The Regulation Mechanism Of PD-1Pathway In Diabetic Retinopathy

Background:Diabetic retinopathy (DR) remains a leading cause of blindness. Occurrence and progression of diabetic retinopathy is related to many factors. It has been reported that the occurrence and progression of DR may be related to hyperglycemia, oxidative stress, the accumulation of polyols and advanced glycation end products, apoptosis, cytokines and so on. There is an accumulating body of evidence that immunological mechanisms play a prominent role in the pathogenesis of DR. Immune response is involved in DR by the finding of autoantibodies and immune cells in the circulation. The upregulation of inflammatory cells and proinflammatory factors is contributed to DR-associated damage. Clinical applications with nonsteroidal anti-inflammatory drugs and corticosteroids in the treatment of DR also indicate that DR is an immune disease. Meanwhile, immunological and genetic factors were involved in DR. In conclusion, DR is a diabetic microvascular complication mainly resulted from immunological and inflammatory response.DR is characterized by an excessive inflammatory response and apoptosis. Activation-induced cell death of lymphocytes contributes to the regulation of cellular immune responses. Activation of lymphocytes is an early and crucial step in the development of antigen-specific immune responses, resulting in production and secretion of cytokines, cell-surface expression, up-regulation of cell adhesion molecules and ligands for molecules expressed on other cells. Helper T cells are important regulatory cells that secrete several cytokines and participate actively in this inflammatory response. According to the pattern of cytokines secreted, the immune response is classified as ThO, Thl(IFN-y-dependent), Th2(IL-4-dependent), Th3and Th17. The activation-induced apoptosis and a detailed characterization of the pattern of lymphocyte cytokine expression in DR are essential for a better understanding of the molecular mechanisms that drive the abnormal inflammatory response.Costimulatory signals have become a hot field in immunological researches. PD-1/PD-Ls is an immunoreceptor belonging to the CD28/B7family. PD-1is mainly induced on T and B cells upon activation. The expression of PD-L1and PD-L2on antigen-presenting cells (APCs) has been widely examined. Compared with PD-L2, PD-L1expressed on APCs can be easily upregulated by various stimulations. PD-1/PD-Ls interactions can regulate the activation of T cells and the secretion of cytokines. As co-inhibitory molecules, PD-1and its ligands have been demonstrated to play an important role in autoimmune disease, organ transplant rejection, microorganism infection, tumor immune escape and so on.Given the importance of PD-1/PD-Ls signal pathway as an immunosuppressive molecule, we hypothesized that it could also be involved in DR. In this study, we will discuss PD-1, PD-L1and PD-L2in patients with diabetic retinopathy using peripheral blood. The expression and functional characteristics of PD-1, PD-L1and PD-L2in DR will be observed and the role of PD-1/PD-Ls signaling pathway in the pathogenesis of DR will be discussed. All by this, we can provide a new theoretical basis for the pathogenesis of DR and new ideas on the prevention and treatment of this disease.Part Ⅰ:The expression of PD-1and its ligands in peripheral blood from patients with diabetic retinopathyObjective:To investigate the expression and its clinical significance of PD-1and its ligands in lymphocytes at mRNA levels from patients with diabetic retinopathy.Methods:Peripheral blood was obtained from41patients with PDR,28patients with NPDR,27patients with DM-NDR and59controls. The mRNA expression of PD-1and its ligands was observed by RT-PCR. The data was analyzed among groups.Results:mRNA of PD-1, PD-L1and PD-L2was detectable in all groups using real time-PCR. The relative expression of PD-1and PD-L1mRNA from patients with PDR was significantly lower than in other groups (PD-1:P=0.004; PD-L1:0.001). mRNA expression of PD-L2was also decreased in the PDR group but did not reach significant difference compared with other groups (P=0.267).Conclusions:A decreased mRNA expression of PD-1and PD-L1in lymphocytes of patients with PDR indicates their involvement in the occurrence of DR. Part Ⅱ:The role of PD-1signal pathway in activation-induced cell death in patients with diabetic retinopathyObjective:To investigate the role of PD-1/PD-Ls signal pathway in the process of AICD in patients with DR, and further explore the mechanism of AICD to provide a theoretical basis for the treatment of DR.Methods:Peripheral blood was obtained from24patients with PDR,15patients with DM-NDR and15controls. Lymphocytes were collected from4ml of peripheral blood, and then cells were stimulated with the PHA at concentration of40μg/ml for48h. Flow cytometry was used to analyze the activation-induced apoptosis and the expression of PD-1, PD-L1and PD-L2. The regulation mechanism of PD-1/PD-Ls signal pathway in AICD in patients with DR was analyzed.Results:Apoptosis of lymphocytes was induced by stimulation with PHA. The results showed that PDR group significantly increased the activation-induced apoptosis compared with DM-NDR and control groups (P=0.037,0.000). The apoptotic cells in DM-NDR group were also increased when compared with the controls (P=0.048). The expression of PD-1protein in lymphocytes and apoptotic lymphocytes of PDR group was higher than DM-NDR and control groups (P=0.042,0.000;.P=0.024,0.000), meanwhile, the expression of PD-1protein in lymphocytes and apoptotic lymphocytes of DM-NDR group was also higher than the controls (P=0.031; P=0.037). On the contrary, PDR group showed a lower frequency of PD-L1expression in lymphocytes and apoptotic lymphocytes compared with DM-NDR and control groups (P=0.000,0.009; P=0.000,0.005), otherwise, DM-NDR group showed a higher frequency of PD-L1expression in lymphocytes and lymphocytes compared with the controls (P=0.035; P=0.003). There was no expression of PD-L2protein in three groups.Conclusions:AICD exists in patients with PDR. PDR have an increased susceptibility to apoptosis with PD-1involvement. But patients with DM-NDR showed higher percentage of PD-L1expression compared to patients with PDR, so we guess that PD-L1may be protect patients with DM from eye damage. Part Ⅲ:The regulation of PD-1signal pathway in Thl/Th2cytokines in patients with diabetic retinopathyObjective:To investigate the regulation of PD-1signal pathway in the expression of Thl/Th2cytokines in patients with DR, and further explore the mechanism of DR from the perspective of cytokines.Methods:Peripheral blood was obtained from15patients with PDR,15patients with DM-NDR and15controls. Lymphocytes were collected from4ml of peripheral whole blood, and then cells were stimulated with50ng/ml PMA,1μg/ml Ionomycin and1.7μg/ml Monensin for4h. Flow cytometry was used to analyze the expression of IFN-y and IL-4. The ratio of Thl/Th2and the relationship between PD-1and Th1/Th2cytokines were analyzed.Results:Under stimulated, the frequency of IFN-y+, IL-4+, PD-1+IFN-γ+and PD-1+IL-4+cells in PDR group was the highest compared with DM-NDR and control groups, respectively. The analysis for the ratio of IFN-y+cells to IL-4+cells revealed that it was markedly higher in PDR group than in control group (P=0.047). However, the ratio of PD-1+IFN-γ+cells to PD-1+IL-4+cells had no significantly differences in each group (P=0.590).Conclusions:The expression of IFN-y and IL-4in PDR group was upregulated, meanwhile, the ratio of Th1/Th2appears imbalance and Th1response may be more important in developing this disease. PD-1is involved in the regulation of Thl and Th2cytokines in patients with PDR but with no relevance in the ratio of Thl/Th2. Part Ⅳ:Correlation analysis of risk factors in patients with diabetic retinopathyObjective:To observe the expression of serum β2-microglobulin, and to analyze the relationship between β2-microglobulin and exploratory variables from patients with PDR, looking for early screening diabetic retinopathy indicators.Methods:The expression of serum β2-microglobulin in patients with PDR was observed. The correlation between β2-microglobulin and a set of exploratory variables, including PD-1+, PD-L1+, AnnexinV+PIPD-1+, AnnexinV+PIPD-L1+, IFN-y+, IL-4+, PD-1+IFN-γ+, PD-1+IL-4+and activation-induced apoptotic cells was investigated in this study. The data of exploratory variables used for the correlation analysis were the results detected by flow cytometry above.Results:The average level of serum β2-microglobulin in patients with PDR was (3.07±1.79) mg/l, which was markedly higher than that in DM-NDR (2.11±0.71) mg/l and control groups (1.73±0.53)(P=0.028,0.000). β2-microglobulin from patients with PDR positively correlated with activation-induced apoptotic (r=0.484,.P=0.017) and IFN-γ+cells (r=0.523, P=0.045), while it negatively correlated with PD-1+(r=-0.515, P=0.034), PD-L1+(r=-0.765, P=0.004), AnnexinV+PI-D-1+(r=-0.625, P=0.010) and IL-4+cells (r=-0.573, P=0.026).Conclusions:Diabetic microangiopathy explains the simultaneous occurrence of typical diabetic glomerulopathy and PDR. The correlation between β2-microglobulin and exploratory variables in patients with PDR may suggest the involvement of PD-1pathway and Th1/Th2cells in the high level of β2-microglobulin, providing a new sight to finding indicators in diabetic microangiopathy.