Huh-7.5 cells (gift from Apath, LLC) and S29 cells, a sub-clone of Huh7 cells that are 1000-fold less permissive to HCV due to decreased expression of CD81 (Russell et al., 2008; gift from Drs. S. Emerson and R. Purcell, National Institutes of Health, USA) were maintained at 37°C with 5% CO₂ in complete medium (CM) containing Dulbecco’s Modified Eagle Medium (DMEM; with high glucose [4.5 g/L] and pyruvate; ThermoFisher Scientific, 11995073), supplemented with 10% heat-inactivated fetal bovine serum (FBS; ThermoFisher Scientific, 10438034) and 0.5% penicillin/streptomycin (Millipore Sigma, P4333-100ML).

THP-1 (gift from Dr. C. Moore, Memorial University), Ramos (gift from Dr. S. Christian via Dr. C. Moore, Memorial University), and Jurkat cells (gift from Dr. M. Grant, Memorial University) were maintained at 37°C with 5% CO₂ in Immune Cell CM containing Roswell Park Memorial Institute 1640 media (RPMI; ThermoFisher, 11875119), supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, and 1X Antibiotic-Antimycotic Solution (Anti-Anti; 100X, Millipore Sigma, A5955).

A cell culture-adapted strain of HCV, known as JFH1_T (Russell et al., 2008; Jones et al., 2011), was used for this study. To generate virus stocks, 1x10⁶ Huh-7.5 cells were seeded in 10-cm culture dishes. Approximately 24 hours later, cells were inoculated at a multiplicity of infection (MOI) of 1 and incubated for 3 hours, after which inoculum was replaced with fresh CM. Culture fluids were harvested four days post-infection (dpi) and virus titre was determined using a limiting dilution focus-forming assay which has been described previously (Wallace et al., 2022).

Previously synthesized DNA plasmids encoding various RNA constructs were linearized by Xbal restriction digest (Xbal GQ, Promega, R6185) and in vitro transcribed using the T7 RiboMAX™ Express large scale RNA production system (Promega, P1320) according to manufacturer instructions. RNA encoding either JFH1_T or JFH1_T ΔGDD (non-replicating RNA; referred to as ΔGDD) or JFH-sgr (sub-genomic replicon, developed by Lohmann et al., 1999) was transfected into cells using DMRIE-C (Thermofisher, 10459-014) as per manufacturer instructions. The RNA encoding JFH1_T results in production of infectious virions. The ΔGDD JFH1_T RNA harbours a three amino acid deletion in the active site of the NS5B polymerase, rendering it incapable of undergoing viral replication as previously described (Jones et al., 2011). The JFH-sgr RNA encodes only the non-structural proteins of HCV and, therefore, does not generate progeny virions, but does undergo viral RNA replication.

To induce pyroptosis as a positive control in Huh-7.5 or S29 cells, lipopolysaccharide from Escherichia coli 0127:B8 (LPS; in vitro LPS, Millipore-Sigma, L5024-10MG) was added to cells at 5 μg/mL and incubated for 3 hours at 37°C. Following incubation with LPS, Nigericin (sodium salt, InvivoGen, tlrl-nig) was added at a concentration of 16.8 μM and left overnight at 37°C. To induce pyroptosis as a positive control in immune cells, LPS was added to cells at 3 μg/mL and incubated for 3 hours at 37°C. Following incubation, Nigericin was added at a concentration of 11.0 μM and left overnight at 37°C. This treatment was always performed the day prior to staining/cell harvest.

Huh-7.5 and S29 cells were trypsinized, pelleted, and resuspended in PBS. RNA was exacted from approximately 1.5 million cells using the RNeasy Plus Mini Kit (Qiagen, 74134) as per the manufacturer’s instructions.

Specific PCR primers were designed to amplify multiple overlapping fragments covering the entire coding sequence of human RIG-I (Supplementary Table 1). RT-PCR was performed in 25 μL reactions using the OneTaq^® One-step RT-PCR Kit (New England Biolabs, E5315S) as followed: 12.5 μL of 2X OneTaq One-step Reaction Mix, 1 μL of 2X OneTaq One-step Enzyme Mix, 1 μL of each forward and reverse primer (10 μM), 8 μL nuclease-free water, and 1.5 μL of RNA. Cycling parameters for all amplicons were as followed: 48°C for 30 min, 95°C for 5 min, 40 cycles of 94°C for 15 sec, 50°C for 30 sec, and 68°C for 75 sec, followed by a final extension at 68°C for 7 min. PCR products were subjected to electrophoresis for visualization, and amplicons were purified using the Monarch^® Spin PCR & DNA Cleanup Kit (New England Biolabs, T1130L) and subjected to Sanger sequencing at The Hospital for Sick Children (Toronto, Canada). Sequences were mapped to the human RIG-I reference sequence (GenBank: NG_046918.1) and translated into the open reading frame using Geneious 11.1.5 (Dotmatics). The RIG-I complete coding sequences for Huh-7.5 and S29s have been deposited in GenBank under accession numbers PX629822 and PX629823, respectively.

Western blotting was performed as described previously (Wallace et al., 2022). Briefly, cells were seeded in 6-well plates and infected with HCV at MOI = 1 or left uninfected the following day. At 3 dpi, cells were harvested and protein extracted. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham/Cytiva, 10600065). Membranes were incubated with antibodies recognizing RIG-I (Alme-1, Adipogen, AG-20B-0009) and β-Actin (C4, Santa Cruz Biotechnology, Inc., sc-47778). Signal was detected using Cytiva Amersham™ ECL Select™ Western Blotting Detection Reagent (FisherScientific, 45000999).

ELISAs to detect and quantify released, extracellular IL-1 β were performed as per manufacturer’s instructions (Human IL-1 β ELISA Kit, Invitrogen, BMS224-2) using a 4-parameter curve fit. Cell culture fluids were collected at 3 dpi, clarified by centrifugation and frozen at -80°C for storage until analysis. Experiments were performed in triplicate, at least twice, and analyzed using a SpectraMax Mini microplate reader (Molecular Devices).

Statistical analyses for flow cytometry and ELISA data were performed using the Analysis ToolPak in Microsoft Excel (2024). For both flow cytometry and ELISA data, one-way ANOVA was used to compare conditions, where p-values< 0.05 were considered statistically significant. All flow cytometry and ELISA statistical analyses were evaluated using at least three independent experiments or two independent experiments performed in triplicate.

Flow cytometry and ELISA data were visualized using Microsoft Excel (2024). The multiple sequence alignment for RIG-I at residue 55 and surrounding region was visualized in R (v4.5.0; R Core Team, 2021) using ggmsa (v1.16.0; Zhou et al., 2022).

RT-qPCR was performed as per (Oraby et al., 2021). Briefly, Huh-7.5 and S29 cells were transfected with JFH-sgr or left untreated. At one hour, one day, two days, three days, and four days following transfection, cells were harvested and RNA isolated. RNA was subjected to RT-qPCR to detect HCV NS5B RNA.

Previously published work demonstrated that HCV induces pyroptosis of infected and uninfected Huh-7.5 cells, the latter of which was shown to be contact-independent (Kofahi et al., 2016; Wallace et al., 2022). Bystander pyroptosis was visualized (Supplementary Figure 1) and the mechanism(s) responsible for bystander pyroptosis was explored. To determine if entry and/or interactions of the virus with cell-surface receptors was sufficient to trigger pyroptosis in the absence of an infectious virus life cycle, HCV stocks were heat- or UV-inactivated prior to addition to cell cultures. Huh-7.5 cells were infected with HCV, heat-inactivated HCV, UV-inactivated HCV, left uninfected or treated with LPS/Nigericin as a positive control. Cells were then left to grow for 3 or 4 days prior to staining, using a probe for active caspase-1 (indicative of pyroptosis) and antibodies against HCV core protein. Cells were visualized via fluorescence microscopy and increased levels of caspase-1 were observed in cells infected with HCV compared to cells left uninfected (Figure 1A). This is accompanied by many caspase-1 positive cells that did not appear to be infected, indicating bystander pyroptosis. To quantify the percentage of cells that were caspase-1⁺, cells were harvested and stained using the caspase-1 probe and analyzed by flow cytometry. As previously reported (Kofahi et al., 2016; Wallace et al., 2022), HCV infection resulted in significantly more caspase-1⁺ cells when compared to cells that were uninfected at either 3 or 4dpi (29.3% (SEM ±3.23) of HCV-infected cells compared to 13.3% (SEM ±2.53) of uninfected, p<0.005, and 62.3% (SEM ±2.3) compared to 14.9% (SEM ±3.48), p<0.0001, respectively; Figures 1B, C). Neither the heat-inactivated nor UV-inactivated HCV were able to productively infect cells (Figure 1A) or induce caspase-1 levels higher or significantly different from that of uninfected cells (Figures 1A–C). To further confirm these findings, S29 cells were also inoculated with HCV, heat-inactivated HCV, UV-inactivated HCV, left uninfected or treated with LPS/Nigericin as a positive control. S29 cells are a sub-clone of Huh7 cells and are ~1000-fold less permissive to HCV infection due to extremely low levels of CD81 expression (Russell et al., 2008), thereby essentially eliminating the possibility of low-level viral replication having an impact on our results. None of the S29 conditions displayed increased active caspase-1 when compared to uninfected cells except for the cells treated with LPS/Nigericin as a positive control (Figure 1D). Taken together, these results indicate that extracellular interactions of HCV with hepatocyte-like cells are insufficient to trigger pyroptosis. These results also eliminate these interactions as potential mechanisms of bystander pyroptosis induction of uninfected cells.

To determine which step of the viral lifecycle is responsible for pyroptosis induction by HCV, Huh-7.5 cells were infected with HCV, transfected with JFH1_T, ΔGDD, JFH-sgr, mock transfected, or left untreated. Cells were then left to grow for 3 or 4 days prior to staining for active caspase-1 and HCV core protein. As demonstrated previously, HCV infection or JFH1_T RNA transfection resulted in an increase of caspase-1 activation with the majority of the cells infected (Figures 2A;1A, D; Kofahi et al., 2016; Wallace et al., 2022). None of the other conditions were sufficient to induce increased levels of caspase-1 activation (Figure 2A). To further confirm these findings, the experiment was repeated but cells were subjected to flow cytometry to quantify the percentage of caspase-1⁺ cells present in each condition at 3 or 4 dpi. On the given day, cells were collected, stained using the caspase-1 probe, and analyzed by flow cytometry. A significantly higher percentage of cells infected with HCV, 29.28% (SEM ±3.23) and 62.4% (SEM ±2.3), were caspase-1⁺ at both 3 and 4 dpi, respectively, in comparison to uninfected cells, 13.3% (SEM ±2.53) and 14.9% (SEM ±3.48), p<0.005 and p<0.0001 at 3 and 4 dpi, respectively (Figures 2B, C). To further elucidate which step of the HCV life cycle is involved with the triggering of pyroptosis, we performed the same experiments again employing S29 cells to remove multiple rounds of infectious virion production as a factor. As expected, transfection of S29 cells with JFH1_T RNA resulted in increased levels of caspase-1 compared to untreated S29 cells, since S29 cells can produce progeny virions when transfected with the positive-sense HCV RNA but additional cells cannot be infected (Figure 2D). We then compared the percentage of caspase-1⁺ cells by flow cytometry and found that S29 cells stimulated with LPS and Nigericin led to the activation of caspase-1 in a significantly higher proportion of the cells (40.36%, SEM ±3.06) when compared to the untreated S29 cells (12.15%, SEM ±0.75), confirming that S29 cells are capable of classical inflammasome activation (p<0.0001). Unexpectedly, we found significantly increased numbers of caspase-1⁺ S29 cells in cultures inoculated with infectious HCV (17.84%, SEM ±0.64) when compared to the uninfected S29 cells (12.15%, SEM ±0.75; p<0.0005; Figure 2E). However, HCV infections are done using stocks of HCV, meaning harvested cell culture fluids, which by nature, includes any soluble factors released from the cells from which the culture fluids were harvested. Interestingly, we found increased numbers of caspase-1⁺ S29 cells induced by transfection with JFH-sgr (22.85%, SEM ±2.61) compared to untreated S29 cells (12.15%, SEM ±0.75; p<0.005; Figure 2E) although we did not see the same effect when Huh-7.5 cells were used (Figure 2B, C). Together, these data clearly demonstrate that the fully infectious HCV life cycle is necessary for pyroptosis induction in infected Huh-7.5 cells. However, this was not the case for S29 cells, where exposure to HCV or transfection with the JFH-sgr were both sufficient to induce pyroptosis. To determine whether this discrepancy was due to differences in RNA replication between the two cell lines, Huh-7.5 and S29 cells were transfected with the JFH-sgr, RNA collected and subjected to RT-PCR for detection of viral NS5B at one hour, and one, two, three, and four days post-transfection. As the average C_T values differed by less than 3.32, which represent a 1 log change in viral RNA load, levels of viral RNA between Huh-7.5 and S29 across each of the sampled timepoints are functionally equivalent. This indicates there is no difference in RNA production between the two cell lines (Supplementary Table 2). Instead, the differences between the two cell lines are likely due to the fact that S29 cells, like parental Huh7 cells and the human RIG-I reference sequence, encode a threonine at position 55 of RIG-I, allowing for RIG-I to function normally (Figure 2F). This is in contrast to Huh-7.5 cells, which have an isoleucine at position 55, which previously studies have shown results in non-functional RIG-I (Figure 2F; Sumpter et al., 2005). Additionally, Huh-7.5 cells appeared to have less RIG-I expression compared to S29 cells (Figure 2G), further supporting that this difference between the two cell lines may explain the differences in pyroptosis induction.

It has been previously documented that the levels of inflammatory cytokines IL-1 β and IL-18 are significantly elevated in the serum of individuals living with HCV (Falasca et al., 2006; Chattergoon et al., 2011; Veenhuis et al., 2016; Burchill et al., 2017; Welsch et al., 2017; Abe et al., 2020; D’Ambrosio et al., 2021; Montaldo et al., 2021). Given the fact that chronic HCV infection is associated with inflammation, and the understanding that hepatocytes are not known to be particularly inflammatory in nature, we investigated whether bystander pyroptosis would also be observed in immune cells co-cultured with HCV-infected hepatocyte-like cells. Since it is well documented that monocytes and macrophages can undergo pyroptosis accompanied by substantial inflammatory cytokine release, we performed an experiment in which Huh-7.5 cells were co-cultured with THP-1 cells (a monocyte-like cell line that can be differentiated into a macrophage-like phenotype). We aimed to further investigate bystander pyroptosis and to determine whether THP-1 cells are susceptible to bystander pyroptosis. For co-culture infections, we inoculated Huh-7.5 cells with HCV or left them uninfected before adding THP-1 cells. Even though THP-1 cells cannot be infected with our HCV isolate, JFH1_T (Sarhan et al., 2012), our experiments (Figure 2) showed that exposure to HCV can trigger pyroptosis of uninfected cells. Therefore, we included a condition in these experiments whereby we exposed the THP-1 cells to HCV. To visualize bystander pyroptosis of THP-1 cells, we differentiated the THP-1 cells into adherent macrophage-like phenotypes using PMA. Cells were stained for caspase-1 or ASC, HCV core protein, and/or CD11b (as a marker of the THP-1 cells) and nuclei were stained using DAPI (Figures 3A–C). Some cells that are CD11b⁺ (the THP-1 cells) also showed caspase-1 activation or ASC speck formation (Figures 3A–C). We also quantified the amount of caspase-1⁺ cells in both HCV-infected and uninfected mono- and co-cultures of Huh-7.5 and THP-1 cells by flow cytometry. As expected, there was a significantly higher percentage of caspase-1⁺ Huh-7.5 cells in samples that were infected with HCV than there was in uninfected Huh-7.5 cells (17.48% (SEM ±0.69) compared to 8.17% (SEM ±0.43); p<0.0005; Figure 3D). The overall percentage of caspase-1⁺ cells was significantly higher in the co-culture of HCV-infected Huh-7.5 and THP-1 cells than it was in the uninfected co-culture condition (9.57% (SEM ±0.3) compared to 7.07% (SEM ±0.47); p<0.05; Figure 3D). When the co-cultures were separated based on CD14⁺ cells (to examine the THP-1 cells specifically), although the overall percentage was low, there were significantly more caspase-1⁺ THP-1 cells when they were co-cultured with HCV-infected Huh-7.5 cells than when co-cultured with uninfected Huh-7.5 cells (15.33% (SEM ±0.24) compared to 12.93% (SEM ±0.21); p<0.005; Figure 3D). The same experimental conditions were used to quantify IL-1 β secretion by ELISA. These data revealed a significant increase in the amount of IL-1 β secreted by THP-1 cells exposed to HCV compared to unexposed cells (0.57 pmol/mL (SEM ±0.57) compared to 20.88 pmol/mL (SEM ±2.43); p<0.005; Figure 3E). Our findings here confirm earlier results that Huh-7.5 cells do not to secrete IL-1 β (Kofahi et al., 2016), even in the context of pyroptosis induction by LPS/Nigericin (Figure 3E). When THP-1 cells were co-cultured with HCV-infected Huh-7.5 cells, we found a significant increase in the amount of IL-1 β when compared to the uninfected co-culture (16.74 pmol/mL (SEM ±2.16) compared to 0.74 pmol/mL (SEM ±0.2), p<0.005). Since Huh-7.5 cells did not secrete IL-1 β, we can be confident that the changes in the levels of IL-1 β secretion in the co-culture can be attributed to pyroptosis induction in THP-1 cells. Together, these data suggest that THP-1 cells are susceptible to bystander pyroptosis when co-cultured with HCV-infected Huh-7.5 cells.

Given our findings that THP-1 cells undergo pyroptosis when co-cultured with HCV-infected Huh-7.5 cells, we were interested to determine whether other immune cell types were also susceptible to bystander pyroptosis in this context. To investigate this, either Ramos cells (B cell line) or Jurkat cells (T cell line) were co-cultured with HCV-infected or uninfected Huh-7.5 cells and caspase-1⁺ cells were quantified by flow cytometry. Caspase-1⁺ immune cells were quantified by gating on either CD19⁺ or CD3⁺ cells for Ramos or Jurkat cells, respectively. Interestingly, there was an increase in the total percentage of caspase-1⁺ cells, although not significant, when Ramos cells were co-cultured with HCV-infected cells compared to those co-cultured with uninfected Huh-7.5 cells (15.51% (SEM ±3.79) compared to 7.24% (SEM ±1.67), p=0.12; Figure 4A). However, when we focused on only the CD19⁺ cells from the co-culture, we did find a significant increase in the percentage of caspase-1⁺ Ramos cells when they were co-cultured with HCV-infected Huh-7.5 cells compared to the Ramos cells co-cultured with uninfected Huh-7.5 cells (3.75% (SEM ±0.44) compared to 1.07% (SEM ±0.35), p<0.05; Figure 4A). When we performed the same experiment with Jurkat cells, we found no significant differences in the percentage of caspase-1⁺ cells when Jurkat cells were exposed to HCV, when they were co-cultured with uninfected or infected Huh-7.5 cells, or when we measured the percentage of cells that were caspase-1⁺CD3⁺ (Figure 4B). The same experimental conditions were also used to evaluate IL-1 β release by ELISA, which showed no significant increase in secretion of IL-1 β in any of the experimental conditions for either Ramos or Jurkat mono- or co-cultures. We were unable to detect any IL-1 β secretion by Ramos cells, even from those treated with LPS/Nigericin, suggesting that Ramos cells do not produce IL-1 β, or it was produced at such low levels that it was undetectable by the assay. A very low amount of IL-1 β secretion was detected from Jurkat cells, suggesting that they do produce low amounts of IL-1 β. However, these amounts were not significantly different between untreated cells and those stimulated with LPS/Nigericin (2.06 pmol/mL (SEM ±0.6) compared to 2.82 pmoL/mL (SEM ±0.14); p=0.28; Figure 4C). IL-1 β secretion by THP-1 cells (previously included in Figure 3E) is included here for comparison purposes, as THP-1 cells secrete large quantities of IL-1 β, especially when stimulated with LPS/Nigericin. Together, these data suggest that there may be an effect of bystander pyroptosis when Ramos cells are co-cultured with HCV-infected Huh-7.5 cells, but future work should investigate this further in primary immune cells.