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

ABSTRACT

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.

KEYWORDS:

• Primary Sjögren’s syndrome
• disease modifying anti rheumatic drugs
• leflunomide
• hydroxychloroquine
• combination therapy

Article highlights

• Stimulation of PBMCs with SEB and TLR-9L induces strong proliferation of T and B cells, as well as production of cytokines and immunoglobulins.

• Leflunomide inhibits in vitro proliferation of lymphocytes, most strongly T lymphocytes and halts production of CXCL13, IFN-α, IFN-γ, and immunoglobulins.

• Hydroxychloroquine in vitro most strongly inhibits proliferation of B-cells, production of CXCL13, IFN-α, and immunoglobulins.

• Their combination additively decreases lymphocyte proliferation and cytokine- and immunoglobulin production.

• Since leflunomide and hydroxychloroquine combination therapy in vitro potently inhibits several immune responses that are key to immunopathology in pSS this drug combination holds promise for treatment of pSS.

• This box summarizes key points contained in the article.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Supplementary material

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Additional information

Funding

This work was supported by ZonMw (Goed Gebruik Geneesmiddelen program), grant number 836021005, and European Research Counsil starting grant to Timothy RDJ Radstake and a grant of the European’s Union Horizon 2020 Research and Innovation Programme (Grant Agreement No 731944).