Associate Professor University of Florida, United States
Introduction: Rheumatoid Arthritis (RA) is a debilitating autoimmune disease affecting ~1% of the population. It is characterized by inflammation of the joint synovium leading to bone and cartilage destruction. There is currently no cure and treatment options entail lifelong management of immunosuppressive drugs. However, these drugs are accompanied by many adverse effects such as increased risk of infection and acute and chronic toxicities. The COVID-19 pandemic has re-invigorated a critical need for new therapies that do not immunocompromise the patient. In this study, we have rationally designed a novel poly (lactic-co-glycolic) acid (PLGA)-based microparticle system, termed REGvac, that upon subcutaneous delivery facilitates de novo generation of tolerogenic dendritic cells (tDC) to mediate antigen-specific tolerance towards rheumatic antigens. This is achieved through delivery of REGvac which is comprised of (1) a non-phagocytosable 30 um PLGA microparticle (MP) encapsulating transforming growth factor β1 (TGF-β1) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and (2) a phagocytosable (1 µm) PLGA MP encapsulating vitamin D3 and the rheumatic antigens, type II collagen and citrullinated fibrinogen. Here, we demonstrate that REGvac treatment is able to induce a tolerogenic phenotype in murine bone marrow-derived dendritic cells and ameliorate RA symptoms in the murine collagen-induced and citrullinated fibrinogen-induced (CIA/FIA) model of RA.
Materials and
Methods: Fabrication of REGvac: Briefly, REGvac was fabricated using double emulsion-solvent evaporation methods. Release kinetics were determined via ELISA or spectrophotometry at various timepoints. Scanning electron microscopy (SEM) images were taken using FEI Scios DualBeam FIB/SEM (Thermo Fisher).
In vitro culture and treatment of bone marrow-derived dendritic cells: Dendritic cells were generated from bone marrow progenitors then treated with either LPS, methotrexate (MTX), or REGvac for 72h, flow cytometry was performed and resolved on an Attune NxT flow cytometer (ThermoFisher).
CIA/FIA induction in DBA/1J mice and REGvac treatment: Rheumatoid Arthritis was induced via immunization with type II collagen (Sigma) and citrullinated fibrinogen (Cayman Chemical) in Complete Freund’s Adjuvant (Sigma). Onset of severe RA initiated therapy with REGvac (2.5 mg s.c.) or methotrexate (2.5 mg/kg i.p.) in the abdominal region. Each paw was evaluated for a score of 0-3, (with 0 being no symptoms, and 3 being most severe erythema and swelling), and summed to give clinical score. Ankle thickness was measured using surgical calipers.
Results, Conclusions, and Discussions:
Results: Fabrication and characterization of REGvac. REGvac fabrication yielded ~1µm and ~30µm MPs with spherical morphology for the small component (Fig. 1A) and large component (Fig. 1B) of REGvac via SEM images and exhibited a delayed release profile for the small MP component of REGvac and a burst release profile for the large MP component of REGvac (Fig. 1C).
REGvac induces a tolerogenic phenotype that resists LPS-induced stimulation in murine BMDCs. Bone marrow-derived dendritic cells (BMDCs) were treated with REGvac, methotrexate, LPS, REGvac + LPS, or methotrexate + LPS for 72 hours and evaluated for surface expression of CD80, CD86 via flow cytometry. REGvac treatment of murine BMDCs suppressed CD80 expression (Fig. 2A), did not induce CD86 expression (Fig. 2B), and bestowed the ability to resist LPS-induced expression of CD80 and CD86.
REGvac treatment ameliorates symptoms in CIA/FIA mice. DBA1/J mice were induced with CIA/FIA. Upon onset of severe RA (clinical score > 6) REGvac, MTX, or PBS interventions were initiated for 8 weeks. REGvac treatment significantly reduced clinical score (Fig. 3A) and ankle thickness (Fig. 3B) after 8 weeks.
Discussion: Our REGvac therapy hinges on delivering rheumatic autoantigens to local dendritic cells in a tolerogenic context. It is important that these tolerogenic dendritic cells loaded with rheumatic peptides are able to retain a regulatory phenotype when exposed to external inflammatory stimuli in order to facilitate mechanisms of tolerance when encountering autoreactive T cells. We show that in vitro REGvac-treated BMDCs are able to retain low expression of CD80 and CD86 when stimulated with LPS, and that REGvac treatment in the CIA/FIA mouse model of inflammatory arthritis is able to ameliorate RA symptoms.
Conclusion: In this study, we demonstrate that REGvac is able to induce a stable tolerogenic phenotype in murine BMDCs that resists subsequent LPS-induced stimulation and ameliorate arthritic symptoms in the CIA/FIA mouse model of RA. These results give evidence that REGvac is able to exert therapeutic effects in vivo through modulation of DCs. Further validation that this microparticle therapy does not compromise the immune system would be a much-needed alternative to current therapies that immunocompromise the patient.
