Towards stratification of patients with Sjögren’s syndrome : Single cell analyses and immune profiling

Primary Sjögren’s syndrome (pSS) is a systemic, chronic autoimmune disorder that is characterized by progressive lymphocytic infiltration in the exocrine glands i.e., the salivary and lacrimal glands, leading to immune-mediated glandular destruction. It mostly affects middle-aged women, making diagnosis of the disease challenging, as the symptoms (primarily dryness of the mouth and eyes and fatigue) are often confused with the side effects of drugs, other comorbidities or aging. Currently there is no cure, as the exact mechanism of the disease pathogenesis is not known, and treatment strategies mainly aim at alleviating the symptoms. Like most autoimmune diseases, pSS progression and phenotype are complex and multi-faceted, with a wide spectrum of clinical manifestations, ranging from local to systemic, including fatal conditions like B cell lymphoma. Patient heterogeneity is a major obstacle to disease management. Therefore, it is imperative to identify potential disease markers that may help in diagnosis, prediction, stratification of the patients and/or identification of new therapeutic targets. The overall aim of this thesis was to study the peripheral blood immune system in pSS, to identify disease-specific immune profiles and potential biomarkers that may help in patient stratification. In paper I, phosphoflow cytometry was used to compare basal and TLR7 and -9 stimulated phosphorylation states in immune cells of pSS patients and healthy donors. Both basal and stimulation-induced phosphorylation differed significantly between pSS patients and healthy individuals, and between patient subgroups. Plasma cytokine levels, measured by Luminex assay, also differed significantly between the patients and controls as well as between patient subgroups, and correlated with autoantibody status and other clinical parameters. In paper II, single cell analysis of peripheral blood immune cells, with special emphasis on intracellular signaling, was done using mass cytometry. We compared the frequencies of different immune cell subsets among the patient subgroups and healthy individuals and analyzed their signaling profiles, upon stimulation with IFNα2b and IFNγ separately. Significant differences in cell frequencies were observed among the SSA- and SSA+ pSS patients and controls, along with increased activation status in many cell types, particularly in the SSA+ subgroup. Upon IFNα2b and IFNγ stimulation, aberrations in phospho-signaling were detected in the various immune cell subsets of the patient subgroups, which were most prominent in the SSA+ pSS patients. In paper III, we used flow cytometry and qPCR to analyze the expression of TAM receptors in various immune cells of pSS patients and healthy controls. Significant differences in the mRNA levels of some of the TAM receptors as well as in cell frequencies were observed between the patients and healthy donors. Differential TAM receptor expressions in the immune cells were detected between the pSS patients and healthy controls, with most of them being expressed at slightly lower levels in the patients. In conclusion, aberrations in cellular compositions, cytokine and TAM receptor levels as well as discrepancies in intracellular signaling pathways were detected in the pSS patients and their subgroups compared to healthy individuals. Further research can shed light on new biomarkers for stratification of patients for personalized treatment. Optimized therapeutic strategies can greatly alter the disease outcome and quality of life.Doktorgradsavhandlin

Bioengineering and Cell-derived Strategies for Salivary Gland Regeneration

Xerostomia (dry mouth symptoms) is a group of incurable debilitating conditions of salivary glands caused by aging, radiation/chemical exposure, or aberrant inflammation in the salivary glands. During this PhD thesis, we aimed to evaluate whether cell-derived strategies (e.g., extracellular vesicles, EVs) could be a potential new therapy to ameliorate salivary gland injury and restore function after radiotherapy or in autoimmune diseases. In addition, we aimed to develop new imaging techniques for both 2D and 3D analysis of larger samples which allows for quantification of disease and regenerative features. Firstly, we constructed an in vivo murine model of 25 Gy irradiation-induced salivary gland damage to evaluate the potential of human dental pulp stem cell (hDPSCs)-derived EVs. EVs were injected 3x weekly via tail vein, beginning immediately after irradiation. Salivary gland function was evaluated 18 days after irradiation using salivary gland flow rate (SFR), gene expression (by qRT-PCR) and histopathology. Next, we tested different methods to generate PCSS using a vibratome and evaluated the slices in terms of viability (by WST-1), gene expression (by qRT-PCR), secreted α-amylase activity (by α-amylase assay kit) and histological/light sheet fluorescence microscopy (LSFM) three-dimensional imaging. Following irradiation, SFR decreased while senescence-associated β-galactosidase-positive cells (via immunofluorescences) and senescence-related genes and secretory-phenotypes (e.g., p21 and MMP3 in qRT-PCR) increased. SFR was unchanged following EVs treatment, but senescence-associated genes and secretory-phenotypes decreased. We also demonstrated that in an animal model of Sjögren’s syndrome, which exhibit dry mouth symptoms, that hDPSCs-EVs could inhibit the acquisition of the senescent phenotype in salivary gland epithelial cells (SGECs) and alleviate the loss of glandular function. EVs were also found to perform these effects through an underlying immunomodulatory mechanism. For PCSS, we developed protocols to produce viable slices of controled thicknesses which retained the ability to secrete functional α-amylase for at least two days in ex vivo culture. Phenotypic salivary gland cell epithelial markers (e.g., Keratin 5 and Aquaporin 5) increased over time in PCSS (by qRT-PCR), indicating the retention of cells that are necessary for salivary glands’ function. We developed workflows to perform LSFM 3D visualization in whole salivary glands as well as the PCSS model. In conclusion, hDPSCs-EVs reduced senescence of salivary gland epithelial cells in both murine irradiation and Sjögren’s syndrome models and may become a promising future for xerostomia patients. For the murine PCSS, we successfully established an executable operating procedure at the methodological level to reliably generate viable and functional murine PCSS and developed new state-of-the-art analytical methods (such as LFSM 3D imaging and qRT-PCR) to increase the diversity of objective tools to evaluate PCSS. Therefore, this work laid the foundation for the future application of other therapies (such as irradiation therapy or EVs therapy) to the PCSS model. Those future applications could include drug screening or mechanism of injury study. At the same time, we developed a sustainable histology process to reduce xylene utilization in histological processing for salivary gland tissue processing. Therefore, this work has developed a set of in vitro and in vivo experiments with state-of-the-art methods to better understand disease mechanisms and to evaluate new therapies for salivary glands

Insights and Perspectives in Rheumatology

This book offers a range of perspectives on pathogenesis, clinical features and treatment of different rheumatic diseases, with a particular focus on some of the interesting aspects of Sjögren's syndrome. It contains detailed and thorough reviews by international experts, with a diverse range of academic backgrounds. It will also serve as a useful source of information for anyone with a passive interest in rheumatology, from the genetic and molecular level, through to the psychological impact of pain and disability

Neutrophil phenotype and function in ocular inflammatory disorders

Ocular inflammatory diseases are the result of the breakdown of protective ocular barriers. Behçet's disease (BD) and Ocular mucous membrane pemphigoid (OcMMP) are chronic inflammatory eye disorders, in which neutrophils have been implicated in their pathogenesis. In this thesis the phenotype and function of neutrophils from patients with these diseases was assessed. Peripheral blood neutrophils were obtained from patients with BD, OcMMP and healthy controls. Percoll isolation was used to isolate all neutrophils, while Ficoll-Hypaque was used to obtain subpopulations. Phagocytic capacity and production of reactive oxygen species (ROS), neutrophil extracellular traps (NETs) and heterogeneity were assessed in all populations. Neutrophils from both patient cohorts showed a reduced phagocytic capacity and ROS production, but greater NET production by total neutrophils compared to cells from healthy controls. Also, patients had elevated numbers of low density and lower number of normal density neutrophils, but showed a similar phenotype as total neutrophils. Granulocytic myeloid-suppressor cells were evident within the Low density neutrophil (LDN) and Normal density neutrophil (NDN) population of both patient cohorts when compared healthy controls. The results suggest that a chronic inflammatory state reduces phagocytosis and ROS production but increases NET production in circulatory neutrophils. Therefore, Further analysis of neutrophil heterogeneity may explain these phenotypic and functional differences

Epigenetics in health and disease : on smoking, multiple sclerosis, and rheumatoid arthritis disease states

The main aim of this thesis was to investigate the genome-wide DNA methylation in tissues associated with the immunopathogenesis of two autoimmune diseases, Rheumatoid Arthritis (RA) and Multiple Sclerosis (MS), as well as smoking-associated methylome patterns in the lung. In Study I, we investigated the methylome of two sets of monozygotic twin pairs, representing two phases of anti citrulline protein antibody (ACPA)-positive RA disease development. The first set included five MZ twin pairs discordant for APCA at risk for developing RA, and the second set included seven pairs discordant for ACPA-positive RA. A differentially methylated region associated with a protocadherin (PCDH) gene proposes a temporal epigenetic connection in the progression from ACPA-positivity to clinical RA. In Study II, we investigated the impact of tobacco smoking on bronchoalveolar lavage (BAL), which mainly consists of tissue-resident alveolar macrophages. We combined methylome and transcriptome data from BAL cells from healthy individuals and provide novel smoking-associated signatures converging to genes involved in migration, signaling and inflammatory response of immune cells. Of note, many of the sites in the smoking-associated methylome signature map to enhancer regions. Our findings propose that the epigenetic landscape of BAL cells is modified in smokers, and that it may involve active demethylation resulting in induced immune-related activities in the lung. Despite strong evidence that cigarette smoking is a risk factor for MS, little research has focused on smoking-associated changes in the lung of MS patients. In Study III, we show that smoking in MS patients resulted in subtle alterations in their methylome related to neuronal processes. Additionally, non-smoking MS patients displayed very discrete changes in transcriptional and translational processes and enhanced cellular motility, supporting findings on lung involvement in the pathogenesis of MS-like disease in animal studies. In conclusion, we demonstrate a non-genetically linked temporal epigenetic connection between ACPA-positivity and clinical RA that may be of interest in future studies. We reveal smoking-associated changes in the epigenetic landscape of BAL cells, and increased activity of immune-related processes in the lung, possibly involving active demethylation. We also present new insights into the impact of cigarette smoking on pulmonary inflammation in the immunopathogenesis of MS

The role of interleukin-21 in type-1 diabetes

Cytokines are an essential component of both normal and aberrant immune responses, such as in autoimmune disease. Interleukin (IL)-21 is a member of the common gamma chain family of cytokines and is adjacent to IL-2 within the strongest non-MHC-linked locus for type-1 diabetes (T1D) susceptibility in non-obese diabetic (NOD) mice. Recent studies demonstrate that IL-21 is necessary for the development of autoimmune disease in several models including T1D in NOD mice. This study explores the critical role of IL-21 in the pathogenesis of T1D. In this study, we demonstrate that the amount of IL-21, but not IL-2, correlated with T1D incidence. Whilst IL-21 is found in high expressing and low expressing allelic forms, IL-2 appears to be kept at a similar level between mouse strains due to differences in mRNA stability. IL-21 is produced in abundance within the autoimmune lesions of the NOD mouse by a novel CD4+ T helper (Th) subset, marked by co-expression of the gut-homing chemokine receptor CCR9. Whilst these CCR9+ IL-21-producing Th cells could be found in healthy mice and humans, they were concentrated in the inflamed pancreas. Critically, the ultimate target of IL-21 is CD8+ T cells whose receptiveness to IL-21 is necessary for the development of diabetes. We also demonstrate successful intervention at a late preclinical stage through neutralisation of IL-21 with IL-21R/Fc. Indeed, when combined with islet allograft transplantation, this therapeutic approach could cure diabetes. We found that the influence of IL-21 on a graft-mounted immune response was robust, as absence of IL-21 signalling was also found to prevent islet allograft rejection. These findings suggest that therapeutic manipulation of IL-21 may serve as a suitable treatment for patients with T1D