Extracellular vesicles from cytokine-treated human pancreatic ductal cells enhance HLA class I expression in beta cells

Abstract

Introduction/objective Type 1 diabetes (T1D) is an autoimmune disease characterized by the loss of insulin-producing beta cells and has no cure. The role of cell-cell interactions in T1D disease progression remains poorly understood. Beta cells develop adjacent to ductal cells and both cell types undergo changes during T1D progression, suggesting a potential intercellular communication. Extracellular vesicles (EVs) are established mediators of intercellular communication, but whether human ductal cells secrete EVs that influence beta cells remains unknown. We hypothesize that exposure of human ductal cells to T1D-associated cytokines alters their EV cargo and that EVs produced by cytokine-exposed human ductal cells modulate gene and protein expression in beta cells. Methods Human exocrine tissues from non-diabetic cadaveric donors were cultured in a 3D suspension to support ductal cell survival. Cells were treated with TNF-α, IL-1β, and IFN-γ or vehicle control for 48 hours and analyzed for proinflammatory-related gene expression and viability by qRT-PCR and Trypan blue exclusion, respectively. EVs from cytokine- and vehicle-treated ductal cells were isolated using size exclusion chromatography and characterized by dot blot, transmission electron microscopy, and nanoparticle tracking analysis. The effects of EVs on EndoC-βH1 human beta cells were assessed with qRT-PCR, RNA-sequencing, and flow cytometry; the effects of EVs on primary human beta cells were assessed with immunofluorescence microscopy. EV cargo was analyzed by proteomics and transcriptomics. Results EVs from cytokine-treated ductal cells were internalized by EndoC-βH1 beta cells, and compared to control ductal EVs, increased HLA Class I gene and protein expression in EndoC-βH1 cells. In primary human beta cells, EVs from cytokine-treated ductal cells, compared to control ductal EVs, also increased expression of HLA Class I. EV cargo from cytokine-treated versus control ductal cells was enriched for transcripts associated with inflammatory and antigen-presentation-related pathways, including CXCL9, CXCL10, CXCL11, IDO1, and GBP4. Conclusion Exposure of T1D-associated cytokines to primary human ductal cells altered cargo of secreted EVs. EVs from cytokine-treated ductal cells upregulated HLA Class I expression in EndoC-βH1 and primary human beta cells. Our results suggest a previously unknown ductal cell communication pathway with a potential implication for modulating beta cells during T1D progression.