monocyte differentiation. Epigenetics is a study that tries to clarify how limited number of genes can define different cell types without affecting the DNA sequence. In the differentiation of monocytes into macrophages and dendritic cells, several epigenetic changes take place. We described gene expression profiles, histone modifications and microRNA profiles in monocytes, monocyte-derived macrophages and dendritic cells. We showed that gene activity and histone modifications are in good correlation. We also described updated microRNAs expression profiles of dendritic cells and macrophages that revealed several upregulated miRNAs, among others novel miR-511. In addition to differentiation, we also described several differentially methylated CpG sites in monocytes of young and elderly individuals during ageing process that may be useful to better understand the role of epigenetics in ageing and provide new approaches to control inflammatory processes in future

Cytotoxic CD8+ Temra cells show loss of chromatin accessibility at genes associated with T cell activation

As humans age, their memory T cell compartment expands due to the lifelong exposure to antigens. This expansion is characterized by terminally differentiated CD8+ T cells (Temra), which possess NK cell-like phenotype and are associated with chronic inflammatory conditions. Temra cells are predominantly driven by the sporadic reactivation of cytomegalovirus (CMV), yet their epigenomic patterns and cellular heterogeneity remain understudied. To address this gap, we correlated their gene expression profiles with chromatin openness and conducted single-cell transcriptome analysis, comparing them to other CD8+ subsets and CMV-responses. We confirmed that Temra cells exhibit high expression of genes associated with cytotoxicity and lower expression of costimulatory and chemokine genes. The data revealed that CMV-responsive CD8+ T cells (Tcmv) were predominantly derived from a mixed population of Temra and memory cells (Tcm/em) and shared their transcriptomic profiles. Using ATAC-seq analysis, we identified 1449 differentially accessible chromatin regions between CD8+ Temra and Tcm/em cells, of which only 127 sites gained chromatin accessibility in Temra cells. We further identified 51 gene loci, including costimulatory CD27, CD28, and ICOS genes, whose chromatin accessibility correlated with their gene expression. The differential chromatin regions Tcm/em cells were enriched in motifs that bind multiple transcriptional activators, such as Jun/Fos, NFkappaB, and STAT, whereas the open regions in Temra cells mainly contained binding sites of T-box transcription factors. Our single-cell analysis of CD8+CCR7loCD45RAhi sorted Temra population showed several subsets of Temra and NKT-like cells and CMC1+ Temra populations in older individuals that were shifted towards decreased cytotoxicity. Among CD8+CCR7loCD45RAhi sorted cells, we found a decreased proportion of IL7R+ Tcm/em-like and MAIT cells in individuals with high levels of CMV antibodies (CMVhi). These results shed new light on the molecular and cellular heterogeneity of CD8+ Temra cells and their relationship to aging and CMV infection

IL-22 Paucity in APECED Is Associated With Mucosal and Microbial Alterations in Oral Cavity

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by recessive mutations in the AIRE gene. The hallmark of the disease is the production of highly neutralizing autoantibodies against type I interferons and IL-22. Considering the importance of IL-22 in maintaining mucosal barrier integrity and shaping its microbial community, we sought to study potential changes in the oral cavity in this model of human IL-22 paucity. We found that besides known Th22 cell deficiency, APECED patients have significantly fewer circulating MAIT cells with potential IL-22 secreting capacity. Saliva samples from APECED patients revealed local inflammation, the presence of autoantibodies against IFN-α and IL-22, and alterations in the oral microbiota. Moreover, gene expression data of buccal biopsy samples suggested impaired antimicrobial response and cell proliferation, both of which are processes regulated by IL-22. Our data complement the knowledge gained from mouse models and support the concept of IL-22 being a critical homeostatic cytokine in human mucosal sites

Koroonaviiruse omikrontüve vastane antikehade kaitse ja T-rakuline immuunvastus pärast BNT162b2-vaktsiini tõhustusdoosi saamist

Eesti Arst 2023; 102(2):11

Genome-wide promoter analysis of histone modifications in human monocyte-derived antigen presenting cells

<p>Abstract</p> <p>Background</p> <p>Monocyte-derived macrophages and dendritic cells (DCs) are important in inflammatory processes and are often used for immunotherapeutic approaches. Blood monocytes can be differentiated into macrophages and DCs, which is accompanied with transcriptional changes in many genes, including chemokines and cell surface markers.</p> <p>Results</p> <p>To study the chromatin modifications associated with this differentiation, we performed a genome wide analysis of histone H3 trimethylation on lysine 4 (H3K4me3) and 27 (H3K27me3) as well as acetylation of H3 lysines (AcH3) in promoter regions. We report that both H3K4me3 and AcH3 marks significantly correlate with transcriptionally active genes whereas H3K27me3 mark is associated with inactive gene promoters. During differentiation, the H3K4me3 levels decreased on monocyte-specific CD14, CCR2 and CX3CR1 but increased on DC-specific TM7SF4/DC-STAMP, TREM2 and CD209/DC-SIGN genes. Genes associated with phagocytosis and antigen presentation were marked by H3K4me3 modifications. We also report that H3K4me3 levels on clustered chemokine and surface marker genes often correlate with transcriptional activity.</p> <p>Conclusion</p> <p>Our results provide a basis for further functional correlations between gene expression and histone modifications in monocyte-derived macrophages and DCs.</p

Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A–producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.Peer reviewe

Cell Specific eQTL Analysis without Sorting Cells

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn's disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus.Peer reviewe

The transcriptional landscape of age in human peripheral blood

Disease incidences increase with age, but the molecular characteristics of ageing that lead to increased disease susceptibility remain inadequately understood. Here we perform a whole-blood gene expression meta-analysis in 14,983 individuals of European ancestry (including replication) and identify 1,497 genes that are differentially expressed with chronological age. The age-associated genes do not harbor more age-associated CpG-methylation sites than other genes, but are instead enriched for the presence of potentially functional CpG-methylation sites in enhancer and insulator regions that associate with both chronological age and gene expression levels. We further used the gene expression profiles to calculate the 'transcriptomic age' of an individual, and show that differences between transcriptomic age and chronological age are associated with biological features linked to ageing, such as blood pressure, cholesterol levels, fasting glucose, and body mass index. The transcriptomic prediction model adds biological relevance and complements existing epigenetic prediction models, and can be used by others to calculate transcriptomic age in external cohorts.Peer reviewe