Altered fractalkine cleavage potentially promotes local inflammation in NOD salivary gland

Introduction: In the nonobese diabetic (NOD) mouse model of Sjögren's syndrome, lymphocytic infiltration is preceded by an accumulation of dendritic cells in the submandibular glands (SMGs). NOD mice also exhibit an increased frequency of mature, fractalkine receptor (CX3C chemokine receptor [CX3CR]1) expressing monocytes, which are considered to be precursors for tissue dendritic cells. To unravel further the role played by fractalkine-CX3CR1 interactions in the salivary gland inflammation, we studied the expression of fractalkine in NOD SMGs. Methods: We studied protein expression using Western blot analysis of whole tissue lysates. Protease activity was measured in salivary gland tissue lysates using fluorimetric substrates. Digestive capacity of enzymes was determined by in vitro incubation of recombinant enzyme and fractalkine, followed by protein staining and Western blot. Results: Fractalkine was detected in salivary glands of both NOD and control mice at all ages. Western blot analysis showed fractalkine cleavage with increasing age, which was more pronounced in NOD mice. This cleavage resulted in a decrease in the 31 kDa form of the protein, and the generation of an approximately 19 kDa band. Furthermore, in NOD animals older than 15

Leflunomide/hydroxychloroquine combination therapy targets type I IFN-associated proteins in patients with Sjögren's syndrome that show potential to predict and monitor clinical response

OBJECTIVES: To assess to what extent leflunomide (LEF) and hydroxychloroquine (HCQ) therapy in patients with primary Sjögren's syndrome (RepurpSS-I) targets type I IFN-associated responses and to study the potential of several interferon associated RNA-based and protein-based biomarkers to predict and monitor treatment. METHODS: In 21 patients treated with LEF/HCQ and 8 patients treated with placebo, blood was drawn at baseline, 8, 16 and 24 weeks. IFN-signatures based on RNA expression of five IFN-associated genes were quantified in circulating mononuclear cells and in whole blood. MxA protein levels were measured in whole blood, and protein levels of CXCL10 and Galectin-9 were quantified in serum. Differences between responders and non-responders were assessed and receiver operating characteristic analysis was used to determine the capacity of baseline expression and early changes (after 8 weeks of treatment) in biomarkers to predict treatment response at the clinical endpoint. RESULTS: IFN-signatures in peripheral blood mononuclear cell and whole blood decreased after 24 weeks of LEF/HCQ treatment, however, changes in IFN signatures only poorly correlated with changes in disease activity. In contrast to baseline IFN signatures, baseline protein concentrations of galectin-9 and decreases in circulating MxA and Galectin-9 were robustly associated with clinical response. Early changes in serum Galectin-9 best predicted clinical response at 24 weeks (area under the curve 0.90). CONCLUSIONS: LEF/HCQ combination therapy targets type-I IFN-associated proteins that are associated with strongly decreased B cell hyperactivity and disease activity. IFN-associated Galectin-9 is a promising biomarker for treatment prediction and monitoring in pSS patients treated with LEF/HCQ.</p

Fatigue in Sjögren's Syndrome: A Search for Biomarkers and Treatment Targets

Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, where patients often suffer from fatigue. Biological pathways underlying fatigue are unknown. In this study aptamer-based SOMAscan technology is used to identify potential biomarkers and treatment targets for fatigue in pSS.Methods: SOMAscan® Assay 1.3k was performed on serum samples of healthy controls (HCs) and pSS patients characterized for interferon upregulation and fatigue. Differentially expressed proteins (DEPs) between pSS patients and HC or fatigued and non-fatigued pSS patients were validated and discriminatory capacity of markers was tested using independent technology.Results: Serum concentrations of over 1,300 proteins were compared between 63 pSS patients and 20 HCs resulting in 58 upregulated and 46 downregulated proteins. Additionally, serum concentrations of 30 interferon positive (IFNpos) and 30 interferon negative (IFNneg) pSS patients were compared resulting in 25 upregulated and 13 downregulated proteins. ELISAs were performed for several DEPs between pSS patients and HCs or IFNpos and IFNneg all showing a good correlation between protein levels measured by ELISA and relative fluorescence units (RFU) measured by the SOMAscan. Comparing 22 fatigued and 23 non-fatigued pSS patients, 16 serum proteins were differentially expressed, of which 14 were upregulated and 2 were downregulated. Top upregulated DEPs included neuroactive synaptosomal-associated protein 25 (SNAP-25), alpha-enolase (ENO1) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Furthermore, the proinflammatory mediator IL36a and several complement factors were upregulated in fatigued compared to non-fatigued pSS patients. ROC analysis indicated that DEPs showed good capacity to discriminate fatigued and non-fatigued pSS patients.Conclusion: In this study we validated the use of aptamer-based proteomics and identified a novel set of proteins which were able to distinguish fatigued from non-fatigued pSS patients and identified a so-called “fatigue signature.

Systemic increase in type I interferon activity in Sjögren's syndrome: a putative role for plasmacytoid dendritic cells

In the salivary glands of primary Sjögren's syndrome (pSjS) patients, type I IFN activity is increased, but systemic levels of type I IFN proteins are rarely detected. This study focused on the systemic activity of type I IFN in pSjS, as well as the role of peripheral plasmacytoid dendritic cells (pDC). Monocytes obtained from pSjS patients showed an increased expression of 40 genes. Twenty-three of these genes (58%), including IFI27, IFITM1, IFIT3 and IFI44, were inducible by type I IFN. pSjS serum had an enhanced capability of inducing IFI27, IFITM1, IFIT3 and IFI44 in the monocytic cell line THP-1, likely due to the action of IFN-beta. This effect could be inhibited by blocking the type I IFN receptor, supporting a high type I IFN bioactivity in pSjS serum. In addition, circulatory pDC showed increased expression of CD40. This expression was correlated to the expression level of the type I IFN-regulated genes IFI27 and IFITM1 in monocytes of the same individual. This study indicates that the increased type I IFN activity observed in pSjS patients is not only a local but also a systemic phenomenon and points to pDC as a possible source of this activit

The kinetics of plasmacytoid dendritic cell accumulation in the pancreas of the NOD mouse during the early phases of insulitis.

In non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, plasmacytoid dendritic cells (pDCs) have a diabetes-promoting role through IFN-α production on one hand, while a diabetes-inhibiting role through indoleamine 2,3-dioxygenase (IDO) production on the other. Little is known about the kinetics and phenotype of pDCs in the NOD pancreas during the development of autoimmune diabetes. While para/peri-insular accumulation of conventional dendritic cells (cDCs) could be observed from 4 weeks of age onwards in NOD mice, pDCs only started to accumulate around the islets of Langerhans from 10 weeks onwards, which is concomitant with the influx of lymphocytes. NOD pancreatic pDCs showed a tolerogenic phenotype as assessed by their high expression of IDO and non-detectable levels of IFN-α and MxA. Furthermore, expression of the pDC-attracting chemokines CXCL10 and CXCL12 was significantly increased in the NOD pancreas at 10 weeks and the circulating pDC numbers were increased at 4 and 10 weeks. Our data suggest that a simultaneous accumulation of IDO(+) pDCs and lymphocytes in the pancreas in 10 weeks old NOD mice, which may reflect both an immunogenic influx of T cells as well as a tolerogenic attempt to control these immunogenic T cells

Increased pDC numbers in the NOD pancreas at 10 weeks of age.

<p>The presence of pDCs in the C57BL/6, NOR and NOD pancreas at the age of 4 and 10 weeks was determined by flow cytometry. Dot plots show the CD11b and PDCA-1 expression on sorted CD45⁺ cells from the pancreas (A). Bar graphs represent the percentage and the absolute number of CD11b⁻PDCA-1⁺ cells in the pancreas at 4 (B–C) and 10 weeks (D–E). Histograms represent the B220, CD80 and CD86 expression on pDCs (CD11b⁻PDCA-1⁺ cells) in the pancreas of 10 weeks (F). Bar graphs represent the geometric MFI of CD80 (G) and CD86 (H) on pDCs. Data are presented as average+SEM, n = 5–6 mice, *p<0.04, ** p<0.01 as determined by the unpaired Mann-Whitney U test.</p

Enhanced IDO and a decreased PD-L1 expression in NOD pancreas pDCs.

<p>Pictures show the immunohistochemical detection of IDO in the pancreas of NOD mice at 4, 10 and 20 weeks of age, magnification 200x (A). The pancreas of NOD mice was stained for Siglec-H (green), IDO (red) and DAPI (blue) by immunofluorescence, magnification 400x (B). Histogram represents the PD-L1 expression on pDCs (CD11b⁻PDCA-1⁺ cells) in the pancreas of C57BL/6, NOR and NOD mice of 10 weeks of age (C). Bar graph represents the geometric MFI of PD-L1 on pDCs (D). Data are presented as average+SEM, n = 5 mice, * p<0.04, ** p<0.01 as determined by the unpaired Mann-Whitney U test.</p