Orally delivered microparticles loaded with all-trans retinoic acid and transforming growth factor β rescue mice from type 1 diabetes development

Abstract

Background and aims: Type 1 diabetes (T1D) is a multifactorial autoimmune disease that develops as a consequence of macrophage and T celldependent pancreatic β cell death. Multiple approaches have been attempted to induce anti-inflammatory/regulatory immune mechanisms that will attenuate disease progression, with little or no beneficial effects. To achieve prolonged stimulation of regulatory immune cells, our aim was to introduce microparticles (MPs) loaded with all-trans retinoic acid (ATRA) and transforming growth factor β (TGF-β). Both molecules are well-known synergistic stimulators of T regulatory cell (Treg) differentiation and stabilization. Materials and methods: Male C57BL/6 mice were treated with multiple low doses of streptozotocin (MLDS) for 5 consecutive days to induce T1D, and with empty MPs or ATRA and TGF-β-loaded MPs (0.1% and 0.03% w/w, respectively) for 10 days (every other day, starting from the first streptozotocin injection). Blood glucose was monitored on a weekly basis and ex vivo analyses of immune cells (flow cytometry, qPCR, immunoblot) were performed and pancreas histology was evaluated 14 days from the beginning of the T1D induction. ANOVA t test was used for statistical analysis and significant change was considered at p<0.05. Results: T1D incidence was significantly lower inATRA/TGF-β MP-treated mice, as was the degree of immune cell infiltration into the pancreatic islets. In Peyer’s patches (PP), ATRA/TGF-β MPs up-regulated the tolerogenic population of dendritic cells (DCs) (CD11c+CD11b-CD103+), while not altering the proportion of mature DCs (CD11c+CD11b+). Additionally, both IL-1β expression and production were reduced in PP, as was the ratio of iNOS/Arginase mRNA expression, reflecting a less inflammatory environment. This was accompanied by a reduction of the proportion of Th1 (CD4+IFN-γ+) and Th17 (CD4+IL-17+) cells and up-regulation of Treg (CD4+CD25highFoxP3+). Lower IL-17 expression within CD4+ cells from PP was in accordance with the observed down-regulation of RORγt mRNA expression (key transcription factor of IL-17). The situation in the pancreatic lymph nodes (PLN) was similar to PP regarding the downregulation of inflammatory Th1 cells. Also, the proportion of Tbet+CD25med cells (T effector cells) was lower, while the proportion of Treg expressing T-bet was increased in PLN, suggesting that these cells specifically mediate the inhibition of Th1 response. Additionally, in response to ATRA/TGF-β MP treatment, the proliferation (Ki67+) of T effector cells was reduced in PLN while Treg proliferated more. Furthermore, ATRA/TGF-β MP treatment favored the presence of CTLA-4+PD1+ and CD39+IL-10+ populations of Treg and thus increased their suppressive activities. Conclusion: ATRA and TGF-β released from MPs successfully ameliorated T1D through the potentiation of tolDC and Treg response and inhibition of Th1 cell differentiation in the draining lymph nodes, thereby blocking the entrance of immune cells into the pancreatic islets and protecting β cells from further destruction.