Role of the Mitochondria in Immune-Mediated Apoptotic Death of the Human Pancreatic β Cell Line βLox5

Abstract

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ0 cells. βLox5 ρ0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

Genetics of New-Onset Diabetes after Transplantation

“…63,64,77 In the absence of Fas-associated protein with death domain-like IL-1b-converting enzyme inhibitory protein, Fas receptors induce apoptosis via both caspase-dependent and independent pathways. 69,[77][78][79] Protracted stimulation of IL-1b promotes apoptosis by downregulating other protective proteins within the b cell. 64,80 In addition, both Fas activation and IL-1b induce mitochondrial dysfunction 77 and in vitro studies of b cells in NODAT have demonstrated a role for mitochondrial dysfunction.…”

In search of a surrogate: engineering human beta cell lines for therapy

“…However, these cell lines have proven to be valuable tools for other in vitro applications, especially immunological studies [6]. Specifically, the adult beta cell-derived βLox5 cell line has been essential in uncovering the role of mitochondria in Fas and proinflammatory cytokine-mediated beta cell death [7]. Additionally, βLox5 cells express relevant T1D autoantigens and, similar to primary islets, display upregulation of major histocompatibility complex (MHC) class I on priming with interferons, making them excellent beta cell surrogates in cell-mediated lymphocytotoxicity assays.…”

Comparative Genetics: Synergizing Human and NOD Mouse Studies for Identifying Genetic Causation of Type 1 Diabetes

“…In a population-based study of 385 Japanese patients (154 male and 231 female) with T1D and 469 age-and sex-matched controls, the methionine-containing allele was associated with protection from T1D (p = 0.017) and a significantly reduced frequency (p = 0.0046) of the T1D-associated auto-antibodies (GAD, IA-2, and IAA). To study the impact of mt-ND2 a on human beta-cell destruction, cytoplasmic hybrid (or cybrid) human beta-cells were generated by fusing mitochondrial DNA-depleted human beta-cells with platelets [57,58], because they are anuclear. Platelet donors were chosen based on their mtDNA haplotype [59][60][61].…”

“…To study the impact of mt-ND2 a on human beta-cell destruction, cytoplasmic hybrid (or cybrid) human beta-cells were generated by fusing mitochondrial DNA-depleted human beta-cells with platelets [57,58], because they are anuclear. Platelet donors were chosen based on their mtDNA haplotype [59][60][61]. Individuals from haplogroup D served as donors for the mt-ND2 a -containing human beta-cell lines, while platelets from donors with haplogroup M9 were fused to produce the mt-ND2 c -containing human beta-cells.…”