Fig 1: Immunoresponsiveness of cell types selected as highly susceptible to HEV infection.(A, B, C) Quantification of transcript expression levels of representatives of the IFN-I/III–related pathway, that is, MXA and ISG15, NF-KB–induced signaling, that is, TNF and IL-6, upon stimulation by agonists of TLR3, that is, addition of polyI:C; RIG-I/MDA-5 cytosolic sensors by transfection of polyI:C LMW and addition of recombinant IFN-β, IFN-λ3, and TNF at the indicated concentrations for 6-hour incubation, determined by RT–qPCR. (A, B, C) Analyses were performed in Huh-7.5 cells (A), HepG2/C3A cells (B), and PLC3 cells (C); bars represent fold expression; means ± SD; each dot represents one independent experiment, n = 4 for treatments including polyI:C addition/transfection and IFN-β treatment, n = 3 for treatment with IFN-λ and TNF; statistical analysis was performed using the paired pairwise Wilcoxon test; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
Fig 2: pDC response against HEV-infected cells.(A, B) Quantification of the transcript expression levels of representatives of IFN-I/III signaling (i.e., MXA, ISG15, OAS2, and IFN-λ1) and NF-KB–related pathway (i.e., TNF and IL-6) determined at 6 d.p.e. of HepG2/C3A (A) and PLC3 (B) cells that were electroporated with 10 μg RNA [HEV cells] or mock-electroporated without RNA [cont cells], in the absence (left panels) or in co-culture with pDCs for 18 h (right panels), as indicated. No pDC and pDC co-culture conditions have been separated into left and right panels as steady-state levels of gene expression are different in these two datasets, and therefore, cross-comparisons between the two panels must be avoided. Bars represent copy number per μg total RNA; means ± SD; each dot represents one independent experiment (n = 3–8). Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (C, D) pDCs were co-cultured with HEV-electroporated PLC3 or HepG2/C3A cells for 18 and 48 h. (C, D) Quantification of IL-29/28A/28B in supernatants of pDCs co-cultured with HEV-replicating HepG2/C3A (C) and PLC3 cells (D); n ≥ 3; statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005. (E, F, G) pDCs were co-cultured with HEV-electroporated PLC3 cells, and their supernatants, in various settings, or treated by inhibitors, as indicated, for 18 h. (E) Quantification of IFN-α in supernatants of pDCs co-cultured with HEV-replicating PLC3 cells [HEV cells] or treated with supernatants from HEV-infected cells [HEV SN] versus in the absence of pDCs [no pDC]; the uninfected [cont] cells were electroporated without HEV RNA and used as a negative control. Bars represent means ± SD, and each dot represents one independent experiment (n = 4). (F) Quantification of IFN-α in supernatants of pDCs in co-culture or separated from HEV-electroporated PLC3 cells by a permeable membrane (0.4 μm) of transwell [transwell setting]. The TLR7 agonist, imiquimod [IMQ], was used as a positive control. (E) Results presented as in (E); n = 4. (G) Co-culture of pDCs and HEV-electroporated PLC3 cells was treated by inhibitors of TLR7 [IRS661], blocking antibodies against αLβ2-integrin and ICAM-1, followed by the quantification of IFN-α in supernatants of the co-cultures. (E) Results presented as in (E); n > 3 independent experiments. Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
Fig 3: Differences in HEV-induced immune signaling among cell types.(A, B) Quantification of the transcript expression levels of TNF determined at 6 d.p.e. in HepG2/C3A (A) and PLC3 (B) cells. Bars represent the copy number per μg total RNA; means ± SD; each dot represents one independent experiment (n = 3–5). Statistical analysis was done using the Wilcoxon rank-sum test with continuity correction; P-values: * ≤ 0.05, ** ≤ 0.005, and *** ≤ 0.0005.
from Cell Signaling Technology for Human TNF-alpha Recombinant Protein