Research ArticleANTIVIRAL IMMUNITY

IFN-κ suppresses the replication of influenza A viruses through the IFNAR-MAPK-Fos-CHD6 axis

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Science Signaling  07 Apr 2020:
Vol. 13, Issue 626, eaaz3381
DOI: 10.1126/scisignal.aaz3381
  • Fig. 1 IFN-κ broadly inhibits IAV replication.

    (A) Body weight change in mice challenged with H7N9 IAV, H9N2 IAV, or PBS expressed as means ± SEM. n = 15 mice for each group. (B) Survival data for mice challenged with H7N9 IAV, H9N2 IAV, or PBS pooled from three independent experiments, each with five mice per group. (C) Expression of Ifn genes in H7N9- and H9N2-infected mice from microarray-based transcriptome analysis of RNA pooled from three lungs for each group. (D) Protein sequence comparison between IFN-κ cloned from A549 cells (IFN-κDual) and the published human IFN-κ sequence, highlighting the two amino acid changes at positions 133 and 164. (E) Western blotting analysis of IAV NP and IFN-κ in A549 cells transfected with empty vector (control) or the indicated IFN-κ construct for 24 hours and then infected with H9N2, H7N9, PR8, or H1N1 for 48 hours. Nontransfected, noninfected A549 cells served as mock control. β-Actin is a loading control. Blots are representative of three independent experiments. Quantitation of the blots in (E) is shown in the Supplementary Materials (fig. S7A).

  • Fig. 2 CHD6 is the key effector molecule mediating the anti-influenza activity of IFN-κ.

    (A) Volcano plots of differentially expressed genes in A549 cells transfected with IFN-κ versus those transfected with IFN-κDual or pSV1.0 empty vector. Dashed vertical lines mark twofold change. Data were generated from n = 3 biological replicates. (B) qRT-PCR analyses of CHD6 expression and some known anti-influenza ISGs in A549 cells that were mock-transfected or transfected with either the IFN-κ or IFN-κDual construct for 24 hours. (C) qRT-PCR analysis of CHD6 transcripts in A549 cells transfected with empty vector (EV) or the IFN-κ construct for 24 hours and then infected with the indicated IAV for 48 hours. (D) qRT-PCR analysis of CHD6 transcripts in CHD6-KO A549 cells. (E) Western blotting analysis of IAV NP, IFN-κ, and CHD6 in wild-type (WT) and CHD6-KO A549 cells either EV-transfected or transfected with IFN-κ construct for 24 hours and then infected with the indicated IAV virus for 48 hours. β-Actin is a loading control. Data in (B) to (D) are means ± SD from three biological replicates. Blots are representative of three independent experiments. Quantitation of the blots (E) is shown in the Supplementary Materials (fig. S7B). *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni post hoc test (B) or unpaired t test (C and D).

  • Fig. 3 IFN-κ requires both IFNAR1 and IFNAR2 to transduce signals for CHD6 induction.

    (A) Western blotting analysis of IFNAR1 and IFNAR2 in IFNAR1-KO and IFNAR2-KO A549 cells generated by CRISPR-Cas9 genomic editing. WT A549 cells served as a positive control. β-Actin is a loading control. (B) qRT-PCR analysis of CHD6 transcripts in WT, IFNAR1-KO, and IFNAR2-KO A549 cells that were either mock-transfected or transfected with the IFN-κ construct for 24 hours and then infected with H9N2 IAV for 48 hours. (C) Western blotting analysis of IAV NP, IFN-κ, and CHD6 in WT, IFNAR1-KO, and IFNAR2-KO A549 cells transfected with the IFN-κ construct as indicated for 24 hours and then infected with H9N2, H7N9, or PR8 virus for 48 hours. Blots are representative of three independent experiments. Data are means ± SD of three biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired t test.

  • Fig. 4 IFN-κ stimulates CHD6 expression through the p38-Fos axis.

    (A) Western blotting analysis of phosphorylated c-Fos (p-c-Fos), total c-Fos, and CHD6 in lysates of A549 cells exposed to purified hIFN-κ-Fc protein for the indicated time. β-Actin is a loading control. (B) Western blotting analysis of p-c-Fos and total c-Fos in A549 cells pretreated with inhibitors of STAT1 (iSTAT1, fludarabine), JNK (iJNK, SP600125), ERK (iERK, SCH772984), p38 (ip38, SB203580), p65 (ip65, curcumenol and PG490), or phosphorylated AKT (ipAKT, triciribine), as indicated for 6 hours, and then stimulated with purified hIFN-κ-Fc protein for 30 min. (C and D) Western blotting analysis of phosphorylated p38 (p-p38), total p38, p-c-Fos, total c-Fos, and CHD6 in A549 cells (C) and BEAS-2B cells (D) pretreated with the p38 inhibitor SB203580, as indicated for 6 hours before exposure to purified hIFN-κ-Fc protein for 30 min. (E) Western blotting analysis of IAV NP, p-p38, and total p38 in A549 cells pretreated with SB203580 for 6 hours, as indicated before exposure to either IFN-κ-Fc or IFN-α2 for 24 hours, and subsequently infected with PR8 virus for 48 hours. (F) Western blotting analysis of phosphorylated STAT1 (p-STAT1), total STAT1, and CHD6 in A549 cells pretreated with iSTAT1 (fludarabine), as indicated for 6 hours before hIFN-κ-Fc exposure for 30 min. (G) Western blotting analysis of IAV NP, p-STAT1, and total STAT1 in A549 cells pretreated with fludarabine, as indicated for 6 hours before exposure to hIFN-κ-Fc for 12 hours, and then infected with PR8 virus for 48 hours. Blots are representative of three independent experiments. Quantitation of the blots in (A), (B), (F), and (G) is shown in the Supplementary Materials (fig. S7, C to F).

  • Fig. 5 IFN-κ is protective against IAV infection in mice.

    Mice were injected intranasally with IFN-κ-Fc protein or PBS 6 hours before intranasal challenge with PR8 virus. (A) Body weight change and survival. n = 12 mice for each group. Body weight change data are means ± SEM. (B) Representative images of H&E-stained lung sections from IFN-κ-Fc and PBS treatment groups at day 1, 2, 3, and 7 after infection (D1, D2, D3, and D7). Quantified pathological scores were calculated on the basis of lung histology data. n = 3 mice for each group. Scale bar, 200 μm. Results are expressed as mean ± SD. (C) Quantification of histochemical staining for p-p38 in lung tissues from the IFN-κ-Fc and PBS treatment groups and representative images. n = 3 mice for each group. Scale bar, 100 μm. Quantification data are expressed as mean ± SD. (D) Quantification of and representative images showing in situ hybridization for CHD6 mRNA in lung tissues from mice treated with PBS or IFN-κ-Fc. Scale bar, 50 μm. Quantification data are expressed as means ± SD (n = 3 for each group). *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired t test.

  • Table 1 Definitions of scores for the inflammatory response in lung tissue.

    The grading of histology sections incorporated evaluations from three categories, including (A) degree of bronchial infiltrate (<25%, 25 to 50%, 50 to 75%, >75%), (B) frequency of inflammatory cells (none, <10%, 10 to 20%,>20%), and (C) degree of parenchymal pneumonia/infiltrate (<25%, 25 to 50%, 50 to 75%,>75%). The total score (ranged from 0 to 9) was set as sum of subscores from (A) to (C). Total score <3, mild; total score 3 to <6, moderate; total score 6 to ≤9, intense.

    Score0123
    Category
    A. Bronchial infiltrate<25%25–50%50–75%>75%
    B. Inflammatory cells infiltrateNone<10%10–20%>20%
    C. Parenchymatous
    pneumonia
    <25%25–50%50–75%>75%

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/626/eaaz3381/DC1

    Fig. S1. IAV-mediated induction of IFNs in human A549 cells.

    Fig. S2. Inhibition of IAV by IFN-κ but not its A549-derived mutants.

    Fig. S3. IFN-κ preferentially stimulates CHD6 expression upon subsequent influenza infection.

    Fig. S4. IFN-κDual largely retains the capability to stimulate STAT1 phosphorylation.

    Fig. S5. Design, purification, and functional analyses of a recombinant IFN-κ protein.

    Fig. S6. Temporal induction of CHD6 in A549 cells responding to different recombinant IFNs.

    Fig. S7. Densitometric quantification of immunoblots.

    Table S1. Primer sequences.

  • This PDF file includes:

    • Fig. S1. IAV-mediated induction of IFNs in human A549 cells.
    • Fig. S2. Inhibition of IAV by IFN-κ but not its A549-derived mutants.
    • Fig. S3. IFN-κ preferentially stimulates CHD6 expression upon subsequent influenza infection.
    • Fig. S4. IFN-κDual largely retains the capability to stimulate STAT1 phosphorylation.
    • Fig. S5. Design, purification, and functional analyses of a recombinant IFN-κ protein.
    • Fig. S6. Temporal induction of CHD6 in A549 cells responding to different recombinant IFNs.
    • Fig. S7. Densitometric quantification of immunoblots.
    • Table S1. Primer sequences.

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