Human Alpha1Antitrypsin For Improvement Of The Autoimmunity And Allograft Rejection In Beta Cell Transplantation

Objective：In this study, an insulinoma cell line, NIT-1, was transfected with human AAT(hAAT), named NIT-hAAT, and was transplanted to the left renal subcapsular spaces of7-week-old female non-obese diabetic (NOD) mice. Cyclophosphamide(CY) was administeredto synchronize and accelerate the development of diabetes. Thus, the immunosuppressive andcytoprotective activity of hAAT in β-cell transplantation was investigated.Methods: In this study, NIT-hAAT was transplanted to the left renal subcapsular spaces of7-week-old female NOD mice (n=22). On the seventh day after transplantation，CY wasadministered to synchronize and accelerate the development of diabetes. Firstly，in order toobserve the hAAT for improvement of type1diabetes，we examined the blood glucose level,diabetes incidence and glucose tolerance testing after cells transplantation from differentgroups.Aim to observe the immune protection of hAAT in vivo, the biopsy of tissues was stainedby hematoxylin and eosin (HE). Histological analysis of insulitis and histology of transplant sitewere detected. Apoptotic cells in islets were detected using a terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL).Then， The immunomodulatoryproperties of NIT-hAAT was investigated.The circulating hAAT levels， the leves of Th1cytokines (TNF-α, IFN-γ) and Th2cytokines (IL-4、IL-10) in serum were detected by ELISA.Besides,The percentage of Th17/Treg cell in CD4+cells were detected by flow cytometry.Results: In present study，the circulating hAAT levels reached approximately164.33-340.33μg/mL within21days after NIT-hAAT transplantation. According to histological analysis，NIT-hAAT transplantation delay the onset of autoimmune diabetes, reduce diabetes incidence,inhibit insulitis and β-cell apoptosis, and dampen transplant site inflammation.Moreover,NIT-hAAT transplantation upregulated Tregs and downregulated Th17cells, shiftedthe balance between Th17cells and protective Tregs. Besides,NIT-hAAT transplantationpromoted secretion of the Th2-related cytokine IL-4and1L-10，decreased the Th1-relatedcytokine IFN-γ and TNF-α， and shifted the Th1/Th2cell ratio toward Th2cells. However, thehAAT level decreased to164.33±8.33μg/mL on day21. No hAAT was detected in the serum byday49of post-transplantation. Since day21of post-transplantation, inflammatory cell infiltration gradually increased in the transplantation site, and insulitis and β-cell apoptosis wereobserved. Similar to the control group and the NIT-1group, NOD mice transplanted withNIT-hAAT became hyperglycemic and diabetic by day49. The hAAT failed to completely blockthe allograft rejection and autoimmunity-mediated islet damage on the later stage oftransplantation.Conclusions: NIT-hAAT cells expressing hAAT in vivo and NIT-hAAT transplantation has adual function by improving islet autoimmunity and protecting transplanted β-cells from allograftrejection. However, hAAT is unable to induce long-term specific immune tolerance and tocompletely block allograft rejection and autoimmunity-mediated islet damage because of a lackof hAAT protein levels in vivo.