Rheumatoid arthritis (RA) is a debilitating autoimmune disease with a well-characterized pro-inflammatory milieu and immune cell infiltration within the joint synovium, causing breakdown of adjacent cartilage and bone [1]. If left unchecked, RA can escalate into systemic issues including interstitial lung disease, cardiovascular disease, lymphoma [2], anemia, osteoporosis, and increased mortality [1]. There is currently no cure and RA affects ~1% of the population [3]. Those afflicted with RA suffer tremendous indirect costs through impaired work performance, disability, and early retirement [4] as well as enormous psychological stress through progressive decline of self-worth and well-being [5].The current anchor drug for RA is the folate inhibitor, methotrexate, and a clinical diagnosis of RA will regularly default to a long-term methotrexate regimen [6]. The therapeutic mechanism of methotrexate in RA is poorly understood. It is generally thought to function through systemic upregulation of anti-inflammatory adenosine signaling, thereby achieving general immunosuppression [7]. Additionally, folate inhibition results in purine depletion, cell cycle arrest, and eventually apoptosis in rapidly proliferating cells [8]. Nonetheless, long-term methotrexate treatment comes with many adverse effects which may manifest in the stomach, liver, blood, kidney, or lungs, and perhaps most notably, increased rate of infection [9]. The pervasive concern that accompanies immunosuppressants is walking the line between undersuppression and oversuppression; a perturbance in either direction results in refractory disease or increased risk of microbial infection [10]. Elucidating therapies that do not immunocompromise the patient will provide much-needed alternatives for the debilitating autoimmune condition of RA. Patients should not have to choose between quality-of-life improvements or risk of serious infection.
Tolerogenic dendritic cells (tDCs) have garnered considerable interest for their ability to mediate antigen-specific tolerance. Generation of tDCs with selected autoantigens allows for precise targeting of only the autoreactive T cells that are propagating autoimmunity. Clinical trials investigating the efficacy of autologous tDC therapy have shown therapeutic efficacy, but are limited by costly ex-vivo culture, expansion, and invasive intra-articular injections [11].
To this end, we have developed a dual poly(lactic-co-glycolic acid) (PLGA) microparticle-based “regulatory vaccine” (REGvac 2.0) to treat RA through the induction of tDCs to mediate antigen-specific tolerance. This is achieved through delivery of (i) a phagocytosable microparticle (MP) encapsulating collagen II and citrullinated fibrinogen peptides, as well as the tolerizing factor, vitamin D3 and (ii) a non-phagocytosable microparticle to dictate the terms of a dendritic cell (DC)-enriched, tolerogenic local environment through sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor β1 (TGF-β1). This system coerces recruitment of immature DCs to the site of microparticle delivery and subsequent de novo generation of tDCs presenting collagen II and citrullinated fibrinogen peptides to facilitate tolerance towards these autoantigens.
We first fabricated the large MP REGvac 2.0 with a large molecular weight (MW) PLGA polymer (~100 kDa) which promotes degradation and a “burst release” profile for the encapsulated factors [12] to promote rapid recruitment of DCs to the site of injection. Conversely, we utilized a low MW PLGA polymer (~10 kDa) for synthesis of the small MP REGvac 2.0 to decrease MP degradation and promote stable encapsulation [12] of vitamin D3, collagen II, and citrullinated fibrinogen for safe delivery into the DCs. We were able to successfully characterize correct shape, size, and release profiles for REGvac 2.0.
We next tested the ability of REGvac 2.0 to promote a tolerogenic DC phenotype in vitro, and whether the resultant tDCs would be able to resist subsequent LPS-induced maturation. We utilized two DC types, murine bone marrow-derived DCs (BMDCs), and human monocyte-derived DCs (mo-DCs). Using flow cytometry, we investigated expression of CD11c, CD80, CD86, and MHCII in REGvac 2.0-treated DCs. Decreased expression of CD80, CD86, and MHCII correlate to a tolerogenic and immature DC phenotype. We demonstrated that REGvac 2.0 treatment was able to downregulate expression of CD80, CD86, and MHCII, in DCs, that was retained when subsequently exposed to LPS.
After validation of REGvac 2.0 in vitro, we utilized the collagen-induced and fibrinogen-induced arthritis (CIA/FIA) mouse model of inflammatory arthritis to investigate whether REGvac 2.0 would be able to exert any therapeutic efficacy and also performed flow cytometry to characterize various T cell compartments within the spleen, inguinal lymph node (LN), and popliteal LN. We found that REGvac 2.0 treatment in the CIA/FIA mice was able to elicit rapid therapeutic effects through a decrease in clinical score as well as ankle thickness. Flow cytometric analysis revealed that REGvac 2.0 treatment decreased DC expression of CD80, CD86, and MHCII in the spleen, inguinal LN, and popliteal LN, and increased expression of CD4+FOXP3+ Treg cells in the inguinal LN and popliteal LN. Additionally, REGvac 2.0-treatment abrogated CD4+Tbet+ Th1 cellular responses in the spleen, inguinal LN, and popliteal LN.
Finally, we sought to test the effects of REGvac 2.0-treatment on protective immunity during an ongoing viral infection. We utilized the mouse adapted strain, Influenza A/Puerto Rico/8/34 H1N1 (PR8), to infect mice that were treated with REGvac 2.0. We found that REGvac 2.0-treatment did not impair the immune response to Influenza A Virus infection compared to the healthy control. This gives evidence that REGvac 2.0-treatment does not impair the immune system’s anti-viral response.
