Study On The Role Of NOD-like Receptors In The Pathogenesis Of Vogt-Koyanagi-Harada (VKH) Syndrome And Behcet’s Disease (BD)

Uveitis involved uveal, vitreous body, retina, retinal vascular which can be classified into various types according to the different positions and involved tissues. Examples of autoinflammatory or autoimmune uveitis entities include Behcet’s disease(BD) and Vogt-Koyanagi-Harada(VKH) syndrome, which are the most common uveitis entities in China and account for 57% of blindness in the population of uveitis patients. BD is a kind of uveitis involved oral ulcers, genital ulcers and polymorphous skin damage but VKH is autoimmune disease characterized by bilateral granulomatous uveitis. The pathogenesis of both BD and VKH syndrome is still not exactly clear and further research into the mechanisms involved may lead to new therapeutic approaches. Recent studies reported that Th1 and Th17 cells play a vital role in the pathogenesis of BD and VKH syndrome, but other cells of the innate and adaptive immune response are probably also involved. Dendritic cells(DCs), in their capacity as antigen-presenting cell(APC) play an important role in the immune response against pathogens, thereby regulating the differentiation and maturation of Th1 and Th17 cells, NOD-like receptors(NLRs) as pattern recognition receptors(PRR) play a vital role in the first line of host defense against pathogens. Several studies reported that an abnormal expression of NOD1 and NOD2 could contribute to a variety of autoimmune diseases such as Crohn’s disease and atopic eczema. Whether the NOD1 and NOD2 pathway could also play a role in autoimmune or autoinflammatory uveitis has not yet been fully investigated and was therefore the purpose of the study presented here.Part ⅠThe expression of NOD1 and NOD2 m RNA in patients with BD and VKH syndromePurposeTo investigate the m RNA expression of NOD1 and NOD2 in patients with BD and VKH syndrome.MethodsPeripheral blood mononuclear cells of VKH patients(including patients with active and inactive disease), BD patients(including patients with active and inactive disease) and healthy controls were isolated from freshly drawn blood by Ficoll-Hypaque density gradient centrifugation. Total RNA of the PBMCs were used to obtain c DNA according to the manufacturer’s instructions. Real-time quantitative PCR was performed using the c DNA to detect the expression of NOD1 and NOD2 in PBMCs.ResultsWe compared the NOD1 and NOD2 m RNA expression in PBMCs from patients with active VKH syndrome, BD and controls. A higher expression of NOD1 was only observed in patients with active VKH syndrome as compared with controls but not in patients with inactive VKH syndrome. A higher expression of NOD2 was observed in patients with active VKH syndrome as compared to patients with inactive VKH syndrome and controls. No differences were observed in active or inactive BD patients.Conclusionswe found that the m RNA expression of NOD1 and NOD2 in PBMCs was higher in active VKH patients but not in patients with BD. The studies indicate significant correlation between the increased m RNA expression of NOD1 and NOD2 with the pathogenesis of VKH syndrome.Part ⅡThe protein expression of NOD1 and NOD2 in patients with BD and VKH syndromePurposeTo investigate the protein expression of NOD1 and NOD2 in patients with BD and VKH syndrome.MethodsPeripheral blood mononuclear cells of VKH patients, BD patients and healthy controls were isolated from freshly drawn blood by Ficoll-Hypaque density gradient centrifugation. Total PBMCs were used to obtain protein according to the manufacturer’s instructions. Western blot was performed to detect the expression of NOD1 and NOD2 in PBMCs.ResultsOur results revealed that patients with active VKH showed higher NOD1 and NOD2 expression as compared with controls but there were no significant differences between patients with BD and controls, which is consistent with the m RNA results.ConclusionsIn this study, we found that the protein expression of NOD1 and NOD2 in PBMCs was higher in active VKH patients but not in patients with BD. The studies indicate significant correlation between the increased m RNA and protein expression of NOD1 and NOD2 with the pathogenesis of VKH syndrome.Part Ⅲ The production of pro-inflammatory cytokines of PBMCs in patients with VKH syndrome and healthy controlPurposeTo investigate production of pro-inflammatory cytokines of PBMCs in patients with VKH syndrome and BD after NOD1 or NOD2 activation.MethodsPeripheral blood mononuclear cells of VKH patients and healthy controls were isolated from freshly drawn blood by Ficoll-Hypaque density gradient centrifugation. Total PBMCs were cultured with i E-dap and MDP according to the manufacturer’s instructions. ELISA was performed to detect the production of pro-inflammatory cytokines in PBMCs.ResultsThe results showed that i E-DAP or MDP stimulation significantly increased the production of IL-6, TNF-α and IL-1β in both groups. Patients with active VKH syndrome showed a higher level of IL-6, TNF-α and IL-1β than healthy individualsConclusionsIn this study, we found that the activation of NOD1 and NOD2 in PBMCs could increase the production of pro-inflammatory cytokines IL-6, TNF-α and IL-1β. The study confirmed that the over expression of NOD1 and NOD2 could influence the function of PBMCs.Part Ⅳ The effect of NOD1 and NOD2 on the function of DCs.PurposeTo investigate the expression of surface markers and production of pro-inflammatory cytokines after NOD1 and NOD2 activation in DCs.MethodsPeripheral blood mononuclear cells of VKH patients and healthy controls were isolated from freshly drawn blood by Ficoll-Hypaque density gradient centrifugation. Magnetic beads were used to separate CD14+ T cells Flow cytometry were used to evaluate the expression of surface markers. ELISA was performed to detect the production of pro-inflammatory cytokines in DCs.ResultsBoth i E-DAP and MDP could increase the expression of DC surface markers(CD40, CD80, CD83, CD86 and HLA-DR) obtained from patients with active VKH syndrome as well as in the healthy controls. The level of IL-6, TNF-α and IL-1β were significantly higher in the i E-DAP or MDP stimulated group both in patients with active VKH syndrome as well as in the controls. Patients with active VKH syndrome showed a higher level of IL-6 and IL-1β than controls with or without stimulation.ConclusionsSurface molecules on DCs are essential for the interaction between the T cell receptor and antigen-associated MHC-II molecules resulting in T cell activation. Pro-inflammatory cytokines produced by DCs also regulate the differentiation of na?ve T cells into Th cell subsets such as Th17 or Th1 cells. We showed that i E-DAP or MDP stimulated DCs to express a higher level of IL-6, TNF-α and IL-1β as compared to controls. Patients with VKH syndrome showed a higher level of IL-6 and IL-1β than controls with or without stimulation. It is most likely that the response seen in PBMCs is mediated by monocytes.Part Ⅴ The effect of NOD1 and NOD2 on the function of CD4+T cellPurposeTo investigate the proliferation of IL-17+CD4+T cells and IFN-γ+CD4+T cells and production of effector cytokines by CD4+ T cells.MethodsPeripheral blood mononuclear cells of VKH patients and healthy controls were isolated from freshly drawn blood by Ficoll-Hypaque density gradient centrifugation. Magnetic beads were used to separate CD4+ and CD14+ T cells. Flow cytometry were used to evaluate the proliferation of IL-17+CD4+T cells and IFN-γ+CD4+T cells. ELISA was performed to detect the production of effector cytokines by CD4+ T cells.ResultsThe results showed that the proportion of both IL-17+CD4+T cells and IFN-γ+CD4+T cells increased significantly in the i E-DAP or MDP stimulated DCs group. Analysis of cell supernatants by ELISA showed that the level of effector cytokines IL-17 and IFN-γ was significantly increased after the stimulation with i E-DAP or MDP as compared to the untreated groupConclusionsThe differentiation and proliferation of Th17 and Th1 cells plays a vital role in the pathogenesis of both VKH syndrome as well as in BD. To verify whether the activation of NOD1 or NOD2 could trigger the T cell-mediated immune response, we co-cultured i E-DAP or MDP stimulated DCs with CD4+T cells. We found that the activation of NOD1 or NOD2 could increase the proportion of IL-17+CD4+T cells and IFN-γ+CD4+T cells and that it induced the secretion of effector cytokines IL-17 and IFN-γ.