Immunoregulatory Effects Of Sinomenine On The Inappropriate T-cell Immune Responses In The Treatment Of Mesangial Proliferative Nephritis

BackgroundExperimental results from inflammatory renal diseases, such as allograft rejection, tubulointerstitial nephritis and glomerulonephritis, have suggested a prominent role of T cells in the injurious renal immune response. It is accepted that signals provide by TCR-peptide-MHC and co-stimulatory molecules are required for the optimal activation of T cells. Without sufficient co-stimulation, T cells are rendered anergy, tolerance or apoptosis. Recently, B7 superfamily molecules, PD-L1, PD-L2 and their receptor PD-1 (programmed death-1) have been identified. Previous studies indicated that the abnormality of PD-1/PD-L pathway might play a role in the development of autoimmune disease, allograft rejection, and various kinds of infection. Treatment aiming at regulating co-stimulatory pathway has now been recognized as a new way of immunotherapy.Inappropriate T cell responses result from not only the abnormality of T cell activation but also the abnormality of T cell differentiation. Immune responses polarized by either Th1 or Th2 subset predominance result in the activation of different inflammatory effector pathways and disease outcomes. T-bet is a newly discovered Th1-specific transcription factor thought to initiate Th1 development, whereas GATA-3 plays a pivotal role in the development of the Th2 phenotype. It has been reported that the T-bet/GATA-3 ratio is representative of the balance between Th1 and Th2 cells. Maintaining the Th1/Th2 balance via the regulation of T-bet/GATA-3 ratio has been recognized as a promising therapy in the treatment of autoimmune diseases.Sinomenine (SN), a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum, has been effectively used for over ten years in the treatment of patients with chronic glomerulonephritis. Although sinomenine is effectively used in the clinic and is gaining international popularity, little is known about its exact mechanisms of action.In this study, patients with mesangial proliferative nephritis (MsPGN), which is one of the most common pathological type of chronic GN, were examined. Both the PD-1/PD-L expression and the balanced state of Th1/Th2 were observed in patients with MsPGN. Then the effect of SN on proteinuria was detected. In order to elucidate the therapeutic mechanism of SN, the effect of SN on the expressions of PD-1/PD-L and T-bet/GATA-3 in peripheral blood mononuclear cells (PBMCs) and the serum levels of interferon-γ(IFN-γ), interleukin (IL)-4, and IL-10 were studied.Patients and methods1. Patients and renal specimensA total of 25 patients with MsPGN and 10 healthy donors were enrolled in this study. Immunohistochemistry was enforced on the paraffin-embedded tissues from renal biopsies of 25 patients with MsPGN. Additionally, ten specimens obtained from pre-transplant renal biopsies and non-tumor kidney tissues from cancer patients served as controls.2. methods(1) In order to observe the expression pattern of PD-1/PD-L in patients with MsPGN, the intrarenal levels of PD-1, PD-L1, and PD-L2 were determined by immunohistochemistry and the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR and flow cytometry.(2) In order to observe if there is an imbalance between Th1 and Th2 cells in patients with MsPGN, the intrarenal levels of T-bet and GATA-3 were determined by immunohistochemistry, the expression levels of T-bet and GATA-3 in PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR, and the serum levels of IFN-γ, IL-4, and IL-10 were studied via ELISA.(3) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on lymphocytes in vitro was studied via real-time RT-PCR and flow cytometry.(4) The effect of SN on the expression levels of T-bet and GATA-3 on lymphocytes in vitro was studied via real-time RT-PCR and Western Blot. Additionally, ELISA was applied to detect the effect of SN on the supernatant levels of IFN-γ, IL-4, and IL-10.(5) To evaluate the clinic effect of SN, blood biochemical parameters and proteinuria were detected at month 0, month 1, and month 3 in the course of treatment with SN.(6) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in vivo was studied via real-time RT-PCR and flow cytometry at 1 month and 3 months after the start of treatment with SN.(7) The effect of SN on the expression levels of T-bet and GATA-3 on PBMCs in vivo was studied via real-time RT-PCR and the serum levels of IFN-γ, IL-4, and IL-10 were studied by ELISA at 1 month and 3 months after the start of treatment with SN.Results1. Positive staining for PD-1 was detected on infiltrating lymphocytes in the interstitial space and PD-L1 was detected in tubular epithelial cells in renal tissues from MsPGN patients. However, the expression of PD-L2 was hardly observed in renal tissues in both MsPGN patients and control. Patients with MsPGN were found to have increased PD-1 and PD-L1 expression in both renal biopsy tissues and PBMCs compared with normal individuals.2. Positive staining for both T-bet and GATA-3 was detected in tubular epithelial cells and infiltrating lymphocytes in the interstitial space. Patients with MsPGN were found to have increased T-bet and depressed GATA-3 expression in both renal biopsy tissues and PBMCs compared with normal individuals. Additionally, MsPGN patients were found to have elevated serum IFN-γvalues and decreased serum IL-10 values when compared with healthy donors.3. The expression of PD-1 and PD-L1 on lymphocytes was suppressed by SN in vitro, and the inhibition effect was concentration dependent.4. The expression of T-bet on lymphocytes was suppressed by SN in vitro, therefore, the T-bet/GATA-3 ratio was markedly decreased. SN was also found to markedly inhibit the secretion of IFN-γ. These effects above were concentration dependent.5. SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects.6. SN could suppresse the expression of PD-1 and PD-L1 on PBMCs from MsPGN patients in vivo, and this effect was more significant at 3 months.7. SN was found to cause a decrease in T-bet expression on PBMCs from MsPGN patients in vivo, resulting in a drop in the T-bet/GATA-3 ratio. SN could also inhibit the secretion of IFN-γ. These effects were more significant at 3 months.ConclusionsSN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects, however, the course of treatment should last for more than 1 month.Increased expression of PD-1 and PD-L1 in renal tissues and PBMCs from MsPGN patients may contribute to the development of MsPGN. Our results indicate that SN has the potential to inhibit the high expression of PD-1 and PD-L1 in PBMCs from MsPGN patients and thereby produce therapeutic effects.A shift toward the Th1 pathway of Th cell activation at both transcriptional level and cytokine level occurs in MsPGN patients, and that SN has the potential to counter this shift in the Th1/Th2 balance and thereby produce therapeutic effects.