Research paper
A novel method for detection of IFN-lambda 3 binding to cells for quantifying IFN-lambda receptor expression

https://doi.org/10.1016/j.jim.2017.03.001Get rights and content

Highlights

  • Novel IFN-λ3 binding assay to quantify IFN-λ receptor expression by flow cytometry.

  • IFN-λ3 binding to the surface of cells can be visualized by imaging flow cytometry.

  • The greater the IFN-λ3 binding, the greater the induction of ISGs.

  • IFN-λ3 gene induction is prolonged compared to IFN-α.

Abstract

Type III interferons (IFN-lambdas) are important antiviral cytokines that also modulate immune responses acting through a unique IFN-λR1/IL-10R2 heterodimeric receptor. Conflicting data has been reported for which cells express the IFN-λR1 subunit and directly respond to IFN-λs. In this study we developed a novel method to measure IFN-λ3 binding to IFN-λR1/IL-10R2 on the surface of cells and relate this to a functional readout of interferon stimulated gene (ISG) activity in various cell lines. We show that Huh7.5 hepatoma cells bind IFN-λ3 at the highest levels with the lowest Kd(app), translating to the highest induction of various ISGs. Raji and Jurkat cell lines, representing B and T cells, respectively, moderately bind IFN-λ3 and have lower ISG responses. U937 cells, representing monocytes, did not bind IFN-λ3 well and therefore, did not have any ISG induction. Importantly, knockdown of IFNLR1 in Huh7.5 cells decreased our binding signal proportionally and reduced ISG induction by up to 93%. IFN-λ3 responsiveness increased over time with maximal ISG responses seen at 24 h for all but one gene. These data confirm our new IFN-λ3 binding assay can be used to quantify IFN-λ receptor surface expression on a variety of cell types and reflects IFN-λ3 responsiveness.

Introduction

Type I and III interferons (IFNs) are induced in host cells in response to a variety of pathogens, and are responsible for the induction of a variety of ISGs leading to inhibition of viral replication and stimulation of the adaptive immune system. The type III IFN family consists of four members: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B) and IFN-λ4 (Kotenko et al., 2003, Sheppard et al., 2003, Prokunina-Olsson et al., 2013, Egli et al., 2014b). Multiple genome wide association studies have demonstrated the importance of the IFNL locus in both IFN-α treatment response and the natural clearance of the hepatitis C virus (HCV) (Ge et al., 2009, Tanaka et al., 2009, Thomas et al., 2009). Type III IFNs have been shown to be important for the defense against other viruses such as rotavirus, norovirus, influenza, herpes simplex virus 2 (HSV-2) and West Nile virus (WNV) (Ank et al., 2006, Ank et al., 2008, Mordstein et al., 2008, Hou et al., 2009, Mordstein et al., 2010, Duong et al., 2014, Lazear et al., 2015, Mahlakoiv et al., 2015, Nice et al., 2015). IFN-λ4 is a unique family member in that it is not expressed in all individuals. The ancestral ΔG allele for single nucleotide polymorphism (SNP) rs368234815 encodes the full length functional IFN-λ4 which correlated with HCV persistence, whereas the TT allele promoted HCV clearance (Prokunina-Olsson et al., 2013).

All type III IFN family members signal through a unique heterodimeric receptor comprised of IFN-λR1 (IL-28RA) and IL-10R2 (Kotenko et al., 2003, Sheppard et al., 2003, Hamming et al., 2013). Unlike the type I IFN receptor (IFNAR1/2) which is ubiquitously expressed, IFN-λR1 is principally expressed on epithelial cells, especially at mucosal surfaces (Meager et al., 2005, Doyle et al., 2006, Sommereyns et al., 2008). Interestingly, gut epithelial cells are unique in that they respond exclusively to type III IFNs (Mordstein et al., 2010, Pott et al., 2011, Mahlakoiv et al., 2015). Specific immune cells also express IFN-λR1. Human plasmacytoid dendritic cells strongly express IFN-λR1 and respond to IFN-λ, but little to no expression of IFN-λR1 has been found on monocytes, natural killer cells and T cells (Ank et al., 2008, Dai et al., 2009, Megjugorac et al., 2009, Witte et al., 2009, Liu et al., 2012, Yin et al., 2012, Dickensheets et al., 2013, O'Connor et al., 2014, de Groen et al., 2015a, Depla et al., 2016, Kelly et al., 2016). IFN-λR1 expression is found however, on monocyte-derived macrophages, leading to IFN-λ1 modulation of toll-like receptor (TLR) responses (Liu et al., 2011). IFNLR1 mRNA is detectable in human B cells, but reports differ on whether B cells directly respond to IFN-λ (Ank et al., 2008, Witte et al., 2009, de Groen et al., 2015b, Kelly et al., 2016). Previously, we demonstrated that IFN-λ3 inhibited B cell antibody production and decreased Th2 responses to H1N1 influenza vaccine antigen (Egli et al., 2014a), but the exact mechanism is still unknown.

Similar to type I IFNs, type III IFNs induce ISGs through activation of JAK1 and TYK2 and phosphorylation of various STAT proteins (Dumoutier et al., 2004, Doyle et al., 2006, Zhou et al., 2007). Microarray studies have focused on hepatocyte cell lines and primary human hepatocytes where it was evident that IFN-λ induced signals were more prolonged compared to IFN-α (Doyle et al., 2006, Marcello et al., 2006, Diegelmann et al., 2010, Thomas et al., 2012, Dickensheets et al., 2013, Bolen et al., 2014, Jilg et al., 2014). One reason for this difference is that ubiquitin-specific peptidase 18 (USP18) creates a negative feedback loop for IFN-α, but not IFN-λ (Sarasin-Filipowicz et al., 2009, Francois-Newton et al., 2011, Makowska et al., 2011).

In this study, we developed a novel assay to measure IFN-λ3 binding to cells using flow cytometry. While many previous studies have measured IFN-λR1 expression by determining mRNA levels, here we demonstrate a direct quantitative approach to detect which cells express the IFN-λ receptor at the cell surface via IFN-λ3 binding. In addition, we investigated how IFN-λ3 binding reflects ISG induction in vitro with four different cell lines including hepatocytes and different immune cells. This new method will be invaluable for identifying cells expressing the IFN-λR and observing how surface the IFN-λR is regulated under varying stimulation conditions.

Section snippets

Cell lines and reagents

Huh 7.5 cells (from Dr. Charles Rice, The Rockefeller University), were cultured in DMEM containing: 10% fetal bovine serum (FBS), 100 U/ml Penicillin, 100 μg/ml Streptomycin, 1 × MEM Non-Essential Amino Acids and 4 mM l-Glutamine (all from Gibco). All other cell lines were cultured in RPMI 1640 containing: 10% FBS, 100 U/ml Penicillin, 100 μg/ml Streptomycin, 10 mM HEPES, 1 × MEM Non-Essential Amino Acids and 2 mM Glutamax (all from Gibco, Thermofisher). All cells were maintained in a humidified

Variable transcript level of IFNLR1 depending on the cell type

One goal of our study was to determine the best method of quantifying IFN-λR1 expression in a variety of cell types. We first compared the relative expression of IFNLR1 mRNA by RT-qPCR. To compare epithelial cells to immune cells we measured IFNLR1 mRNA levels in 4 different human cell lines: Huh7.5 (hepatocellular carcinoma), Raji (Burkitt's lymphoma (B cells)), Jurkat (T cell leukemia) and U937 (monocytes). Huh7.5 cells had the highest relative IFNLR1 (membrane form, mIFNLR1) mRNA expression,

Discussion

In this study we have developed a new assay to quantify IFN-λ3 binding and IFN-λR expression at the cell surface by utilizing a C-terminal his-tagged IFN-λ3. We visualized IFN-λ3 binding to the cell surface via imaging flow cytometry and related the levels of IFN-λ3 binding to the magnitude of the ISG responses in each cell type tested. Lastly, we thoroughly compared the timing of IFN-λ3 responsiveness in multiple cell types confirming that in general IFN-λ3 ISG induction increases over time.

Conflict of interest

Authors have no conflict of interest.

Acknowledgments

We wish to thank Dr. Rob Ingham, Dr. Chris Power and Dr. Charles Rice for generously providing cell lines, Dr. Aja Rieger for assistance with Amnis Imagestream mark II data acquisition and analysis and everyone at the Flow Cytometry core at the University of Alberta. This work has been supported by the Li Ka Shing Institute of Virology, a Canadian Excellence Research Chair (CERC) grant (213965), for which MH is the holder, and a Canadian Institutes of Health Research (CIHR) grant (MOP-126142)

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