IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo

Abstract

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-alpha/beta (type I IFN) and IFN-lambda (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-alpha/beta and IFN-lambda systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-lambda. In the brain, IFN-alpha/beta was readily produced after infection with various RNA viruses, whereas expression of IFN-lambda was low in this organ. In the liver, virus infection induced the expression of both IFN-alpha/beta and IFN-lambda genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-alpha/beta and IFN-lambda to be compared. The response to IFN-lambda correlated with expression of the alpha subunit of the IFN-lambda receptor (IL-28R alpha). The IFN-lambda response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-lambda in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-alpha/beta was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-lambda system probably evolved to specifically protect epithelia. IFN-lambda might contribute to the prevention of viral invasion through skin and mucosal surfaces.

Figure 1. Relative expression of the various IFN-α subtypes in MHV-A59 infected livers.

IFN-α coding sequences were amplified by RT-PCR using a primer mixture designed to amplify equally the different murine IFN-α subtypes . PCR products were then cloned and individual clones were sequenced. The histogram shows the percentage of sequences from 2 mice (50 and 53 sequences) corresponding to each IFN-α subtype.

Figure 2. Quantification of IFN-λ, IFN-α5, and IFN-β transcripts in virus-infected brains and livers.

Histograms show the number of IFN cDNA copies per 10⁶ β-actin cDNA copies, determined by real-time PCR, after reverse transcription of RNA extracted from the brain and liver of mice infected with different RNA viruses, in different experimental settings (see Table 2). A, B, F, G, H: mean and standard deviation of groups of mice. C–E: data from individual mice. Background amplification in mock-infected mice (not shown) was less than 1 copy of IFN-β or IFN-λ, and less than 10 copies of IFN-α5 cDNA, per 10⁶ copies of β-actin cDNA.

Figure 3. Expression of luciferase, in vivo, after i.m. electroinjection of an expression plasmid.

Left panel: picture of a mouse showing luciferase expression in the tibialis muscle of the right leg. Right panel: follow-up of luciferase expression in vivo (arbitrary units), in two mice electroinjected with 10 µg of plasmid DNA (pCS41) expressing the firefly luciferase gene and in one mouse electroinjected with 0.5 µg of pCS41 plasmid DNA and 10 µg of plasmid DNA expressing IFN-α6T.

Figure 4. OASl2 expression in different tissues after electroinjection of plasmids coding for IFN-α or IFN-λ.

OASl2 transcripts detected by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-α6T/D78N, MuIFN-λ3 or the empty vector (mock), in 7 week-old FVB/N mice. Results are expressed as OASl2 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.

Figure 5. Mx1 gene expression induced by systemic IFN-α and IFN-λ, in organs of IFNAR1-positive and IFNAR1-deficient mice.

Mx1 gene transcription was analyzed by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-λ3 or the empty vector (mock) in 6 week-old Mx1-positive mice (2 BALB.A2G-Mx1 and 2 B6.A2G-Mx1 mice, grouped as Mx1/WT mice) or in 8 week-old Mx1/IFNAR1-KO mice. Results are expressed as Mx1 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.

Figure 6. IL-28Rα expression exhibits tissue specificity.

IL-28Rα and IFNAR1 expressions were determined 7 days after electroinjection of plasmids coding for MuIFN-λ3 (right) or MuIFN-α6T (center), or after electroinjection of an empty vector (left), in 6 week-old BALB.A2G-Mx1 mice. IL-28Rα and IFNAR1 expressions were measured by real-time RT-PCR. Results are expressed as means and SD of the ratio between IL28Rα and IFNAR1 cDNA copies.

Figure 7. IFN-α and IFN-λ responding cells in the kidney.

Mx1 expression, detected by immunohistochemistry (white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. Sections of the kidney from: A. control Mx1/WT mouse electroinjected with the empty vector. Note that few cells (mostly endothelial cells) were weakly Mx1-positive. B. control Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-α6T. C–E–G: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. D–F–H: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3.

Figure 8. IFN-α and IFN-λ responding cells in the kidney adipose tissue and in the brain.

Mx1 expression, detected by immunohistochemistry (as white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. A–C–E: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. B–D–F: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3. A–B: sections showing the kidney adipose tissue. C–D: brain sections showing the choroid plexus of the 4th ventricle. E–F: Higher magnification of the choroid plexus. G: Cartoon showing the structural organization of the choroid plexus.

Figure 9. Correlation between the relative responsiveness of organs to IFN-λ and the relative expression of IL-28Rα and IFNAR1.

A. Relative functional influence of IFN-λ over IFN-α in various organs. The histogram shows, for each organ, the ratio between OASl2 expression in response to circulating IFN-λ and OASl2 expression in response to circulating IFN-α (mean of two mice), 7 days after electrotransfer of the expression plasmids. B. Relative expression, measured by real-time RT-PCR, of IL-28Rα and IFNAR1 (mean and SD from 5 mice).