Single cell based phosphorylation profiling identifies alterations in toll-like receptor 7 and 9 signaling in patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and −9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and −9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways.publishedVersio

Aberrant signaling of immune cells in Sjögren's syndrome patient subgroups upon interferon stimulation

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, characterized by mononuclear cell infiltrates in the salivary and lacrimal glands, leading to glandular atrophy and dryness. Patient heterogeneity and lack of knowledge regarding its pathogenesis makes pSS a difficult disease to manage. Methods: An exploratory analysis using mass cytometry was conducted of MAPK/ERK and JAK/STAT signaling pathways in peripheral blood mononuclear cells (PBMC) from 16 female medication free pSS patients (8 anti-Sjögren’s syndrome-related antigen A negative/SSA- and 8 SSA+) and 8 female age-matched healthy donors after stimulation with interferons (IFNs). Results: We found significant differences in the frequencies of memory B cells, CD8+ T central and effector memory cells and terminally differentiated CD4+ T cells among the healthy donors and patient subgroups. In addition, we observed an upregulation of HLA-DR and CD38 in many cell subsets in the patients. Upon IFNα2b stimulation, slightly increased signaling through pSTAT1 Y701 was observed in most cell types in pSS patients compared to controls, while phosphorylation of STAT3 Y705 and STAT5 Y694 were slightly reduced. IFNγ stimulation resulted in significantly increased pSTAT1 Y701 induction in conventional dendritic cells (cDCs) and classical and non-classical monocytes in the patients. Most of the observed differences were more prominent in the SSA+ subgroup, indicating greater disease severity in them. Conclusions: Augmented activation status of certain cell types along with potentiated pSTAT1 Y701 signaling and reduced pSTAT3 Y705 and pSTAT5 Y694 induction may predispose pSS patients, especially the SSA+ subgroup, to upregulated expression of IFN-induced genes and production of autoantibodies. These patients may benefit from therapies targeting these pathways.publishedVersio

Psoriasis in Norway: A Prescription-based Registry Study of Incidence and Prevalence

Epidemiological data on psoriasis in Norway are limited. The aim of this study was to provide objective national data on the incidence and prevalence of psoriasis. Patients registered in the Norwegian Prescription Database with a diagnostic code indicating psoriasis vulgaris on prescriptions were included. During the period 2004 to 2020, 272,725 patients received prescription for psoriasis vulgaris in Norway. During the period 2015 to 2020, 84,432 patients received prescription for psoriasis vulgaris for the first time. In 2020, 71,857 (97.7%) patients received topical, 7,197 (9.8%) conventional systemic and 2,886 (3.9%) biological medication for psoriasis vulgaris. In the period 2015 to 2020, the point prevalence of psoriasis was 3.8–4.6% and the incidence was 0.29–0.25%. Norway is divided into 4 geographical health regions. A latitudinal difference was observed between the 4 regions, highest in Northern Norway. In the incidence population, median age was 47–53 years and males comprised 46–50%. In this study of psoriasis vulgaris, prevalence in Norway was higher than in earlier reports from other countries. There was a small female predominance regarding incidence and prevalence; however, men had more prescriptions for systemic treatment. Prescriptions for psoriasis vulgaris showed a stable level, with a trend of increasing use of biological medication during the study period

Systemic inflammation in psoriasis: Circulating immune cells and cytokines

Psoriasis is a common, chronic inflammatory skin disease associated with arthritis and multiple comorbidities. Autoantigens in the skin elicit a response in cytotoxic T cells, leading to local inflammation and recruitment of Th1 and Th17 cells from the blood. There is a complex immunological interplay between cytokines and cells from the innate and adaptive immune system, creating self-sustaining amplification loops. Increased levels of inflammatory cytokines and cells have been detected in blood from psoriasis patients. This notion, together with mechanistic similarities in establishment of psoriatic and atherosclerotic plaques, probably contributes to the increased prevalence of cardiovascular disease in psoriasis patients, however, this link needs to be further elucidated. No cure for psoriasis exists, and treatments aim at amelioration of symptoms. If topical treatments or UV-light are not effective enough, systemic medication including methotrexate, ciclosporin, fumarate or acitretin can be tried. Biological drugs specifically targeting the key cytokines TNF, IL-12/23 and IL-17 are available if conventional treatment is insufficient or contraindicated. However, these newer drugs are not accompanied by similarly precise laboratory analyses to aid selection of a specific drug for individual patients. As adverse events and loss of effect can be encountered, the switching from original to cheaper biosimilar drug has been controversial. The overall aim of this thesis was to study the blood immune system in psoriasis during active inflammation and treatment with biological drugs, in the search for disease specific immune signatures and biomarkers. In Study I, Luminex® Technology was used to investigate if serum cytokine levels could reflect psoriasis activity. In Study II, we compared impact of switching from original TNF inhibitor infliximab to biosimilar CT-P13 in psoriasis patients, both evaluating clinical parameters and effect on peripheral blood cells and their intracellular signalling, measured by phosphoflow cytometry. In Study III, single cell analysis of blood immune subsets, with special emphasis on the T cell lineage and intracellular signalling, was explored by use of mass cytometry. In all studies, clinical parameters including Psoriasis Area and Severity Index and Dermatological Life Quality Index were incorporated in analyses. The results indicate that cytokine and single cell analysis of blood can be useful methods for describing the complex systemic immunological picture in psoriasis. In Study I, logistic regression revealed higher risk of having severe psoriasis with increased IL-17A. Increase of IL-2 positively correlated with improvement of PASI and DLQI. Moreover, increase of IL-5, IL-10, IL-12, IL-22 and GM-CSF correlated with treatment effect. In Study II, intracellular phosphorylation levels in peripheral blood mononuclear cells were increased in psoriasis patients compared to healthy controls. This increased signalling activity decreased during continued treatment with infliximab, but did not completely normalize despite clinical remission. Switching from original to biosimilar infliximab did not affect laboratory findings, like cell abundance and phosphorylation levels, or clinical parameters. Study III revealed that biological therapy of psoriasis facilitated a shift in the balance of Th1 and Th2 cells in blood, transition from naïve/effector to memory predominance, reduction of circulating Th17, Th22, Th9 and CD8 cells and enhancement of inhibitory PD-1 expression on T cells. In the monocyte compartment, changes in favor of reduced cardiovascular risk were observed. Intracellular phosphorylation of blood immune cells was higher in psoriasis patients compared to healthy controls and in non-responders to treatment compared to responders. In conclusion, multiple aberrancies in circulating cells and cytokines were detected in patients with severe psoriasis, confirming that systemic inflammation is a trait of psoriasis. Further research can highlight the role of cytokines and peripheral blood mononuclear cells as potential tools for stratification of patients for personalized treatment. Optimized therapeutic strategies might alter the chronic course of psoriasis with positive implications on quality of life and long-term comorbidities

Single cell based phosphorylation profiling identifies alterations in toll-like receptor 7 and 9 signaling in patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is associated with polymorphisms and mRNA expression profiles that are indicative of an exaggerated innate and type I IFN immune response. Excessive activation potential of signaling pathways may play a role in this profile, but the intracellular signaling profile of the disease is not well characterized. To gain insights into potentially dysfunctional intracellular signaling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signaling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, as well as samples during a 4 h time period following activation of Toll-like receptor (TLR) 7 and 9. Expression levels of MxA, IFI44, OAS1, GBP1, and GBP2 in PBMC were analyzed by real-time PCR. Cytokine levels in plasma were determined using a 25-plex Luminex-assay. Principal component analysis (PCA) showed that basal phosphorylation profiles could be used to differentiate pSS patients from healthy donor samples by stronger intracellular signaling pathway activation in NK and T cells relative to B cells. Stimulation of PBMC with TLR7 and −9 ligands showed significant differences in the phosphorylation profiles between samples from pSS patients and healthy donors. Including clinical parameters such as extraglandular manifestations (EGM), we observed stronger responses of NF-κB and STAT3 S727 in B cells from EGM-negative patients compared to EGM-positive patients and healthy controls. Plasma cytokine levels were correlated to the basal phosphorylation levels in these patients. In addition, 70% of the patients had a positive IFN score. These patients differed from the IFN score negative patients regarding their phosphorylation profiles and their plasma cytokine levels. In conclusion, we here report increased signaling potentials in peripheral B cells of pSS patients in response to TLR7 and −9 stimulation through STAT3 S727 and NF-κB that correlate with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of STAT3 S727 and NF-κB signaling could therefore benefit from therapies targeting these pathways

Aberrant signaling of immune cells in Sjögren's syndrome patient subgroups upon interferon stimulation

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, characterized by mononuclear cell infiltrates in the salivary and lacrimal glands, leading to glandular atrophy and dryness. Patient heterogeneity and lack of knowledge regarding its pathogenesis makes pSS a difficult disease to manage. Methods: An exploratory analysis using mass cytometry was conducted of MAPK/ERK and JAK/STAT signaling pathways in peripheral blood mononuclear cells (PBMC) from 16 female medication free pSS patients (8 anti-Sjögren’s syndrome-related antigen A negative/SSA- and 8 SSA+) and 8 female age-matched healthy donors after stimulation with interferons (IFNs). Results: We found significant differences in the frequencies of memory B cells, CD8+ T central and effector memory cells and terminally differentiated CD4+ T cells among the healthy donors and patient subgroups. In addition, we observed an upregulation of HLA-DR and CD38 in many cell subsets in the patients. Upon IFNα2b stimulation, slightly increased signaling through pSTAT1 Y701 was observed in most cell types in pSS patients compared to controls, while phosphorylation of STAT3 Y705 and STAT5 Y694 were slightly reduced. IFNγ stimulation resulted in significantly increased pSTAT1 Y701 induction in conventional dendritic cells (cDCs) and classical and non-classical monocytes in the patients. Most of the observed differences were more prominent in the SSA+ subgroup, indicating greater disease severity in them. Conclusions: Augmented activation status of certain cell types along with potentiated pSTAT1 Y701 signaling and reduced pSTAT3 Y705 and pSTAT5 Y694 induction may predispose pSS patients, especially the SSA+ subgroup, to upregulated expression of IFN-induced genes and production of autoantibodies. These patients may benefit from therapies targeting these pathways