Ethyl Pyruvate Stimulates Regulatory T Cells and Ameliorates Type 1 Diabetes Development in Mice

Abstract

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic β-cells, while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP), a stable pyruvate derivate and certified inhibitor of an alarmin–high mobility group box 1 (HMGB1), exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D, EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence, reduced the infiltration of cells into the pancreatic islets and preserved β-cell function. Apart from reducing HMGB1 expression, EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b−CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4, secreted TGF-β, and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells), but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently, suggesting that they readily suppressed IFN-γ-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall, our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation, proliferation, their suppressive capacity, and recruitment into the pancreas.

Ethyl Pyruvate Promotes Proliferation of Regulatory T Cells by Increasing Glycolysis

“…As we have previously shown, EP can act as an anti-inflammatory agent by inhibiting the action of effector T cells and by promoting the regulatory phenotype of dendritic and T cells [ 8 , 18 ]. EP enhanced Treg proportions and their suppressive ability when applied simultaneously with streptozotocin-induced type 1 diabetes in C57BL/6 mice.…”

“…EP enhanced Treg proportions and their suppressive ability when applied simultaneously with streptozotocin-induced type 1 diabetes in C57BL/6 mice. In addition, our results indicated that EP could partially replace the activity of TGF-β during Treg differentiation in vitro [ 8 ]. Currently, we have shown that EP stimulates Treg proliferation in vitro rather than differentiation.…”

“…This is based on the increased number of divided Treg after the addition of EP, while the number of undivided Treg was comparable to Treg differentiated in the presence of conventional stimulation. The effect of EP seems to be specific for Treg, since it did not affect the number of Th1 and Th17 during differentiation from conventional CD4 + T cells [ 8 ]. Similarly to the in vitro effect, EP applied in vivo through the oral route was stable, reached the intestine, and increased the proportion of Treg in both the lamina propria and the Peyer’s patches.…”

“…Treg suppressive capacity depends upon their production of versatile inhibitory mediators and the inhibition of T cell activation by cell-to-cell contact [ 32 ]. We have shown that in vitro EP stimulates the expression of CTLA-4, PD-1 and GITR [ 8 ], as well as IL-10 and perforin expression. These results indicate that EP-treated Treg possess an increased suppressive capacity, as IL-10 is known to down-regulate Th1 and Th17 activity.…”

“…Further, EP acts as a potent inhibitor of nuclear factor-κB (NF-κB), which is commonly considered as a transcription factor for pro-inflammatory cytokines [ 2 , 7 ]. Our previous study [ 8 ] demonstrated that during the development of type 1 diabetes in mice, EP skewed the immune response towards anti-inflammatory, i.e., in vivo administration of EP favored tolerogenic dendritic cells and regulatory T cells (Treg) in the pancreatic lymph nodes and pancreatic infiltrates. Moreover, we showed that EP stimulated Treg proliferation and migration to the inflamed tissue and increased Treg suppressive properties in an animal model of type 1 diabetes [ 8 ].…”