IFN-ε is constitutively expressed by cells of the reproductive tract and is inefficiently secreted by fibroblasts and cell lines

Abstract

Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-α or IFN-β are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-ε was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-ε expression after viral infection and the constitutive expression of IFN-ε by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-ε precursors were inefficiently processed and secretion of IFN-ε was minimal. Analysis of chimeric constructs produced between IFN-ε and limitin (IFN-ζ) showed that both the signal peptide and the mature moiety of IFN-ε contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-ε in transfected cells suggested that IFN-ε and chimeric proteins were defective for progression through the secretory pathway. IFN-ε did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-ε in specialized cells and the poor processing of IFN-ε precursor in fibroblasts and cell lines, we hypothesize that IFN-ε secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.

Figure 1. Plasmid constructs.

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. 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. 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.

Figure 2. In vivo expression of IFN-ε.

A–C. RT-qPCR analysis of Mengo virus replication (A), Ifnb expression (B) and Ifne expression (C) in the brain, spinal cord and heart of Mengo virus-infected mice. Histograms show the mean ± SD of Mengo virus cDNA copies per 10⁴ β-actin cDNA copies (n=3). ND: not detected. NS: non significant. D–E. RT-qPCR data showing the expression of Ifne in organs collected from uninfected female (D) and male (E) C57BL/6 mice. Each column refers to an individual sample and indicates the number of Ifne cDNA copies per 10⁶ β-actin cDNA copies.

Figure 3. Activity of FLAG-tagged and untagged mouse IFNs.

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 U test).

Figure 4. Processing of the IFN-ε precursor in transfected cells.

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. 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.

Figure 5. IFN-ε signal peptide is not fully functional.

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. 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. 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. 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).

Figure 6. IFN-ε does not act intracellularly.

FACS analysis of cells transduced for 4 days with bicistronic lentiviral constructs co-expressing mCherry and the indicated IFN. pTM945 is the empty vector expressing mCherry alone. A. Representative FACS dot plot. B. Table showing the mean and standard deviation of the percentage of infected cells in the total cell population (data from 3 infection experiments).