Induction of an antiviral state and attenuated coxsackievirus replication in type III interferon-treated primary human pancreatic islets

Abstract

Type III interferons (IFNs), also called lambda interferons (IFN-λ), comprise three isoforms, IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B). Only limited information is available on their expression and biological functions in humans. Type I and type II IFNs protect human pancreatic islets against coxsackievirus infection, and this is important since such viruses have been proposed to play a role in the development of human type 1 diabetes. Here we investigated whether type III IFN is expressed during infection of human islet cells with coxsackievirus and if type III IFN regulates permissiveness to such infections. We show that human islets respond to a coxsackievirus serotype B3 (CVB3) infection by inducing the expression of type III IFNs. We also demonstrate that islet endocrine cells from nondiabetic individuals express the type III IFN receptor subunits IFN-λR1 and IL-10R2. Pancreatic alpha cells express both receptor subunits, while pancreatic beta cells express only IL-10R2. Type III IFN stimulation elicited a biological response in human islets as indicated by the upregulated expression of antiviral genes as well as pattern recognition receptors. We also show that type III IFN significantly reduces CVB3 replication. Our studies reveal that type III IFNs are expressed during CVB3 infection and that the expression of the type III IFN receptor by the human pancreatic islet allows this group of IFNs to regulate the islets' permissiveness to infection. Our novel observations suggest that type III IFNs may regulate viral replication and thereby contribute to reduced tissue damage and promote islet cell survival during coxsackievirus infection.

Fig 1

Human pancreatic islets express type III IFN mRNA upon infection with CVB3. (A) Human pancreatic islets from nine donors were infected with CVB3 (4 × 10⁴ PFU/islet), and the expression of IFN-λ1 and IFN-λ2 mRNA was measured at 24 and 48 h p.i. using real-time RT-PCR. The IFN-λ mRNA expression levels are presented relative to that of GAPDH. The expression of IFN-λ1 and IFN-λ2 mRNAs was below the detection limit in uninfected control islets (C_T values of ≥35 [data not shown]). (B) Titers (PFU/ml) of infectious virus particles were measured in culture supernatants harvested from infected islets from the same donors at 24 h and 48 h p.i. using the plaque assay technique. Data are presented as log₁₀(PFU/ml), mean ± SD. **, P < 0.01, 24 h versus 48 h for both panels A and B, Wilcoxon matched pairs test.

Fig 2

Human pancreatic islets express the type III IFN receptor. (A) mRNA expression levels of type III IFN receptor subunits IFN-λR1 and IL-10R2 were quantified in human pancreatic islet cells from seven donors using real-time RT-PCR. The expression levels were compared with those observed in two hepatic cell lines and four human PBMC donors. The receptor mRNA expression levels are presented relative to that of GAPDH. Data are presented as means ± SDs for islets and PBMCs and as means from three independent cell preparations for the cell lines. (B to M) Expression of each component of the type III IFN receptor (IFN-λR1 and IL-10R2) was assessed in parallel with islet hormone immunostaining in pancreas sections from five nondiabetic individuals, to determine their respective cellular localization. Representative images from a single pancreas section are shown, and arrows are used to illustrate relevant cells in each panel. (B to D) Sections were stained with anti-insulin (B), anti-IFN-λR1 (C), and both anti-insulin and anti-IFN-λR1 (merged image) (D). (E to G) Sections were stained with anti-insulin (E), anti-IL-10R2 (F), and both anti-insulin and anti-IL-10R2 (merged image) (G). (H to J) Sections were stained with antiglucagon (H), anti-IFN-λR1 (I), and both antiglucagon and anti-IFN-λR1 (merged image) (J). (K to M) Sections were stained with antiglucagon (K), anti-IL-10R2 (L), and both antiglucagon and anti-IL-10R2 (merged image) (M).

Fig 3

Expression profile of ISGs after type III IFN treatment. (A) Human pancreatic islets from three donors were stimulated with IFN-λ1 (100 ng/ml), IFN-λ2 (100 ng/ml), or IFN-α (1,000 U/ml) or mock treated with buffer alone for 6 h; thereafter, mRNA expression of 86 ISGs was assessed by real-time RT-PCR. The mRNA expression levels were normalized to the expression level of GAPDH (individual genes; RIG-I and TLR3) or five housekeeping genes (genes analyzed using PCR array) and calculated relative to untreated control islets from the same donor. For each gene, a 2-fold-or-higher-increased expression in at least two out of the three donors was used as a criterion to classify the gene as being increasingly expressed following IFN stimulation. For each gene, the mean of the fold induction values for the three donors ± SD is shown as log₁₀(fold induction). (B) Venn diagram of genes demonstrating increased expression after IFN treatment, showing the numbers of overlapping and individually expressed genes upon IFN-λ1, IFN-λ2, or IFN-α treatment.

Fig 4

Type III IFN-treated human pancreatic islets upregulate the expression of proteins involved in antiviral defense and virus recognition. Human pancreatic islets from two donors were treated with IFN-λ1 (100 ng/ml), IFN-λ2 (100 ng/ml), or IFN-α (1,000 U/ml) for 24 h, and the protein expression levels of MDA5, MxA, and RIG-I were measured using Western blotting. Actin was used as a loading control. One representative experiment out of two is shown.

Fig 5

Type III IFN inhibits CVB3 protein expression and replication. (A) Human islets from five donors were treated with IFN-λ1 (100 ng/ml, n = 5), IFN-λ2 (100 ng/ml, n = 5), or IFN-α (1,000 U/ml, n = 4) for 24 h prior to infection with CVB3 (4 × 10⁴ PFU/islet). Total protein was isolated at 48 h p.i. Five micrograms of protein from each sample was added to SDS-polyacrylamide gels, and the expression of VP-1 was measured using Western blotting. Actin was used as a loading control. One representative experiment out of five is shown. (B) Supernatant was collected at 0, 24, and 48 h p.i., from experiments described for panel A, and viral titers (PFU/ml) were determined using plaque assay. Data are presented as log₁₀(PFU/ml), means ± SDs of five independent experiments; ***, P < 0.001, versus untreated infected islets, one-way ANOVA with Bonferroni correction.