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.

Leflunomide-hydroxychloroquine combination therapy in patients with primary Sjögren's syndrome (RepurpSS-I): a placebo-controlled, double-blinded, randomised clinical trial.

BACKGROUND: Primary Sjögren's syndrome is a systemic autoimmune disease characterised by secretory gland dysfunction, for which no effective therapy is available. Based on the complementary properties of leflunomide and hydroxychloroquine in inhibiting activation of key immune cells in primary Sjögren's syndrome, we aimed to evaluate the clinical efficacy and safety of leflunomide-hydroxychloroquine combination therapy in patients with primary Sjögren's syndrome. METHODS: We did a placebo-controlled, double-blinded, phase 2A randomised clinical trial in patients with primary Sjögren's syndrome at the University Medical Center Utrecht (Utrecht, Netherlands). Eligible patients were aged 18-75 years, had a European League Against Rheumatism (EULAR) Sjögren's syndrome disease activity index (ESSDAI) score of 5 or higher, and a lymphocytic focus score of 1 or higher in labial salivary gland biopsy specimens. Patients were randomly assigned (2:1) with block randomisation (block size of six) to receive leflunomide 20 mg and hydroxychloroquine 400 mg daily or placebo for 24 weeks. The primary endpoint was the between-group difference in change in ESSDAI scores from 0 to 24 weeks, adjusted for baseline ESSDAI score. Patients were analysed according to the intention-to-treat principle. This study is registered with EudraCT, 2014-003140-12. FINDINGS: Between March 7, 2016, and Nov 30, 2017, 37 patients were screened, of whom 29 patients (28 women and one man) were enrolled. 21 patients were assigned to receive leflunomide-hydroxychloroquine and eight patients were assigned to receive placebo. One patient in the placebo group required high-dose prednisone to treat polymyalgia rheumatica at week 13 and was excluded from the primary analysis. From 0 to 24 weeks, the mean difference in ESSDAI score, adjusted for baseline values, in the leflunomide-hydroxychloroquine group compared with the placebo group was -4·35 points (95% CI -7·45 to -1·25, p=0·0078). No serious adverse events occurred in the leflunomide-hydroxychloroquine group and two serious adverse events occurred in the placebo group (hospital admission for pancreatitis and hospital admission for nephrolithiasis). The most common adverse events in the leflunomide-hydroxychloroquine group were gastrointestinal discomfort (11 patients [52%] vs two [25%] in the placebo group), modest transient increases in alanine aminotransferase (ten [48%] vs one [13%]), and short episodes of general malaise and shivering (nine [43%] vs one [13%]). INTERPRETATION: Leflunomide-hydroxychloroquine was safe and resulted in a clinical response in patients with primary Sjögren's syndrome. These results warrant further evaluation of leflunomide-hydroxychloroquine combination therapy in larger clinical trials. FUNDING: ZonMw.

Plasmacytoid DCs From Patients With Sjögren's Syndrome Are Transcriptionally Primed for Enhanced Pro-inflammatory Cytokine Production.

Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each n = 31) and performed RNA-sequencing, after which data-driven networks and modular analysis were used to identify robustly reproducible transcriptional "signatures" of differential and co-expressed genes. Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of in vitro activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon in vitro stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.

Additive immunosuppressive effect of leflunomide and hydroxychloroquine supports rationale for combination therapy for Sjögren's syndrome.

Objective: Effective treatment for primary Sjögren's syndrome (pSS) is not available. pSS immunopathology involves a variety of immune-cells and dysregulated pathways; targeting several pathways instead of only one could therefore be effective. Treatment with leflunomide (LEF) and hydroxychloroquine (HCQ) might be successful given their unique immunosuppressive properties. We aimed to study the in vitro effects of LEF, HCQ and their combination on T- and B-cell proliferation, cytokine and immunoglobulin production by activated PBMCs. Methods: PBMCs of six healthy individuals and nine pSS patients were stimulated with superantigen and TLR9 agonist to mimic the hallmark features. LEF, HCQ and their combinations were tested at clinically observed concentrations and proliferation, cytokine and immunoglobulin production were measured. Results: TCR/TLR9 activation of PBMCs induced strong proliferation of T and B-cells and production of CXCL13, IFN-α, IFN-γ, IgG and IgM. LEF dose-dependently inhibited all measured parameters, where HCQ potently and dose-dependently decreased B cell proliferation, CXCL13, IFN-α, IgG and IgM production. At different concentration combinations, HCQ and LEF inhibited several immune hallmark features more potently than each single compound. Conclusion: A combination of LEF and HCQ at clinically applicable concentrations additively inhibits immune activation, supporting a potential implementation of this drug combination in pSS treatment.

Influence of geolocation and ethnicity on the phenotypic expression of primary Sjögren's syndrome at diagnosis in 8310 patients: a cross-sectional study from the Big Data Sjögren Project Consortium.

OBJECTIVES: To analyse the influence of geolocation and ethnicity on the clinical presentation of primary Sjögren's syndrome (SjS) at diagnosis. METHODS: The Big Data Sjögren Project Consortium is an international, multicentre registry designed in 2014. By January 2016, 20 centres from five continents were participating. Multivariable logistic regression analyses were performed. RESULTS: We included 7748 women (93%) and 562 men (7%), with a mean age at diagnosis of primary SjS of 53 years. Ethnicity data were available for 7884 patients (95%): 6174 patients (78%) were white, 1066 patients (14%) were Asian, 393 patients (5%) were Hispanic, 104 patients (1%) were black/African-American and 147 patients (2%) were of other ethnicities. SjS was diagnosed a mean of 7 years earlier in black/African-American compared with white patients; the female-to-male ratio was highest in Asian patients (27:1) and lowest in black/African-American patients (7:1); the prevalence of sicca symptoms was lowest in Asian patients; a higher frequency of positive salivary biopsy was found in Hispanic and white patients. A north-south gradient was found with respect to a lower frequency of ocular involvement in northern countries for dry eyes and abnormal ocular tests in Europe (OR 0.46 and 0.44, respectively) and Asia (OR 0.18 and 0.49, respectively) compared with southern countries. Higher frequencies of antinuclear antibodies (ANAs) were reported in northern countries in America (OR=1.48) and Asia (OR=3.80) while, in Europe, northern countries had lowest frequencies of ANAs (OR=0.67) and Ro/La (OR=0.69). CONCLUSIONS: This study provides the first evidence of a strong influence of geolocation and ethnicity on the phenotype of primary SjS at diagnosis.

Genetic disorders in complement (regulating) genes in patients with atypical haemolytic uraemic syndrome (aHUS).

BACKGROUND: Atypical HUS (aHUS) is thought to be caused by predisposing mutations in genes encoding complement (regulating) proteins, such as Factor H (CFH), Factor I (IF), membrane co-factor protein (MCP) and Factor B (FB), or by auto-antibodies against CFH (alphaFH) in combination with a homozygous polymorphic deletion of the genes encoding Complement Factor H-related 1 and 3 (DeltaCFHR1/3). The clinical impact of this knowledge is high, as it might be a prognostic factor for the outcome of renal transplantations and kidney donations. METHODS: Mutational screening, by means of PCR and DNA sequencing, is performed in the above-mentioned genes in a group of 72 aHUS patients. Also, the presence of alphaFH and DeltaCFHR1/3 was tested in patients and controls. RESULTS: In 23 patients, a genetic aberration in at least one gene or the presence of alphaFH was found. A heterozygous mutation was observed in CFH in nine patients, in IF in seven patients and in MCP in three patients. No mutations were observed in FB. Seven patients presented alphaFH, of whom five also carried DeltaCFHR1/3. Three patients carried a combined mutation (two patients: IF and MCP; one patient: IF, alphaFH and DeltaCFHR1/3). A significant difference between patients and controls was detected for the presence of all three associated polymorphisms in CFH. CONCLUSIONS: Genetic abnormalities or the presence of alphaFH were detected in 31.9% of the aHUS patients. Furthermore, bigenic mutations were present, indicating that routine DNA mutation analysis of all complement factors associated with aHUS is important.