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

Epidemiological profile and north-south gradient driving baseline systemic involvement of primary Sjogren's syndrome

Objective: To characterize the systemic phenotype of primary Sjögren’s syndrome at diagnosis by analysing the EULAR-SS disease activity index (ESSDAI) scores. Methods: The Sjögren Big Data Consortium is an international, multicentre registry based on worldwide data-sharing cooperative merging of pre-existing databases from leading centres in clinical research in Sjögren’s syndrome from the five continents. Results: The cohort included 10 007 patients (9352 female, mean 53 years) with recorded ESSDAI scores available. At diagnosis, the mean total ESSDAI score was 6.1; 81.8% of patients had systemic activity (ESSDAI score ≥1). Males had a higher mean ESSDAI (8.1 vs 6.0, P &lt; 0.001) compared with females, as did patients diagnosed at &lt;35 years (6.7 vs 5.6 in patients diagnosed at &gt;65 years, P &lt; 0.001). The highest global ESSDAI score was reported in Black/African Americans, followed by White, Asian and Hispanic patients (6.7, 6.5, 5.4 and 4.8, respectively; P &lt; 0.001). The frequency of involvement of each systemic organ also differed between ethnic groups, with Black/African American patients showing the highest frequencies in the lymphadenopathy, articular, peripheral nervous system, CNS and biological domains, White patients in the glandular, cutaneous and muscular domains, Asian patients in the pulmonary, renal and haematological domains and Hispanic patients in the constitutional domain. Systemic activity measured by the ESSDAI, clinical ESSDAI (clinESSDAI) and disease activity states was higher in patients from southern countries (P &lt; 0.001). Conclusion: The systemic phenotype of primary Sjögren’s syndrome is strongly influenced by personal determinants such as age, gender, ethnicity and place of residence, which are key geoepidemiological players in driving the expression of systemic disease at diagnosis.

Repurposing established drugs for new indications: leflunomide and hydroxychloroquine combination therapy for Sjögren’s syndrome

The immunopathology of pSS is characterized by broad immune-activation involving a wide variety of immune cells. Effective therapeutic options are lacking. Considering the complex interplay of involved immune cells, the hypothesis to this thesis is that successful inhibition of pSS immune-activation requires a combination of drugs targeting both overlapping and distinct immunopathological pathways, jointly resulting in broad immune inhibition. The combination of the cDMARDs leflunomide and hydroxychloroquine may hold promise of clinical efficacy. In this thesis, the efficacy and safety of leflunomide/hydroxychloroquine therapy in pSS patients was investigated. An overview of the current knowledge on pSS immunopathology is provided in Chapter 2, and the place of cDMARDs in its treatment is described. Given the upregulation of potential additive pathways in pSS, synergism between the different cell-types involved is likely to occur. Consequently, combining drugs targeting several pathways, for example cDMARDs leflunomide and hydroxychloroquine, seems reasonable. In Chapter 3, we showed in an in vitro system mimicking pSS pathology that at different concentration combinations leflunomide and hydroxychloroquine inhibited several immune hallmark features more potently than each single compound. A placebo-controlled, double-blinded randomized trial investigating leflunomide/hydroxychloroquine therapy in pSS patients was performed (Chapter 4). The study included 29 patients with active disease (EULAR Sjögren’s Syndrome Disease Activity Index, ESSDAI score ≥ 5). 21 patients received leflunomide/hydroxychloroquine therapy, 8 patients were assigned to placebo treatment. Disease activity reflected by ESSDAI, the primary endpoint, significantly decreased upon leflunomide/hydroxychloroquine compared to placebo. Also other clinical parameters such as patient reported outcome ESSPRI-score and saliva output improved. Histological assessment by parotid biopsies supported these positive findings. Leflunomide/hydroxychloroquine combination therapy proved to be safe and well-tolerated. Response to therapy could be predicted with high accuracy by a model comprising ten circulating proteins. In Chapter 5, five different IFN- associated biomarkers (IFN-score in whole blood and PBMCs, MxA in whole blood, CXCL10 and Galectin-9 in serum) in patients treated with either leflunomide/hydroxychloroquine or placebo were assessed and their potential for treatment monitoring was investigated. Patients responding to the therapy showed strong early decreases of MxA and Galectin-9 levels from week 8 onwards. Subsequent ROC analysis revealed good predictive values for early changes in (delta) MxA and Galactin-9 levels after 8 weeks, the latter outperforming the former. In Chapter 6 we focused on Galectin-9 as a biomarker that reflects the IFN signature in patients with SLE, APS and pSS. The findings of this chapter are in line with previous findings showing that Galectin-9 is an easy to measure and accurate biomarker for the IFN signature in SLE and APS patients and correlates with disease activity. Usage of Galectin-9 as a biomarker for the IFN signature needs further confirmation. Data from this thesis indicate that at the least Galectin-9 might play an additional role in monitoring of pSS immunopathology and disease activity. Leflunomide/hydroxychloroquine treatment induced a decline in Galectin-9 levels, parallel with changes in B cell hyperactivity, ESSDAI and ESSPRI, and markers of IFN activity. This warrants further study of Galectin-9 as a biomarker for disease monitoring in pSS

Repurposing established drugs for new indications: leflunomide and hydroxychloroquine combination therapy for Sjögren’s syndrome

The immunopathology of pSS is characterized by broad immune-activation involving a wide variety of immune cells. Effective therapeutic options are lacking. Considering the complex interplay of involved immune cells, the hypothesis to this thesis is that successful inhibition of pSS immune-activation requires a combination of drugs targeting both overlapping and distinct immunopathological pathways, jointly resulting in broad immune inhibition. The combination of the cDMARDs leflunomide and hydroxychloroquine may hold promise of clinical efficacy. In this thesis, the efficacy and safety of leflunomide/hydroxychloroquine therapy in pSS patients was investigated. An overview of the current knowledge on pSS immunopathology is provided in Chapter 2, and the place of cDMARDs in its treatment is described. Given the upregulation of potential additive pathways in pSS, synergism between the different cell-types involved is likely to occur. Consequently, combining drugs targeting several pathways, for example cDMARDs leflunomide and hydroxychloroquine, seems reasonable. In Chapter 3, we showed in an in vitro system mimicking pSS pathology that at different concentration combinations leflunomide and hydroxychloroquine inhibited several immune hallmark features more potently than each single compound. A placebo-controlled, double-blinded randomized trial investigating leflunomide/hydroxychloroquine therapy in pSS patients was performed (Chapter 4). The study included 29 patients with active disease (EULAR Sjögren’s Syndrome Disease Activity Index, ESSDAI score ≥ 5). 21 patients received leflunomide/hydroxychloroquine therapy, 8 patients were assigned to placebo treatment. Disease activity reflected by ESSDAI, the primary endpoint, significantly decreased upon leflunomide/hydroxychloroquine compared to placebo. Also other clinical parameters such as patient reported outcome ESSPRI-score and saliva output improved. Histological assessment by parotid biopsies supported these positive findings. Leflunomide/hydroxychloroquine combination therapy proved to be safe and well-tolerated. Response to therapy could be predicted with high accuracy by a model comprising ten circulating proteins. In Chapter 5, five different IFN- associated biomarkers (IFN-score in whole blood and PBMCs, MxA in whole blood, CXCL10 and Galectin-9 in serum) in patients treated with either leflunomide/hydroxychloroquine or placebo were assessed and their potential for treatment monitoring was investigated. Patients responding to the therapy showed strong early decreases of MxA and Galectin-9 levels from week 8 onwards. Subsequent ROC analysis revealed good predictive values for early changes in (delta) MxA and Galactin-9 levels after 8 weeks, the latter outperforming the former. In Chapter 6 we focused on Galectin-9 as a biomarker that reflects the IFN signature in patients with SLE, APS and pSS. The findings of this chapter are in line with previous findings showing that Galectin-9 is an easy to measure and accurate biomarker for the IFN signature in SLE and APS patients and correlates with disease activity. Usage of Galectin-9 as a biomarker for the IFN signature needs further confirmation. Data from this thesis indicate that at the least Galectin-9 might play an additional role in monitoring of pSS immunopathology and disease activity. Leflunomide/hydroxychloroquine treatment induced a decline in Galectin-9 levels, parallel with changes in B cell hyperactivity, ESSDAI and ESSPRI, and markers of IFN activity. This warrants further study of Galectin-9 as a biomarker for disease monitoring 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

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

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