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IFN-α Induces Heterogenous ROS Production in Human β-Cells

ABSTRACT: Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors, including viral infection. We investigated the impact of interferon alpha (IFN-α), a cytokine produced during the immune response to viral infection or the presence of un-edited endogenous double-stranded RNAs, on human beta cell physiology. Intravital microscopy on transplanted human islets using a beta cell-selective reactive oxygen species (ROS) biosensor (RIP1-GRX1-roGFP2), revealed a subset of human beta cells that acutely produce ROS in response to IFN-α. Comparison to Integrated Islet Distribution Program (IIDP) phenotypic data revealed that while all donors had functional islets, donors with lower BMI and HbA1c had more ROS accumulating cells. In vitro IFN-α treatment of human islets similarly elicited a heterogenous increase in superoxide production that originated in the mitochondria. To determine the unique molecular signature predisposing cells to IFN-α stimulated ROS production, we flow sorted human islets treated with IFN-α. RNA sequencing identified genes involved in inflammatory and immune response in the ROS-producing cells. Importantly, rapid stimulation of key antiviral response genes by IFN-α was dependent on mitochondrial ROS elevation. Comparison with single cell RNA-Seq datasets available through the Human Pancreas Analysis Program (HPAP) showed that genes upregulated in ROS-producing cells are enriched in beta cells from nondiabetic and autoantibody positive donors rather than T1D donors. Combined, these data suggest that IFN-α stimulates mitochondrial ROS production in healthy human beta cells, potentially predicting a more efficient antiviral response if the processes to respond to the ROS elevation are intact.

ORGANISM(S): Homo sapiens

PROVIDER: GSE318506 | GEO | 2026/02/27

REPOSITORIES: GEO

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Project description:Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic β-cells, insulin insufficiency, and hyperglycemia. Receptor interacting protein kinase 1 (RIPK1) is a multifunctional regulator of cell fate with kinase and scaffolding functions. We previously identified RIPKs as regulators of beta-cell cytotoxicity in vitro, and Ripk1 expression is increased in islets from aged non-obese diabetic (NOD) mice and β-cells from T1D donors. We hypothesized that RIPK1 regulates cytokine- and autoimmune-mediated β-cell demise in a mouse model of T1D. Using NIT-1 beta-cells derived from NOD mice, we tested control (CTL) and Ripk1 gene-edited (Ripk1delta, CRISPR/Cas9 KO) beta-cells treated with TNFα+IFNγ or co-cultured with self-reactive splenocytes from diabetic NOD mice. We performed live cell imaging, RNAseq, kinome profiling, and flow cytometry, tested small molecule RIPK1 inhibitors, and evaluated resistance to beta-cell autoimmunity in vivo. We found TNF alpha +IFN gamma increase RIPK1 phosphorylation, caspase 3/7 activity, and cell death in NIT-1 CTL cells. In contrast, cytotoxicity was blocked with small molecule RIPK1 inhibition or in NIT-1 Ripk1delta cells. Co-labeling of caspase 3/7 activation and cell death in single cells revealed protection from caspase-dependent and -independent death in Ripk1Δ cells. RNAseq uncovered differential cell death-, immune-, and identity-related gene expression between genotypes, and kinome profiling identified changes in MAPK, Eph, JAK, and other kinase activity that were associated with protection from cell death. Furthermore, NIT-1 Ripk1delta cells were protected from destruction following co-culture with self-reactive splenocytes in vitro, and NIT-1 implant graft luminescence was 399-fold greater in Ripk1delta versus CTL cells 19 days post splenocyte administration in vivo. Collectively, our findings indicate RIPK1 regulates beta-cell fate in a model of autoimmune diabetes via actions on gene expression and kinase signaling. Additional studies are needed to characterize RIPK1 signaling in primary islets and human beta-cells, and to determine whether RIPK1 can be targeted to protect beta-cells in T1D. In this data set, NIT-1 cells (control and Ripk1 KO), vehicle treated or treated with IFN-gamma and TNF alpha were used for kinome profiling.

Project description:Aims/Hypothesis: Type 1 diabetes (T1D) is an autoimmune disease marked by the destruction of beta cells in the pancreatic islets, with an incomplete picture of disease pathogenesis/progression and a lack of definitive cure. A recent finding linked pancreatic ductal cells of T1D donors with elevated levels of human leukocyte antigen (HLA) Class II molecules; however, the causal relationship and functional significance of this finding remain unknown. Because HLA Class II molecules are typically expressed by professional antigen presenting cells (APCs), this raises the possibility of ductal cells functioning as non-professional APCs. In this study, we test the hypothesis that non-T1D ductal cells are responsive to T1D-associated proinflammatory cytokines, TNF-α, IL-1β, and IFN-γ, and can act as non-professional APCs. Methods: Pancreatic exocrine cells, after the removal of islets, were obtained from cadaveric donors without apparent diseases and with appropriate consent. Cells were cryopreserved, thawed into a defined culture medium tailored to support ductal cell survival in a 3D suspension culture system. Ductal cells were exposed to various doses of cytokines for 48 hours and analyzed for gene and protein expression, using qRT-PCR, bulk-RNAseq, flow cytometry and Western blot analyses. Functional ability of cytokine-treated ductal cells to present an exogenous autoantigen (glutamate decarboxylase 65kD isoform [GAD65]) to T cells was tested using a GAD65-specific autoreactive CD4+ T cell clone (named BRI4.13) isolated from a T1D donor. Results: We found that within 48 hours a combination of TNF-α, IL-1β, and IFN-γ stimulated the expression of HLA Class II, co-stimulatory, and antigen-processing molecules at mRNA and protein levels in non-T1D ductal cells. Bulk RNAseq analysis showed that cytokines most significantly upregulated biological pathways in “antigen processing and presentation” and “T1D”. When cytokine-treated ductal cells were pulsed with an exogenous GAD65 peptide and cocultured with the BRI4.13 T cells, these T cells became activated and proliferated. Unexpectedly, we also found ~1% of KRT19+ ductal cells express GAD protein endogenously. Conclusions/interpretation: These results demonstrate that non-diseased primary ductal cells are sensitive to TNF-α, IL-1β, and IFN-γ stimulation, and can upregulate APC molecules and function in antigen presentation to autoreactive CD4+ T cells. To the best of our knowledge, our results provide the first evidence demonstrating antigen presentation by non-T1D human ductal cells to T cells, which implicate ductal cells in contributing to T1D progression.

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IFN-α Induces Heterogenous ROS Production in Human β-Cells

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