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Corrado Cilio

Professor

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Human enteroviral infection impairs autophagy in clonal INS(832/13) cells and human pancreatic islet cells

Author

  • Anya Wernersson
  • Luis Sarmiento
  • Elaine Cowan
  • Malin Fex
  • Corrado M. Cilio

Summary, in English

Aim/hypothesis: Human enteroviral infections are suggested to be associated with type 1 diabetes. However, the mechanism by which enteroviruses can trigger disease remains unknown. The present study aims to investigate the impact of enterovirus on autophagy, a cellular process that regulates beta cell homeostasis, using the clonal beta cell line INS(832/13) and human islet cells as in vitro models. Methods: INS(832/13) cells and human islet cells were infected with a strain of echovirus 16 (E16), originally isolated from the stool of a child who developed type 1 diabetes-associated autoantibodies. Virus production and release was determined by 50% cell culture infectious dose (CCID50) assay and FACS analysis. The occurrence of autophagy, autophagosomes, lysosomes and autolysosomes was detected by western blot, baculoviral-mediated expression of microtubule-associated protein light chain 3 (LC3)II-GFP and LysoTracker Red, and quantified by Cellomics ArrayScan. Autophagy was also monitored with a Cyto-ID detection kit. Nutrient deprivation (low glucose [2.8 mmol/l]), amino acid starvation (Earle’s Balanced Salt Solution [EBSS]) and autophagy-modifying agents (rapamycin and chloroquine) were used in control experiments. Insulin secretion and the expression of autophagy-related (Atg) genes and genes involved in autophagosome–lysosome fusion were determined. Results: E16-infected INS(832/13) cells displayed an accumulation of autophagosomes, compared with non-treated (NT) cells (grown in complete RPMI1640 containing 11.1 mmol/l glucose) (32.1 ± 1.7 vs 21.0 ± 1.2 μm2/cell; p = 0.05). This was accompanied by increased LC3II ratio both in E16-infected cells grown in low glucose (LG) (2.8 mmol/l) (0.42 ± 0.03 vs 0.11 ± 0.04 (arbitrary units [a.u.]); p < 0.0001) and grown in media containing 11.1 mmol/l glucose (0.37 ± 0.016 vs 0.05 ± 0.02 (a.u.); p < 0.0001). Additionally, p62 accumulated in cells after E16 infection when grown in LG (1.23 ± 0.31 vs 0.36 ± 0.12 (a.u.); p = 0.012) and grown in media containing 11.1 mmol/l glucose (1.79 ± 0.39 vs 0.66 ± 0.15 (a.u.); p = 0.0078). mRNA levels of genes involved in autophagosome formation and autophagosome–lysosome fusion remained unchanged in E16-infected cells, except Atg7, which was significantly increased when autophagy was induced by E16 infection, in combination with LG (1.48 ± 0.08-fold; p = 0.02) and at 11.1 mmol/l glucose (1.26 ± 0.2-fold; p = 0.001), compared with NT controls. Moreover, autophagosomes accumulated in E16-infected cells to the same extent as when cells were treated with the lysosomal inhibitor, chloroquine, clearly indicating that autophagosome turnover was blocked. Upon infection, there was an increased viral titre in the cell culture supernatant and a marked reduction in glucose-stimulated insulin secretion (112.9 ± 24.4 vs 209.8 ± 24.4 ng [mg protein]–1 h–1; p = 0.006), compared with uninfected controls, but cellular viability remained unaffected. Importantly, and in agreement with the observations for INS(832/13) cells, E16 infection impaired autophagic flux in primary human islet cells (46.5 ± 1.6 vs 34.4 ± 2.1 μm2/cell; p = 0.01). Conclusions/interpretation: Enteroviruses disrupt beta cell autophagy by impairing the later stages of the autophagic pathway, without influencing expression of key genes involved in core autophagy machinery. This results in increased viral replication, non-lytic viral spread and accumulation of autophagic structures, all of which may contribute to beta cell demise and type 1 diabetes. [Figure not available: see fulltext.]

Department/s

  • Diabetes - Immunovirology
  • EXODIAB: Excellence in Diabetes Research in Sweden
  • Diabetes - Molecular Metabolism
  • EpiHealth: Epidemiology for Health

Publishing year

2020-11

Language

English

Pages

2372-2384

Publication/Series

Diabetologia

Volume

63

Issue

11

Document type

Journal article

Publisher

Springer

Topic

  • Endocrinology and Diabetes

Keywords

  • Autophagy
  • Beta cells
  • Enterovirus
  • Insulin secretion
  • Lysosomes
  • Type 1 diabetes
  • Viral replication
  • Virus spread

Status

Published

Research group

  • Diabetes - Immunovirology
  • Diabetes - Molecular Metabolism

ISBN/ISSN/Other

  • ISSN: 0012-186X