Non-conventional interferons : characterization of interferon epsilon (IFN-ε) and specificities of the interferon lambda (IFN-λ) response in the liver

Interferons (IFNs) are a family of class II cytokines playing a role of mediators against pathogens in vertebrates. They have antiviral activity, as well as antiproliferative and immunomodulatory activities. IFNs are grouped into three classes and called type I, II and III IFNs, according to their functions, amino acid sequences, and receptor usage. There is a great multiplicity among IFN genes, and the objective of this thesis was to better understand why such a multiplicity exists. Among type I IFNs, IFN-α and IFN-β are the most well-known and act both locally and systemically to control viral infection. However, other type I IFNs are less characterized and appear to be devoted to the protection of specific tissues or cells. This is the case for IFN-ε. The first part of this work was based on an observation made by Caroline Sommereyns in our laboratory who noticed that supernatants from cells transfected with a pcDNA3-IFN-ε plasmid had no antiviral activity. We analyzed IFN-ε expression in vivo and noticed its constitutive expression in mouse reproductive organs. Surprisingly, IFN-ε expression was not induced upon viral infection. Using plasmid constructs expressing FLAG-tagged IFN-ε and chimeric constructs produced between IFN-ε and limitin (IFN-ζ), a mouse type I IFN, we also noticed that the cleavage of the IFN-ε signal peptide was inefficient in various cell lines, and that both the signal peptide and the mature moiety of IFN-ε contributed to this poor processing. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cell lines and fibroblasts allowed us to highlight a probable defect in the progression of the cytokine through the secretory pathway. These observations led us to the hypothesis that IFN-ε secretion is tightly regulated and that its secretion may require a co-factor specifically expressed in cells of the reproductive organs, securing the system against secretion of this IFN from other cells. The second part of this work focuses on type III IFNs, which were discovered only 10 years ago. This group of cytokines comprises four subtypes: IFN-λ1 to -λ4. Type III IFNs signal through a heterodimeric receptor composed of two chains: IFNλR1, which is specific to IFN-λ, and IL-10R2, which is shared by other IL-10 related cytokines. Unlike the type I IFN receptor, which is expressed ubiquitously, IFNλR1 is preferentially expressed by epithelial cells. Recently, IFN-λ1 entered phase 3 clinical trials as a candidate drug against hepatitis C virus (HCV) infection. Because of its epithelial specificity, IFN-λ is expected to be a good alternative to type I IFNs for the treatment of some viral diseases, as less side effects are expected due to the more restricted range of IFN-λ target cells. Surprisingly, the mouse liver responds poorly to IFN-λ, in spite of the epithelial nature of hepatocytes. Here, we found that, although mouse hepatocytes can respond to IFN-α, they do not respond to IFN-λ. Instead, the response to IFN-λ in mouse liver was restricted to choliangocytes, the epithelial cells forming the bile ducts. Next, we used a model of chimeric mice that were transplanted with human hepatocytes to show that human but not mouse hepatocytes are responsive to IFN-λ under identical experimental conditions in vivo. Finally, we have investigated the polarization of the IFN-λ response in epithelial cells. We found that both the apical and the basolateral poles of epithelial cells were responsive to IFN-λ in vitro, suggesting that the IFN-λ receptor is equally distributed between the apical and basolateral sides of these cells. On the contrary, IFN-α mainly acts on the basolateral side of epithelia. Our observations suggest that IFN-λ may allow a more localized response if secreted in the lumen of some organs.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Interferon-λ in the context of viral infections: Production, response and therapeutic implications

Interferon (IFN)-λ forms the type III IFN family. Although they signal through distinct receptors, type I (IFN-α/β) and type III IFNs elicit remarkably similar responses in cells. However, in vivo, type III and type I IFN responses are not fully redundant as their respective contribution to the antiviral defense highly depends on virus species. IFN-λ is much more potent than IFN-α/β at controlling rotavirus infection. In contrast, clearance of several other viruses, such as influenza virus, mostly depends on IFN-α/β. The IFN-λ receptor was reported to be preferentially expressed on epithelial cells. Cells responsible for IFN-λ production are still poorly characterized but seem to overlap only partly IFN-α/β-producing cells. Accumulating data suggest that epithelial cells are also important IFN-λ producers. Thus, IFN-λ may primarily act as a protection of mucosal entities, such as the lung, skin or digestive tract. Type I and type III IFN signal transduction pathways largely overlap, and cross talk between these IFN systems occurs. Finally, this review addresses the potential benefit of IFN-λ use for therapeutic purposes and summarizes recent results of genome-wide association studies that identified polymorphisms in the region of the IFN-λ3 gene impacting on the outcome of treatments against hepatitis C virus infection. © 2014 S. Karger AG, Basel

Hintner, Michael

(1842 - 1900), Bildhaue

Antiviral Type I and Type III Interferon Responses in the Central Nervous System.

The central nervous system (CNS) harbors highly differentiated cells, such as neurons that are essential to coordinate the functions of complex organisms. This organ is partly protected by the blood-brain barrier (BBB) from toxic substances and pathogens carried in the bloodstream. Yet, neurotropic viruses can reach the CNS either by crossing the BBB after viremia, or by exploiting motile infected cells as Trojan horses, or by using axonal transport. Type I and type III interferons (IFNs) are cytokines that are critical to control early steps of viral infections. Deficiencies in the IFN pathway have been associated with fatal viral encephalitis both in humans and mice. Therefore, the IFN system provides an essential protection of the CNS against viral infections. Yet, basal activity of the IFN system appears to be low within the CNS, likely owing to the toxicity of IFN to this organ. Moreover, after viral infection, neurons and oligodendrocytes were reported to be relatively poor IFN producers and appear to keep some susceptibility to neurotropic viruses, even in the presence of IFN. This review addresses some trends and recent developments concerning the role of type I and type III IFNs in: i) preventing neuroinvasion and infection of CNS cells; ii) the identity of IFN-producing cells in the CNS; iii) the antiviral activity of ISGs; and iv) the activity of viral proteins of neurotropic viruses that target the IFN pathway

Human but not mouse hepatocytes respond to interferon-lambda in vivo

The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-γ) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-γ. In contrast, the IFN-γ response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-γ response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-γ. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-γ response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-γ. The lack of mouse hepatocyte response to IFN-γ was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-γ. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-γ in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-γ in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment. © 2014 Hermant et al

Activity of FLAG-tagged and untagged mouse IFNs.

<p>Histograms show the log₂ of antiviral activities detected in the supernatant of Neuro2A cells collected 24h after transfection of the pcDNA3 derivatives expressing the indicated FLAG-tagged (+) or untagged (-) IFNs or after transfection of the corresponding empty vectors (pcDNA3). Antiviral activities are presented relative to those of culture medium. NS: non significant (Mann–Whitney <i>U</i> test).</p

IFN-ε signal peptide is not fully functional.

<div><p>A. Signal peptides predicted for IFN-ε and limitin. Predicted signal peptides are indicated in bold letters. Related amino acids around the putative cleavage site are framed.</p> <p>B. Western blot analysis of cell lysates from Neuro2A cells that were transfected for 24h with pcDNA3 derivatives expressing FLAG-tagged IFN-ε, IFN-ε(Δsp), lim-ε, ε-lim or limitin. Cells were harvested in laemmli buffer twenty-four hours post-transfection.</p> <p>C. Histograms showing, for the indicated constructs, the proportion of cells where IFN colocalizes mostly with the Golgi or with the endoplasmic reticulum. Means and SD of countings from 4 immunostainings. For each counting, n = ± 200 for IFN-α, lim-ε, limitin and ε-lim; n = 100 for IFN-ε.</p> <p>D. Immunofluorescent detection of FLAG-tagged IFNs in HeLa cells transfected with plasmids expressing the indicated tagged IFNs. IFNs appear in green. The WGA lectin was used to detect glycosylated proteins and to highlight the Golgi network (red).</p></div

Processing of the IFN-ε precursor in transfected cells.

<div><p>Western blot analysis of IFN-α and IFN-ε processing in total protein extracts of Neuro2A cells transfected for 24h with pcDNA3 derivatives expressing FLAG tagged IFNs.</p> <p>A. Mouse IFN-αA and IFN-ε detection in the presence or absence of brefeldin A. Arrowheads point to the two bands detected for IFN-ε.β-actin detection was used as loading control. B. Detection of mouse IFN-αA and IFN-ε along with corresponding proteins expressed without signal peptide (Δsp). C. Human IFN-ε and mouse IFN-β detection in 293T cells before and after treatment with N-glycosidase F.</p></div

Plasmid constructs.

<div><p>A. pcDNA3 derivatives expressing FLAG-tagged or untagged IFNs. In these plasmids, IFN coding regions (frames) are cloned downstream of the cytomegalovirus promoter (pCMV). Restriction sites used for cloning IFN reading frames are shown. FLAG tag coding sequences were added downstream of the last IFN codon as indicated in C. IFN-αA(Δsp) and IFN-ε(Δsp) are constructs where the region encoding the signal peptide of the IFN precursor was deleted. lim-ε: chimeric IFN precursor with the signal peptide of limitin and the mature moiety of IFN-ε.ε-lim is the converse chimeric precursor with the signal peptide of IFN-ε and the mature moiety of limitin. Human IFN-ε with or without the signal peptide are indicated hIFN-ε and hIFN-ε(Δsp). Note that the various elements on these graphic representations are not to scale.</p> <p>B. Lentiviral vectors. In these vectors, transcription of the IFN gene is driven by the cytomegalovirus promoter. The IRES sequence from TMEV allows co-expression of the cloned coding sequence with the red fluorescent protein mCherry.</p> <p>C. Sequence of the IFN-FLAG junctions. X represents the last amino acid of IFN. The linker sequence between IFN and FLAG (bold letters) is AAA for ε-lim and limitin and TAA for the other constructs.</p></div

Primers.

a<p>Sequence kindly provided by Professor Stephan Brand, University Hospital Munich-Grosshadern, University of Munich, Germany.</p>b<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087906#pone.0087906-OConnor1" target="_blank">[32]</a>.</p>c<p>(f) forward primer; (r) reverse primer.</p>d<p>For RT-qPCR, annealing reactions were performed at 63°C for influenza A virus and at 60°C for mOASl2, hOAS1, hMxA and β-actin.</p