Immune Phenotyping Of Patients With Acute Vogt-Koyanagi-Harada Syndrome Before And After Glucocorticoids Therapy

Background:Vogt-Koyanagi-Harada(Vogt-Koyanagi-Harada,VKH)syndrome is an autoimmune uveitis that seriously threatens the vision of young adults.It is accompanied by symptoms including depigmentation,multi-tissue,and multi-organ damage to the inner ear,nervous system,and skin.At present,the treatment of VKH syndrome is largely dependent on GC therapy,supplemented by immunodepressants and biological agents.Although widely used with acceptable efficacy,GC therapy also has some drawbacks,such as side effects,a long therapeutic procedure and poor patient compliance.Therefore,further investigation is warranted to provide a better understanding for the underlying pathogenic mechanism of VKH syndrome,and to develop new immunotherapy methods that can reduce the occurrence of complications.This research was therefore designed and implemented for this purpose.In the past few decades,extensive investigations have been conducted regarding the pathogenesis of VKH syndrome.It is increasingly recognized that an autoimmune response against melanocytes might be attributable to the occurrence of this disease.Early studies found that in vitro stimulation of lymphocytes from VKH syndrome patients with crude melanocyte extracts resulted in the activation and proliferation of these cells,which then exhibited significant cytotoxicity against melanocytes.Subsequently,the pathogenic role of Th1 cells was discovered in experimental autoimmune uveitis(EAU),a classic animal model of human uveitis.Recently,Th17 effector cells have aslo been shown to be involved in the pathogenesis of EAU as well as clinical uveitis.It has been reported that activated T lymphocytes are the main cell type during choroidal inflammation in patients with VKH syndrome,and choroidal melanocytes express the major histocompatibility complex class II(HLA-DR)molecule,indicating that cell-mediated immunity is involved in the pathogenesis of ocular diseases.In addition to T cells,other immune cells also play an important role in the pathogenesis of VKH syndrome,including NK cells,B cells and myeloid cells.Antigen presenting cells(APC)are essential for the activation of T cells,and monocytes are considered to be the major professional APCs in peripheral blood.Various costimulatory molecules expressed on monocytes interact with corresponding ligands on T cells,thus provide a complete signal which is necessary for T cell activation.In addition,the pro-inflammatory cytokines secreted by APC,such as IL-6,IL-1βand IL-23,are essential for the differentiation of Th17 cells.However,the role of these cells and molecules in the pathogenesis of VKH syndrome has not been systematically elucidated.Objective:Profiling the phenotypic changes of peripheral immune cells in VKH patients before and after GC treatment to indentify potential pathogenic immune cell populations and molecules of VKH syndrome,and to find new targets for the treatment of this autoimmune disease.Methods:25 peripheral blood samples of VKH syndrome patients before GC therapy,16peripheral blood samples of VKH syndrome patients after GC therapy for seven consecutive days,and 21 gender-and age-matched peripheral blood samples of healthy controls(HCs)were collected.The frequency and phenotype of immune cell subpupolations were determined by flow cytometry and intracellular staining;Alternatively,Luminex and ELISA assay were used to detect the content of inflammatory cytokines in the plasma;RNA sequencing was performed for peripheral blood and were analyzed to find differentially expressed genes in VKH syndrome patients before and after GC therapy,followed by the verification of sequencing results by q RT-PCR and in vitro cell culture experiments.Confocal laser microscopy was used to identify monocyte subpopulations and the content of ROS in different monocyte subpopulations was compared.Results:1.Significant differencies were found in peripheral lymphocytes between VKH syndrome patients and healthy subjects in terms of quantity and quality.Compared with healthy controls,VKH syndrome patients harbor more T cells(especially activated T cells),and more CD27~+Ig D~+B cells and monocytes.2.GC therapy reduced the percentage of T cells and NK cells,and inhibited NK cell activation as well as T cell differentiation.After GC treatment,the distribution of monocyte subpopulations in the peripheral blood of VKH syndrome patients also changed markedly.The frequencies and numbers of CD16~-monocytes and CD56~+ monocytes were increased significantly,whereas the proportion and number of CD16~+ monocytes were markedly reduced.Collectively,GC therapy mainly affects the activation,proliferation of lymphocytes and the distribution of monocyte subtypes.3.RNA sequencing results revealed that apoptosis-related genes in peripheral blood of patients with VKH syndrome were down-regulated and inflammation-related genes were up-regulated;GC treatment of VKH syndrome may function through inhibiting lymphocyte activation,proliferation and differentiation,and promoting the apoptosis of immune cells.Additionally,NOS3 may play a key role in the occurrence of VKH syndrome and the therapeutic effects of GC treatment.4.Ex vivo examination of the apoptosis of PBMC by flow cytometry found that GC treatment resulted in more apoptosis in PBMCs from VKH syndrome patients. Furthermore,upon in vitro stimulation with dexamethasone,the proportion of apoptotic cells in PBMC from both healthy subjects and VKH syndrome patients was markedly increased,which is consistent with the findings in RNA sequencing (upregulation of pathways related to apoptosis after GC treatment).5.Confocal laser microscopy confirmed that CD56~+monocytes are a bona fide monocyte subpopulations that express both CD14 and CD56.Compared with CD56~- monocytes,CD56~+monocytes are more capable of producing ROS.Conclusion:Our findings suggest that advanced activation and differentiation,as well as dysregulated numbers of peripheral lymphocytes are the major immunological features of VKH syndrome.GC therapy with MP not only inhibits T cell activation directly,but also affects monocyte subsets,which might combinatorically result in the inhibition of the pathogenic immune response.