Coxsackievirus-B4 Infection of Human Primary Pancreatic Ductal Cell Cultures Results in Impairment of Differentiation into Insulin-Producing Cells

Abstract

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Coxsackievirus-B4 Infection Can Induce the Expression of Human Endogenous Retrovirus W in Primary Cells

“…Human pancreatic cells were harvested from brain-dead adults in agreement with the French law and ethical committee of our institution. The exocrine fraction was extracted and processed as described previously [29][30][31]. Briefly, pancreatic cells were seeded in six-well plates in Dulbecco's Modified Eagle Medium (DMEM) containing 3 g/L glucose, 10% fetal bovine serum (FBS) (Gibco, Thermofisher Scientific, Courtaboeuf, France), 1% insulin transferrin selenium (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and 1% penicillin/streptomycin (Gibco), as well as 50 µg/mL Geneticin (G418, Sigma-Aldrich) to limit fibroblast overgrowth.…”

Coxsackievirus B Type 4 Infection in β Cells Downregulates the Chaperone Prefoldin URI to Induce a MODY4-like Diabetes via Pdx1 Silencing

“… 43 , 44 , 45 , 46 More important, recent findings suggest that CVB4 infection of human primary pancreatic ductal cell cultures impairs differentiation into insulin-producing cells. 47 We therefore checked for the presence of polyhormonal cells (secreting glucagon and insulin), a phenomenon partly mediated by the loss of PDX1. 43 Significantly, confocal images of Uri1 flox/flox ; Pdx1-cre pancreatic islets showed increased polyhormonal cells ( Figures 3 K and 3L).…”

Persistence of Coxsackievirus B4 in Pancreatic β Cells Disturbs Insulin Maturation, Pattern of Cellular Proteins, and DNA Methylation

“…We previously demonstrated the persistence of CV-B4 in human pancreatic islets [ 28 ] in human primary ductal pancreatic cells [ 38 ] and in a human β cell line (1.1 B4) produced by fusion of human pancreatic β cells with a human pancreatic duct cell line (Panc-1) [ 29 ]. This latter model of β cell does not produce insulin; therefore, it is not suitable for investigating the impact of virus persistence on the production of this hormone.…”

“…In this model of acute infection, a decrease in insulin secretion was demonstrated [ 39 ]. Recently, it was shown that insulin mRNA expression and C-peptide levels were inhibited by persistent CV-B4 E2 infection in human primary pancreatic ductal cells differentiated into ICA in vitro [ 38 ]. Furthermore, Yin et al reported that persistent infection of human pancreatic islets with the strain CV-B4 VD2921 did not affect the proinsulin and insulin content of β cells [ 40 ].…”