Flow cytometry based analyses as a tool in biomarker discovery for patient stratification in primary Sjögren’s syndrome
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Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltrates of exocrine glands, notably the salivary and lacrimal glands combined with immune-mediated glandular destruction. The disease is chronic, disabling and there is no cure. Diagnosis of the disease is difficult, with the symptoms of the disease (dryness of the mouth and eyes, and fatigue) frequent in the population as a side effect of many drugs, other co-morbidities or aging. Many cases of pSS are hence misclassified or go unidentified. Like other autoimmune diseases pSS progression and phenotype are heterogeneous with many clinical presentations limited to local manifestations, while others develop extraglandular manifestations as well as life threatening conditions such as B cell lymphomas. New disease markers for pSS that are specific for diagnosis or useful to predict disease development have the potential to radically change how we treat, diagnose and define the disease. The work contained in this thesis focused on the use of flow cytometry based assays in the search for disease markers for the identification and stratification of pSS patients. In paper I we assessed a multiplex flow cytometry protocol used for the measurement of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells for inter-assay precision for experimental variables (phospho-protein measured, cell type and stimulant). In addition, three different blood collection tubes were assessed for their effect on basal and induced intracellular signaling in different cell subsets. The method showed a high level of precision with median coefficients of variation under 10 %, while the use of heparin as an anti-coagulant was superior in retaining immune cell responsiveness compared to citrate. Citrate strongly affected NK cell responses to stimuli, while CPT based isolation methods were associated with higher basal phosphorylation. In papers II and III the flow cytometry protocol presented in paper I was used to compare basal and IFNα or TLR7 and -9 stimulation induced phosphorylation states in immune cells from pSS patients and healthy individuals. Both basal and induced phosphorylation differed significantly between pSS patients and healthy individuals, while induced phosphorylation also differed between by patient sungroups. In paper IV, we compared immune cell quantities in peripheral blood of patients with pSS and healthy individuals, and associated changes with clinical manifestations of the disease. Primary Sjögren’s syndrome patients displayed decreased absolute counts of diverse subtypes of lymphocytes and increases of monocytes and granulocytes compared to healthy individuals. Greater decreases of lymphocytes were associated with differing patient phenotype. In conclusion analysis of both intracellular signaling pathways and cell quantification are promising techniques for the identification of biomarkers that could be used in diagnosis and stratification of pSS.
Has partsPaper I: Davies, R., Vogelsang, P., Jonsson, R., Appel, S. An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells. J Immunol Methods 436: 58-63 (2016). The article is available at: http://hdl.handle.net/1956/15584
Paper II: Davies, R., Hammenfors, D., Bergum, B., Vogelsang, P., Gavasso, S., Brun, J.G., Jonsson, R., Appel, S. Single cell based phosphorylation profiling identifies alterations in Toll-like receptor 7 and -9 signaling in patients with primary Sjögren’s syndrome. Full text not available in BORA.
Paper III: Davies, R., Hammenfors, D., Bergum, B., Vogelsang, P., Gavasso, S., Brun, J.G., Jonsson, R., Appel, S. Aberrant cell signaling in peripheral blood mononuclear cells upon interferon alpha stimulation in patients with primary Sjögren’s syndrome associates with type I interferon signature. Full text not available in BORA.
Paper IV: Davies, R., Hammenfors, D., Bergum, B., Jakobsen, K., Vogelsang, P., Brun, J.G., Bryceson, Y., Jonsson, R., Appel, S. Patients with primary Sjögren’s syndrome have alterations in absolute quantities of various peripheral leukocyte populations. Full text not available in BORA.