Defective proinsulin handling modulates the MHC I bound peptidome and activates the inflammasome in β-cells

Abstract

ABSTRACTBackgroundHow immune-tolerance is lost to pancreatic β-cell peptides triggering autoimmune type 1 diabetes is enigmatic. We have shown that loss of the proinsulin ER chaperone glucose-regulated protein (GRP) 94 leads to mishandling of proinsulin, ER stress and activation of the inducible proteasome. We hypothesize that inadequate ER proinsulin folding capacity relative to biosynthetic need may lead to an altered β-cell MHC-I bound peptidome and inflammasome activation, sensitizing β-cells to immune attack.MethodsWe used INS-1E cells with or without GRP94 knockout (KO), or in the presence or absence of GRP94 inhibitor PU-WS13 (GRP94i, 20µM), or exposed to proinflammatory cytokines interleukin (IL)-1β or IFNγ (15 pg/ml and 10 ng/ml, respectively) for 24 hours. RT1.A (rat MHC I) expression was evaluated using flow cytometry. The total RT1.A-bound peptidome analysis was performed on cell lysates fractionated by reverse phase high performance liquid chromatography (RP-HPLC) followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NALP1, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and (pro) IL-1β expression and secretion were investigated by Western blotting.ResultsGRP94 KO increased RT1.A expression in β-cells as did cytokine exposure compared to relevant controls. Immunopeptidome analysis showed increased RT1.A-bound peptide repertoire in GRP94 KO/i cells as well as in the cells exposed to cytokines. The GRP94 KO/cytokine exposure groups showed partial overlap in their peptide repertoire. Notably, proinsulin-derived peptides diversity increased among the total RT1.A peptidome in GRP94 KO/i along with cytokines exposure. NALP1 expression was upregulated in GRP94 deficient cells along with decreased IκBα content while proIL-1β cellular levels declined, coupled with an increased secretion of mature IL-1β. Our results suggest that limiting β-cell proinsulin chaperoning enhances RT1.A expression, alters the MHC-I peptidome including proinsulin peptides and activates inflammatory pathways, suggesting that stress impeding proinsulin handling may sensitize β-cells to immune-attack.