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. 2012 Sep;90(8):774-83.
doi: 10.1038/icb.2011.109. Epub 2012 Jan 17.

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent

Philippa Hillyer  1 , Viraj P Mane, Lynnsie M Schramm, Montserrat Puig, Daniela Verthelyi, Aaron Chen, Zeng Zhao, Maria B Navarro, Kevin D Kirschman, Srikant Bykadi, Ronald G Jubin, Ronald L Rabin
Affiliations
Free PMC article

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent

Philippa Hillyer et al. Immunol Cell Biol. 2012 Sep.
Free PMC article

Erratum in

  • Immunol Cell Biol. 2013 Oct;91(9):593

Abstract

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-β and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.

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Figures

Figure 1
Figure 1
IFN-α and IFN-λ primer probe sets are highly specific. For each IFN-α and IFN-λ subtype, 1.0 pM of cDNA was amplified with its specific primer/probe set, and each nonspecific primer/probe set. The difference in cycle numbers between the target cDNA template and best amplified nonspecific template for each primer/probe set demonstrates a minimum of ∼1000-fold specificity.
Figure 2
Figure 2
The expression pattern of IFN mRNA and protein differs with each TLR ligand. (a) PBMC were stimulated with each TLR ligand and the cells were harvested for qRT-PCR analysis. The geometric means of the peak responses to poly I:C (8 h), LPS (4 h), imiquimod and CpG and unstimulated control (16 h each) from six donors are shown in log10 scale as a function of expression of the HKG UBC (ΔCq, left), or as copy number μg–1 RNA (right). IFN-α subtypes are ordered according to the phylogenetic plot of amino acid sequence similarity shown in Supplementary Figure 4. (b–e) IFN-α, -β and -λ proteins were measured by ELISA, and detectable protein levels were plotted as a function of gene expression. (b) The expression of each IFN-α subtype in copy number μg–1 RNA was added, and the sum was plotted against protein expression. (c) Correlation of transcript (sum of all subtypes) and protein levels of IFN-α at 8h post-stimulation with poly I:C and CpG oligonucleotides. (d) Levels of IFN-β transcript and protein expressed by PBMC at 16 and 24 h in response to CpG oligonucleotides correlate. (e) Levels of IFN-λ (sum of IFN-λ1, -λ2 and λ3) transcript and protein expressed by PBMC at 8, 16 and 24 h in response to poly I:C correlate.
Figure 3
Figure 3
PBMC expression of type I IFN and ISG correlates after stimulation through TLR7 and TLR9. The expression of each type I IFN subtype in copy number μg–1 RNA was added and the sum was plotted on the X axis against expression (ΔCq) of the ISG IRF7 (Y axis, top) and MX1 (bottom). Type I IFN expression correlates with either of the ISG after stimulation with imiquimod (diamond) or CpG (circle), but not LPS or poly I:C (triangle and square, respectively). For IFN vs IRF7 after stimulation with imiquimod: R2=0.87; after stimulation with CpG: R2=0.87. For IFN vs MX1 after stimulation with imiquimod: R2=0.81; after stimulation with CpG: R2=0.83. Data shown are from PBMC from the three donors in which the cells were not frozen.
Figure 4
Figure 4
Myeloid cells primarily express a limited profile of IFN in response to LPS and poly I:C. (a) Monocytes and mDC were harvested from the same donor, and aliquots of the monocytes were differentiated into MDDC and MDM. Each cell type was stimulated for 4 h with LPS, poly I:C, and in the case of the MDM, CpG oligonucleotides. IFN expression is shown in log10 scale as a function of expression of HKG SDHA for mDC and MDDC, and UBC for monocytes and MDM (ΔCq, top), or copy number μg–1 RNA (bottom) in log10 scale. Note the difference in scale of each graph. (b) Compiled expression profiles shown as the geometric mean of copy number μg–1 RNA for monocytes (eight donors), MDDC and MDM (five donors each). (c) Expression of IFN-α1, -β and λ1 (ΔCq) among a set of donors, each of which is represented by a different symbol, and tested for statistical significance (vs control); P-values are shown at bottom of each graph after Bonferroni's correction and are italicized if tested but not statistically significant; NT=not tested. (d) MDM expresses IFN-α10 and -α14 in response to CpG; independent statistical analysis of the expression of three IFN-α subtypes that the radial plots suggest are expressed by MDM.
Figure 5
Figure 5
Expression by pDC of all type I IFN in response to CpG. pDC were purified and stimulated with poly I:C, imiquimod, CpG or GpC control. (a) IFN expression is shown in log10 scale as a function of expression of the HKG SDHA (ΔCq, left) or copy number μg–1 RNA (right). Although all IFN-α subtypes are expressed in response to CpG, the pDC from this donor failed to express IFN-α7 in response to imiquimod. (b) Expression (ΔCq) of IFN by seven donors and statistical analysis (with Bonferroni's correction) of types I and III IFN by pDC in response to CpG, imiquimod and poly I:C vs control as in Figure 4. Identical symbols in Figures 4 and 5 indicate that the cells are from the same donor.
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