Exploration Of The Role And Mechanism Of Peripheral Blood Mononuclear Cell In Glaucoma-Induced Retinal Ganglion Cell Damage

Research Objective:Primary open-angle glaucoma(POAG),a multifactorial disease,recognizes the pivotal role of immune cells,particularly T lymphocytes,in its pathological progression.This perspective has been increasingly acknowledged by researchers.Our previous studies revealed an elevated expression of T lymphocytes in the retinas of mice with glaucoma.Further investigations discovered that transferring T lymphocytes,rather than B lymphocytes,from mice with glaucoma to healthy mice led to a loss of Retinal Ganglion Cells(RGC)in the recipients.However,direct scientific evidence regarding the role of immune cells,especially T lymphocytes,in glaucoma patients is still lacking.To fill this research gap,we conducted a series of experiments involving Peripheral Blood Mononuclear Cells(PBMC)obtained from patients with POAG.By transferring these PBMC into mice,we aimed to verify a direct link between leukocytes in the peripheral blood of glaucoma patients and glaucoma-associated optic nerve damage.Additional research focused on T lymphocytes,a crucial component of PBMC,aiming to elucidate the causes of RGC damage in glaucoma patients.Furthermore,we explored whether the augmentation of Regulatory T cells(Treg)possessing immunosuppressive functions could provide protection for RGC under high intraocular pressure conditions.Methods:The first part of the experiment aimed to investigate the differences in T lymphocytes within PBMCs between glaucoma patients and healthy individuals.POAG patients constituted the glaucoma group,and patients without glaucoma who were visiting the ophthalmology department were used as a control group.Both groups followed the same inclusion and exclusion criteria.PBMCs were extracted from 10 ml of peripheral venous blood taken from each patient.Surface antibodies labeled CD3,CD4,CD8,CD45RA,CD11a,CD49d,CCR4,CD73,CD27,CCR7,CX3CR1,CD28,PD-1,TIM-3,CXCR5,CD38,CD226,anti-CXCR3,CD95,TCRγδ,CD39,KLRG1,CD127,CD25,CD45,CD56,and HLA-DR were used to stain the cells.In addition,CD45RA,CD8,CD3,CCR7,TCRγδ,CD25,CD45,TNF,IL-10,IL-21,IL-17A,IFNy,IL-13,Granzyme B,CD 137,CD 154,and CD4 antibodies were used to analyze cell function,revealing differences in T lymphocytes between glaucoma patients and the control group.The second part of the experiment confirmed the correlation between PBMCs and RGC damage.3.5x106 peripheral blood mononuclear cells were collected from each glaucoma patient and healthy control,then transferred into NOD.Cg-Prkdcscid I12rtml Wjl/SzJ mice via tail vein injection to establish humanized glaucoma mouse models.Subsequent observations were made of the damage to the mouse retinal ganglion cells.The third part of the experiment explored the use of Treg cells for negative regulation of immune responses to reduce RGC damage in glaucoma mice.Conditional gene knockout technology was used to knock out the inhibitory receptor lag3 on Treg cells in Lag3fl/fl.Foxp3CreERT2 mice.The change in the quantity of Treg cells lacking lag3 expression and its effect on the survival of retinal ganglion cells in glaucoma mice were evaluated.Results:First part results:Flow cytometry analysis of PBMCs showed that POAG patients had a higher frequency of CD4+T cells producing pro-inflammatory cytokines and a lower frequency of CD4+T cells expressing anti-inflammatory cytokines.They also had fewer fully differentiated CD4+and CD8+T cells.When stimulated,the PBMCs of glaucoma patients were able to generate a higher proliferation response.Second part results:In the recipient mice that received PBMCs from glaucoma patients,there was a reduction in RGC in the central retina compared to the control group.This RGC reduction was negatively correlated with the donor patient’s visual field and retinal ganglion cell layer thickness,but there was no significant statistical correlation with patient intraocular pressure.Third part results:Tamoxifen injection temporarily increased the number of Treg cells in the peripheral lymph nodes and spleen of Lag3fl/fl.Foxp3CreERT2 mice.After intraocular pressure increased,the number of Treg cells could improve the visual function of glaucoma mice and reduce axonal damage to the RGCs.However,increasing the number of regulatory T cells prior to increased intraocular pressure did not prevent RGC damage in the mice.Conclusion:The humanized mouse model accurately reflects the direct role of PBMC in the pathological process of glaucoma.PBMC from glaucoma patients is closely associated with RGC loss in mice and is also related to the clinical staging of the disease course.This could be due to the pro-inflammatory changes in T lymphocytes,especially CD4+cells,within glaucoma patients.Suppressive T-cell activity could potentially offer visual preservation benefits for some patients with glaucoma,providing a new direction for clinical treatment.However,its specific function and time of onset still require further research.