Expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of Echovirus: implication for post-virus islet autoimmunity

Abstract

Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

Figure 1. Virus-induced cytopathic effect in primary human pancreatic islets cells.

A. Uninfected islet. B. Islets infected with the E4 isolate 5 days post infection. C. Islets infected with E16 isolates 3 days post infection. D. Islets infected with E30 isolates 3 days post infection. The figure is representative of seven islet donors.

Figure 2. Viral titers of the clinical strains of E16, E30 strains and E4 in the culture medium of infected primary human islets during 3 days post-infection.

Aliquots of the culture medium were withdrawn day 0 and day 3. Virus titers were obtained using the cell culture infectious dose 50 (CCID50) titration methods. The results are shown as the means ± SD from experiments performed in triplicate.

Figure 3. Dynamic release of insulin in primary human islet cells after perifusion with glucose (1.67, 16.7, and 1.67 mmol/L) at three days after infections with clinical strains of E4, E16 and E30.

Data are presented as means ± SD and were based on observations from at least three donors. *P<0.05 between groups (minutes 42 to 102); Kruskal-Wallis test.

Figure 4. Innate immunity gene expression in primary human islets cultured after three days of infection with clinical strains of E4, E16 and E30.

Gene expression levels are presented as mRNA expression relative to expression of the housekeeping gene 18(2^_dct). Data are presented as means ± SD and were based on observations from at least three donors. ^† P<0.05, between all groups (Islets infected with E4, E16, E30 and uninfected controls). *P<0.05, Islets infected with E16 and E30 were compared to E4-inoculated islets and the uninfected control.

Figure 5. Dendrograms showing phylogenetic relationships between sequenced E30 isolated during the Cuban epidemic of aseptic meningitis in 2001 and the E30 isolate of the GenBank sequence database.

The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Strains of E30 known to be highly destructive of primary human insulin producing beta cells are shown in the tree by small black triangles.