Inhibition of Type III Interferon Expression in Intestinal Epithelial Cells—A Strategy Used by Coxsackie B Virus to Evade the Host’s Innate Immune Response at the Primary Site of Infection?

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  Figure 1. Intestinal epithelial cells (IECs) express Coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF) and can be infected by Coxsackievirus B3 (CVB3) in a dose-dependent manner. (a) The expression levels of the enterovirus receptors Coxsackie and adenovirus (CAR) and decay-accelerating factor (DAF) were assessed in CaCo-2 and HT-29 IECs by flow cytometry. At least 50,000 cells were screened in each experiment (CaCo-2 n = 7, HT-29 n = 7, HeLa n = 4). Shown are means ± SD. (b) CaCo-2 and (c) HT-29 cells were infected with 10−5 to 10−2 multiplicity of infection (MOI) CVB3 for 24 h and virus replication in the cells and supernatant was measured 24 h p.i. by standard plaque assay. Data are presented as mean plaque forming unites (PFUs) ± SD from three independent experiments per cell line. Significant increases in virus titers were observed in all pair-wise comparisons (p < 0.05, one-way ANOVA).

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  Figure 2. Intestinal epithelial cells (IECs) respond to Type I and III interferons (IFNs) through the up-regulation of interferon stimulated genes (ISGs) at the mRNA and protein level. (a) IFNλ receptor subunits mRNA (IFNLR1/IL-28Rα and IL-10RB) were detected by real-time PCR in IECs and human pancreatic islets (a positive control; n = 2 donors). Data are presented as the means ± SD from three independent experiments per cell line. (b–d) IECs were treated with vehicle, IFNλ1, IFNλ2 (both at concentration of 100 ng/mL), or IFNα (1000 U/mL) for 6 h or 24 h and then RNA (b,c) or protein (d) were extracted. (b,c) The expression of ISGs (protein kinase regulated by dsRNA, PKR; 2′,5′-oligoadenylate synthetase 2, OAS-2; myxovirus resistance protein 1, MXA; interferon-induced 17 kDa protein, ISG15; and inducible nitric oxide synthase, iNOS) were measured by real-time PCR in (b) CaCo-2 and (c) HT-29 cells. Data are presented as the mean ± SD from at least three independent experiments per cell line. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by one-way ANOVA or Student’s t-test. n.d., none detected. (d) Myxovirus resistance protein 1 (MxA) protein levels were examined by Western blot.…

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  Figure 3. Type I and III interferons (IFNs) protect against Coxsackievirus B3 (CVB3) infection in intestinal epithelial cells (IECs). (a) CaCo-2 and (b) HT-29 cells were treated with vehicle, IFNλ1, IFNλ2 (both at concentration of 100 ng/mL), or IFNα (1000 U/mL) for 24 h and were then infected with CVB3 at a multiplicity of infection (MOI) of 10−4 for 24 h. Virus titers in cells and supernatants were measured 24 h p.i. by standard plaque assay. Data are presented as the mean plaque forming units (PFUs) ± SD from at least four independent experiments per cell line. Significant decreases in virus titers were observed in all treatments when compared to the vehicle alone (*** p < 0.001; one-way ANOVA).

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  Figure 4. Polyinosinic:polycytidylic acid (poly (I:C)) transfection, but not Coxsackievirus B3 (CVB3) infection, induces the expression of interferons (IFNs) in intestinal epithelial cells (IECs). (a) The expression of the pattern recognition receptors toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA-5), and retinoic acid-inducible gene 1 (RIG-I) were confirmed in CaCo-2 and HT-29 cells at the mRNA level by real-time PCR. (b) CaCo-2 or (c) HT-29 cells were treated with 10 ng/mL or 30 ng/mL poly (I:C) (i) or transfected with 10 ng/mL poly (I:C) (ii) or infected with 0.01 or 1 plaque forming units (PFU)/mL CVB3 (iii) for 3 h or 6 h. The expression of the type I or III IFNs at the mRNA level was measured by real-time PCR. Data presented as the mean ± SD from at least three independent experiments per cell line (* p < 0.05 and ** p < 0.01; Student’s t-test with Welch’s correction or one-way ANOVA).

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  Figure 5. Coxsackievirus B3 (CVB3) interferes with proteins involved IFN production. (a–c) Intestinal epithelial cells were mock infected (C-0 h) or infected with CVB3 (multiplicity of infection, MOI = 1; V-0 h–V-8 h) and protein was extracted at 0, 2, 4, 6, and 8 h p.i. The presence of eukaryotic initiation factor 4G (eIF4G; a), mitochondrial antiviral signaling protein (MAVS; b) and TIR-domain-containing adaptor-inducing interferon-β (TRIF; c) were detected by Western blot and β-actin was used as a loading control. Representative images from a minimum of two experiments with similar results.