IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection

Abstract

Early, vigorous intrahepatic induction of interferon (IFN)-stimulated gene (ISG) induction is a feature of hepatitis C virus (HCV) infection, even though HCV inhibits the induction of type I IFNs in vitro. To identify the cytokines and cells that drive ISG induction and mediate antiviral activity during acute HCV infection, type I and III IFN responses were studied in (1) serial liver biopsies and plasma samples obtained from 6 chimpanzees throughout acute HCV infection and (2) primary human hepatocyte (PHH) cultures upon HCV infection. Type I IFNs were minimally induced at the messenger RNA (mRNA) level in the liver and were undetectable at the protein level in plasma during acute HCV infection of chimpanzees. In contrast, type III IFNs, in particular, interleukin (IL)-29 mRNA and protein, were strongly induced and these levels correlated with ISG expression and viremia. However, there was no association between intrahepatic or peripheral type III IFN levels and the outcome of acute HCV infection. Infection of PHH with HCV recapitulated strong type III and weak type I IFN responses. Supernatants from HCV-infected PHH cultures mediated antiviral activity upon transfer to HCV-replicon-containing cells. This effect was significantly reduced by neutralization of type III IFNs and less by neutralization of type I IFNs. Furthermore, IL-29 production by HCV-infected PHH occurred independently from type I IFN signaling and was not enhanced by the presence of plasmacytoid dendritic cells.

Conclusion: Hepatocyte-derived type III IFNs contribute to ISG induction and antiviral activity, but are not the principal determinant of the outcome of HCV infection.

Fig. 1

ISG induction in the liver during acute HCV infection parallels viremia. (A) Serum HCV RNA titers (filled circles) and ALT levels (gray-colored area) were previously reported^, and are shown for reference purposes. HCV^{+ /−} above each graph indicates qualitative nested RT-PCR (for chimpanzee A3A025) and TMA results (for all other chimpanzees). Presence or absence of viremia was assessed for 77 weeks (not shown) to confirm outcome of infection. (B–D) Intrahepatic mRNA levels of the ISGs IFIT1 (B), MX1 (C), and CXCL11 (D), correlate with serum HCV RNA titers.

Fig. 2

Type I IFNs are minimally, but type III IFNs are robustly induced during acute HCV infection. (A) Serum HCV RNA titers (filled circles) and ALT levels (gray-colored area) were previously reported^, and are shown for reference purposes. (B–D) Intrahepatic type I (B) and III IFN (C) mRNA levels were determined by qPCR, normalized to endogenous references, and presented as fold-increase over preinfection levels. Type III (D), but not type I, IFNs (not shown) were detectable by ELISA in plasma.

Fig. 3

ISG response of PHH to dsRNA is associated with weak type I, but strong type III IFN induction. PHHs were trans-fected with polyI:C to mimic intracellular viral RNA. ISG (A) and IFN mRNA (C) and protein levels (B and D) were determined by qPCR in transfected cells and by ELISA in culture supernatants. mRNA levels were normalized to endogenous references and presented as fold-increase over pretrans-fection levels. Mean and standard error of the mean are shown (A and C).

Fig. 4

ISG response of PHH to HCV infection is associated with weak type I, but strong type III IFN induction. PHHs were infected with HCV at an MOI of 0.4–2.7. ISG mRNA (A) and protein (B) levels as well as IFN mRNA (C) and protein (D) levels are presented as in Fig. 3. (E and F) HCV RNA levels in PHHs were compared to ISG (E) and IFN levels (F). Mean and standard error of the mean are shown (A, C, and E).

Fig. 5

Type III IFNs produced by HCV-infected PHH inhibit HCV replication. (A) Recombinant type I (IFN-αA2 and IFN-β; 500 pg/mL) and type III (IL-28A, IL-28B, and IL-29; 100 ng/mL) IFNs decreased luciferase activity of Huh7-HCV-Luc cells, and this effect was blocked by Ab-mediated neutralization of the respective cytokines. An isotype control Ab did not change RLU values (not shown). (B) Recombinant type I and III IFNs mediated a dose-dependent antiviral effect when added for 30 hours to Huh7-HCV-Luc cells. (C) Supernatant of HCV-infected PHH, harvested 72 hours postinfection and containing an average of 526 pg/ mL of type III IFN proteins, reduced HCV replication of Huh7-HCV-Luc cells by 50%. NAbs against IL28A, IL-28B, and IL-29 (type III IFN) reversed antiviral activity to a significantly greater extent than NAbs against IFN-α and IFN-β (type I IFN; P = 0.0244). Statistical analysis: mean plus standard error of the mean, paired Wilcoxon’s signed-rank test. RLU, relative light units; Sup, supernatant; Nabs, neutralizing Abs.

Fig. 6

IL-29, but not IL-28, is produced in a type I IFN-independent manner in HCV-infected hepatocytes. Treatment of PHH before and during HCV infection with NAb against IFN-α and IFN-β reduced the level of secreted IL-28, but not IL-29, in most PHH cultures. Statistical analysis: paired Wilcoxon’s signed-rank test. Nab, neutralizing Ab.

Fig. 7

Production of IL-29 by PHH is independent of direct contact with pDCs. (A) Experimental strategy: PHHs were infected with HCV for 30 hours, followed by medium change and a 24-hour coculture with or without pDCs or transwells. (B) IFN-α secretion depended on direct contact between pDCs and HCV-infected PHHs. CXCL10 and CXCL11, but not IL-29, secretion was enhanced by PHH/pDC contact. PHH/ pDC coculture in the absence of HCV infection did not induce cytokines (not shown). Mean and standard error of the mean are shown for CXCL10 and CXCL11.