Molecular events that render pancreatic beta cells targets of T lymphocytes

© 2012 Dr. Dhana Govind GorasiaType 1 Diabetes (T1D), which affects approximately 140000 people in Australia, is an autoimmune disease that results from specific destruction of insulin-producing pancreatic beta cells. Individuals suffering from T1D have to take exogenous insulin to survive. Studies using a non obese diabetic (NOD) mouse model have revealed that multiple immune cells, such as macrophages, B cells and T lymphocytes infiltrate the islets and destroy beta cells via different effector mechanisms. One group of identified mediators of beta cell death is pro-inflammatory cytokines, such as IFN-γ, TNF-α and IL-1β, which are produced by the infiltrating immune cells. In this study, the effects of a combination of pro-inflammatory cytokine treatments on beta (NIT-1) and alpha (αTC-1) cell lines as well as isolated mouse islets were assessed using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). A total of 58 proteins in NIT-1 cells and 55 in αTC-1 cells were differentially expressed when exposed to different combinations of cytokines. Protein identification by mass spectrometry (MS) revealed that the major difference in the protein expression between cytokine treated NIT-1 and αTC-1 cells were stress induced proteins and free radical scavenging enzymes. Stress induced phosphoprotein 1 and heat shock proteins were observed to be altered in cytokine treated NIT-1 cells and free radical scavenging enzymes such as superoxide dismutase 2 (SOD2) and peroxiredoxin 3 were observed to be upregulated in cytokine treated αTC-1 cells. These data are indicative of NIT-1 cells exhibiting cellular stress upon cytokine treatment, reflecting insufficient defense against oxidative stress. In contrast, αTC-1 cells were able to scavenge free radicals hence did not exhibit cellular stress under the same conditions. To investigate if these pathways were also important in cytokine responses of purified islets, the effect of IFN-γ, IL-1β and TNFα on protein expression of islets was examined. Subcellular fractionation was performed to increase the coverage of the islet proteome. Treatment of islets with cytokines caused an expression change in 254, 236 and 154 proteins in the cytosol, membrane/organelle and nuclear fraction, respectively. Protein identification by MS showed that the expression levels of heat shock proteins were altered and also SOD2 levels were upregulated. Peroxiredoxin levels remained unaltered indicating an ineffective defense response against free radicals and subsequent cellular stress within islets. Proteomics interrogation of cytokine treated beta cells also revealed changes in the levels of Endoplasmic Reticulum (ER) foldases which may affect the folding of proinsulin in the ER. In this study, NIT-1 cells expressing Flag tagged proinsulin were generated in order to determine which foldases interact with proinsulin, and therefore play a role in proinsulin folding. Co-immunoprecipitation studies revealed that BiP, DnajB11 and PDIA6 together with other ER foldases associated with proinsulin. PDIA6 had not previously been associated with disulfide bond formation in proinsulin. The foldases that interact with misfolded proinsulin were also explored. NIT-1 cells expressing Flag tagged proinsulin with the Akita mutation (C96Y) were generated. Co-immunoprecipitation studies revealed that the majority of chaperones that interacted with wild type proinsulin also interacted with misfolded Akita proinsulin except for P58IPK which only interacted with misfolded proinsulin. Interestingly, more PDIA6 was associated with the misfolded form compared to wild type proinsulin. These results indicate a possible involvement of PDIA6 in the reduction of disulfide bonds in misfolded proinsulin, thereby enabling misfolded proinsulin to enter the ER associated degradation (ERAD) pathway. Pro-inflammatory cytokines are also known to cause an upregulation of MHC class I molecules on the beta cell surface. IFN-γ causes an upregulation of tapasin levels, which is a chaperone involved in MHC class I synthesis and is known to play a role in the editing of peptides presented by MHC class I molecules. In this study, the effect of tapasin upregulation on antigen processing and presentation in the absence of cytokines was examined. NIT-1 cells overexpressing tapasin were generated. Tapasin upregulation had no effect on the expression of MHC class I molecules on the cell surface. Peptides presented by MHC class I molecules were analysed in both NIT-1 and NIT-1 cells overexpressing tapasin. No changes were observed in the sequences of peptides presented on MHC class I molecules in the presence of elevated tapasin. Nevertheless, NIT-1 peptidome was established. Thus tapasin on its own was shown not to have a profound effect on the type of antigen presented on MHC class I molecules