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Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus–infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1. Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Several genetic variants in the human IFNL3/IFNL4 locus have been associated with reduced hepatic fibrosis despite poor clearance of HCV infection. We expanded this analysis to cirrhosis, a more advanced stage of fibrosis. In 2931 individuals with chronic HCV, the IFNL4 genotype that generates IFN-λ4 was associated with protection from cirrhosis (OR=0.65, p=0.012, adjusted for age and sex). The IFNL4 genotype affects the production of IFN-λ4 and, additionally, may affect expression levels of IFN-λ3, making it difficult to delineate the individual contribution of these IFNs. To address this, we established HepG2-based cell models engineered to inducibly express either IFN-λ3 or IFN-λ4. Using RNA-seq based expression profile generated in these hepatic cell lines, we explored the global transcriptome of liver tumors (n=373) from The Cancer Genome Atlas to identify transcription factor networks affected by these IFNs in the liver. Several networks, including IRF1, were upregulated by IFN-λ4 more strongly than by IFN-λ3. Intracellular expression of IFN-λ4 but not of IFN-λ3 also led to potent IRF1-dependent antiproliferative effects. Live cell imaging revealed that IFN-λ4 was poorly secreted, mainly accumulated in lysosomes, and caused apoptosis, suggesting increased ER-stress via the misfolded protein response. Knockdown of DNA damage-inducible transcript 3 (DDIT3), an ER-stress response effector, significantly attenuated the antiproliferative effects of IFN-λ4. This novel interplay of enhanced IRF1 signaling coupled with intracellular accumulation and induction of ER stress by IFN-λ4 may have complex consequences on liver homeostasis during chronic HCV infection but also mediate anti-cirrhotic phenotypes.

IFN-λ4 is a type-III interferon with strong clinical significance in humans. Only individuals who carry the ΔG allele of a genetic variant rs368234815-TT/ΔG are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear hepatitis C virus (HCV) infection, however the mechanisms are not fully understood. Here, we used RNA-seq to evaluate the effects of IFN-λ4 and IFN-λ3 expression in inducible hepatic cell lines (HepG2) with and without the receptor (IFNLR) required for signaling of both interferons. Pathway analysis showed that despite being poorly secreted, IFN-λ4 expression induced a stronger ISG response compared to IFN-λ3. IFN-λ4 expression was also associated with reduced proliferation and increased expression of genes involved in the unfolded protein response (UPR). Subcellular localization analysis revealed that IFN-λ4 was accumulated in the Golgi and lysosomes, suggesting it is misfolded and targeted for elimination. Furthermore, intrinsic but not extrinsic expression of IFN-λ4 inhibited cell proliferation via cell cycle arrest leading to apoptosis; both indicative of UPR outcomes. In conclusion, our results show that in addition to inducing ISGs, IFN-λ4 expression induces UPR, contributing to inhibition of cell proliferation. This could have important consequences for conditions such as HCV and fibrosis, where IFN-λ4 expression has been implicated.

HCV infection and cirrhosis are among the strongest risk factors for developing liver cancer. Impaired HCV clearance is strongly associated with a dG allele of a genetic variant, rs368234815-TT/dG; this allele creates an open reading frame for interferon lambda 4 (IFNL4). The dG allele has also been linked to reduced risk of cirrhosis. Despite strong association of HCV and cirrhosis with liver cancer, no link between rs368234815-TT/dG and liver cancer has been reported. To evaluate potential association of rs368234815-TT/dG with liver cancer, we explored resources of The Cancer Genome Atlas (TCGA). We used a genetic variant rs12980275-A/G located downstream of IFNL3 gene as a TCGA-genotyped proxy for rs368234815-TT/dG. Using multivariate statistical models, we observed that liver cancer patients homozygous for rs12980275-G allele (corresponds to rs368234815-dG allele) had less cirrhosis but reduced survival. Tumor transcriptome analysis showed association of rs12980275-G allele with immunosuppressed tumor microenvironment in non-cirrhotic patients. By deconvoluting tumor infiltrating immune cell signatures, we observed lower levels of CD8+ and follicular helper T cells, but increased M1 macrophages in non-cirrhotic patients with rs12980275-G allele. Transcriptome analysis also identified metallothioneins (MTs), which are involved in removal of reactive oxygen species in the liver, to be differentially expressed according to rs12980275 genotypes. Using hepatoma cell lines, we show that MTs are induced by IFNL4. Our results suggest an association of IFNL3/IFNL4 locus with both an immunosuppressed tumor microenvironment in liver cancer, and increased expression of MTs which could contribute to reduced fibrosis.

The BCR serves as both signal transducer and Ag transporter. Binding of Ags to the BCR induces signaling cascades and Ag processing and presentation, two essential cellular events for B cell activation. BCR-initiated signaling increases BCR-mediated Ag-processing efficiency by increasing the rate and specificity of Ag transport. Previous studies showed a critical role for the actin cytoskeleton in these two processes. In this study, we found that actin-binding protein 1 (Abp1/HIP-55/SH3P7) functioned as an actin-binding adaptor protein, coupling BCR signaling and Ag-processing pathways with the actin cytoskeleton. Gene knockout of Abp1 and overexpression of the Src homology 3 domain of Abp1 inhibited BCR-mediated Ag internalization, consequently reducing the rate of Ag transport to processing compartments and the efficiency of BCR-mediated Ag processing and presentation. BCR activation induced tyrosine phosphorylation of Abp1 and translocation of both Abp1 and dynamin 2 from the cytoplasm to plasma membrane, where they colocalized with the BCR and cortical F-actin. Mutations of the two tyrosine phosphorylation sites of Abp1 and depolymerization of the actin cytoskeleton interfered with BCR-induced Abp1 recruitment to the plasma membrane. The inhibitory effect of a dynamin proline-rich domain deletion mutant on the recruitment of Abp1 to the plasma membrane, coimmunoprecipitation of dynamin with Abp1, and coprecipitation of Abp1 with GST fusion of the dyanmin proline-rich domain demonstrate the interaction of Abp1 with dynamin 2. These results demonstrate that the BCR regulates the function of Abp1 by inducing Abp1 phosphorylation and actin cytoskeleton rearrangement, and that Abp1 facilitates BCR-mediated Ag processing by simultaneously interacting with dynamin and the actin cytoskeleton.

IFN-λ4, a recently discovered type-III interferon (IFN), is genetically regulated, and has clinical significance because of its association with impaired clearance of hepatitis C virus (HCV) infection. Type-III IFNs play a critical role in the innate immune response to viral infections. Although IFN-λ4 induces a similar antiviral profile as other type-III IFNs, its specific contribution to the innate immune response is unclear. Delineating the functional properties of IFN-λ4 that distinguish it from other type-III IFNs may give insights into its unique role during HCV, and other infections. Here we compared antiviral responses induced by all four type-III IFNs in HepG2 cells. We show that IFN-λ4 attained 50% peak antiviral activity by 4 hours while other type-III IFNs were only at 20% of peak activity at this time. The rapid antiviral response induced by IFN-λ4 was followed by a sharp decline to less than 25% of peak activity within 24 hours, while activity of other type-III IFNs were still over 60% from peak. By comparing antiviral activity of IFN-λ4 and IFN-λ3 against Sendai virus infection in primary human hepatocytes, we show that IFN-λ4 had stronger antiviral activity than IFN-λ3 early in the course of infection, but comparable activity at later time points. We also show that despite its poor secretion, IFN-λ4 was significantly more potent than IFN-λ3 in inducible cell lines engineered to express either protein. In summary, we show that IFN-λ4 is more active during the early stages of viral infection compared to other type-III IFNs, which maybe a clinically important window for restricting viral infection in the host, and affecting disease outcome.