The Protective Role Of Regulatory T Cells In The Mice Subjected To Traumatic Brain Injury

Backgroud: Traumatic brain injury(TBI)is one of the leading causes of death and severe disabilityworldwide,especially in the young.Although many interventions are applied atdifferent time points post-injury,it remains a major challenge to promote long-terrecovery in these patients.Following the initial trauma,inflammatory responses canaggravate brain damage,mainly via the infiltration of leukocytes and the activation ofmacrophages.Although these cells are essential for debris clearance and tissue remodeling,their overactivation results in the release of a large number of cytotoxic molecules that eventually damage neurons,the extracellular matrix and endothelial cells.Tregs are key endogenous immune-regulatory cells that accommodate the inflammatory response.Because of their potent immunomodulatory properties,much of the stroke literature has focused on Tregs.For example,Li et al.demonstrated that the intravenous transfer of Tregs exerts a neuroprotective effect by suppressing neutrophil-derived matrix metallopeptidase 9 and reducing the subsequent proteolytic damage of the blood–brain barrier.Liesa et al.showed that boosting Tregs reduced microglial cell activation and neurotoxic cytokine secretion and that the main mediator of these effects was IL-10.Furthermore,in in vitro experiments,Tregs inhibited the pro-inflammatory effects of macrophages and promoted macrophage differentiation toward an anti-inflammatory phenotype.However,few studies have explored the effects of Tregs on TBI.Object: In this study,we determined if the increasion in Tregs could offer beneficial effects by suppressing the inflammatory response and reducing the degradation of tight junction proteins.Then,we explored the effects of exosomes derived from Tregs on the activated BV2 cells in vitro.Methods:(1)The TBI mice received 3 consecutive days to induce a rapid expansion of Tregs.The M1 and M2 microglia relative factors were detected using immunofluorescent staining and western blot.(2)The pure Tregs were isolated from the spleen by magnetic-activated cell sorting,followed by transplanted intracerebroventricuarly 2 hours post-injury.PKH67 was used to label and track the Tregs after transfer.BBB permeability was measured by the extravasation of Evans Blue.The expression of inflammatory cytokines IL-10,YM-1,arginase-1,i NOS,CD11 b and MCP-1 and tight junction proteins ZO-1,CLN5 and occludin were assessed by immunoblots and q RT-PCR.(3)Differential centrifugation was used to isolated the exosomes from the medium.Immunoblot and ELISA were used to detect the expression of inflammatory cytokines.Results:(1)Treatment with IL-2C dose-dependently increased the number of Tregs,which could enter the central nervous system.And an increase in Tregs was associated with the polarization of macrophages from the M1 to the M2 phenotype.(2)The Tregs labeled with PKH67 was detected in the contusion area.Adoptive transfer of Tregs reduced the expression of M1-relatve cytokines,while increased the expression of M2-relative factors.And the transplanted Tregs could reduce the permeability of Evans Blue,as well as the degradation of blood-brain barrier.(3)The exosomes derived from the Tregs could be uptaken by the BV2 cells,eventually suppressing the expression of inflammatory cytokines.Conclusion: The increasion in Tregs could promote the polarization of macrophages from M1 to M2 phenotype and reduce the degradation of blood-brain barrier,as well as the permeability of BBB.Furthermore,the effects of Tregs suppressing the inflammatory response were mediated partly by the release of exosomes.