CD4+CD25+ regulatory T (Treg) cells participate in immunologic homeostasis by active suppression of inappropriate immune responses and are able to inhibit a variety of autoimmune and inflammatory diseases. Treg cells inhibit the activation of autoreactive T cells and suppress organ-specific autoimmunity. The mechanisms of Treg cells involved in the regulation of experimental autoimmune encephalomyelitis (EAE) are not well understood. Recent studies have shown a direct involvment of Treg cells in the natural resolution of EAE within the central nervous system (CNS) and a strong correlation between their migration pattern and their ability to control inflammatory responses. However, the molecular mechanisms controlling the migration of Tregs in inflamed brain are not known. P-selectin glycoprotein ligand-1 (PSGL-1) and alpha (1,3) fucosyltransferases (FucT), enzymes that catalyze the glycosylation of PSGL-1 and control its functionality, are molecules involved in the migration of leukocytes in sites of inflammation. The GOAL of this study was to determine the role of PSGL-1 in the migration of Treg cells in mice with EAE.
METHODS: Active and transfer EAE were performed in WT/C57Bl/6, FucT-VII and PSGL-1 deficient (FucT-VII-/- and PSGL-1-/-) mice using MOG35-55 peptide. CD4+CD25+ cells were obtained by magnetic cell sorting. Flow cytometry and ImageStream technology were used to determine the expression and distribution of adhesion molecules and binding capacity to P-selectin and E-selectin chimeras. Migration properties of WT, FucT-VII-/- and PSGL-1-/- Tregs were determined with in vivo migration assays using 3H-glycerol-labeled Tregs. Intravital microscopy experiments were performed in order to determine the ability of WT, FucT-VII-/- and PSGL-1-/- Tregs to interact with inflamed brain endothelium.
RESULTS: Encephalitogenic T cells produced from FucT-VII-/- and PSGL-1-/- mice transferred a significantly more severe disease that WT T cells. We observed no significant differences in the expression of adhesion molecules and IL-4 and IFN-\u3b3 production of autoreactive T cells from PSGL-1 and FucT-VII deficient mice. However, co-cultures with CD4+CD25+ Tregs and effector cells showed that deficiency of PSGL-1 and FucT-VII leads to a marked decrease of suppression capacity of Tregs. Interestingly, activated CD4+CD25+ Tregs have increased expression of functional PSGL-1 and a significantly higher suppressor activity in vitro when compared to na\uefve cells. In addition, activated Tregs display increased migration capacity in inflamed brain in mice with EAE. Both activated and na\uefve WT Treg cells preferentially migrated into the CNS in the pre-clinical phase of active EAE, than at disease onset. Treg cells from FucT-VII-/- and PSGL-1-/- mice present decreased migration ability to inflamed CNS when compared to WT Tregs. Moreover, intravital microscopy experiments showed a dramatic decrease of adhesive interactions in inflamed brain microcirculation in FucT-VII-/- and PSGL-1-/- Tregs. Finally, Tregs deficient of PSGL-1 and FucT-VII displayed a reduced capacity to suppress active EAE when compared to WT cells.
CONCLUSION: Our data demonstrate that PSGL-1 and the fucosylation of its glycans by FucT-VII are involved in the suppression mediated by CD4+CD25+ Treg cells in MOG-induced EAE. Moreover, in addition to a role in cell-cell contact required for efficient suppression, our results suggest a key role of PSGL-1 and FucT-VII activity in the recruitment of CD4+CD25+ cells into the brain of mice with EAE