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Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease

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Science  22 Apr 2016:
Vol. 352, Issue 6284, pp. 463-466
DOI: 10.1126/science.aaf3926
  • Fig. 1 Monocytes from old humans have impaired IFN but otherwise intact cytokine responses.

    Human monocytes from young (age 20 to 30 years; n = 33) or old (age 65 to 89 years; n = 20) healthy donors were infected with A/PR/8/34 influenza virus at a multiplicity of infection of 10 for 12 hours, after which cell supernatants were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) to measure (A) IL-6, (B) IL-1β, and (C) IFN-β. Data are pooled from two independent experiments and are presented as means ± SEM. (D) RNA was isolated from PR8-infected monocytes and was analyzed by quantitative polymerase chain reaction (qPCR) to measure relative gene expression. Gene expression data were arranged in a heat map to identify age-related defects. **P < 0.01; linear mixed-effects models adjusted for covariates, P values from the post hoc t statistics are Hochberg adjusted.

  • Fig. 2 RLR and TLR7 induce Mx1 expression and protection after influenza A virus infection.

    Wild-type, Casp1/11–/–, Tlr7–/–, Mavs–/–, and Tlr7–/–Mavs–/– mice carrying functional Mx1 alleles were infected intranasally (i.n.) with 100 PFU of influenza virus A/PR/8/34. (A) Weight loss and (B) survival were monitored for 14 days (n = 5 to 9 mice per group). (C) Bronchoalveolar lavage (BAL) was collected 48 hours after infection (n = 5 to 7 mice per group), and levels of IFN-β were measured by ELISA. Data are means ± SEM. (D) Mice were sacrificed on day 2 and lungs were fixed in 4% paraformaldehyde. Tissue was embedded in paraffin, sectioned for staining with a rabbit antibody to mouse Mx1, and visualized by fluorescence microscopy [red, Mx1; blue, counter-staining of nuclei with 4′,6-diamidino-2-phenylindole (DAPI)]. Samples are representative of 3 to 5 mice per group. ****P < 0.0001; **P < 0.01; one-way analysis of variance (ANOVA); log-rank (Mantel-Cox).

  • Fig. 3 In the absence of TLR7 and MAVS, deletion of Casp1/11 rescues mice from lethality after influenza A virus infection.

    Wild-type, Casp1/11–/–, Tlr7–/–, Mavs–/–, Tlr7–/–Mavs–/–, and Tlr7–/–Mavs–/–Casp1/11–/– mice carrying functional Mx1 alleles were infected (i.n.) with 100 PFU of influenza virus A/PR/8/34. (A) Weight loss and (B) survival were monitored for 14 or 16 days, respectively (n = 4 to 10 mice per group). (C) BAL was collected and viral titer was measured (n = 4 to 8 mice per group) on day 4 after infection. Data are means ± SEM. (D) Tlr7–/–Mavs–/– (n = 21), Tlr7–/–Mavs–/–Casp1/11–/– (n = 4), and Myd88−/−Mavs−/− (n = 18) mice carrying functional Mx1 alleles were infected (i.n.) with 100 PFU of A/PR/8/34 influenza virus. A separate group of Tlr7–/–Mavs–/– (n = 8) was treated with 100 mg/kg of Anakinra intraperitoneally every 12 hours starting on day –1. Survival was monitored for 2 weeks after infection. Data are pooled from two separate experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; one-way ANOVA; log-rank (Mantel-Cox).

  • Fig. 4 Failure to induce type I IFNs and viral control is sufficient to promote bacterial bloom in the lung.

    (A) Mice carrying functional Mx1 alleles were infected (i.n.) with 100 PFU of influenza virus A/PR/8/34. A group of Tlr7–/–Mavs–/– mice was treated with either a neutrophil-depleting antibody to Ly6G) or recombinant DNase (Pulmozyme) intraperitoneally, daily starting on day –1. Survival was monitored for 14 days (n = 8 to 13 mice per group). (B) Lungs were harvested on day 8, stained with a rabbit antibody to histone H3 (citrulline R2 + R8 + R17) and visualized by fluorescence microscopy (red, H3Cit; blue, counter-staining of nuclei with DAPI). Samples are representative of 3 to 6 samples per group. (C to E) Nasal wash samples collected on day 8 were (C) plated to determine bacterial load and (D) analyzed by qPCR for relative expression levels of 16S rDNA (n = 4 to 5 per group). Data are means ± SEM. (E) The average relative abundance of bacterial genera of greater than 1% abundance from naïve wild-type (n = 3), wild-type (n = 1), Tlr7–/–Mavs–/– (n = 5), and Tlr7–/–Mavs–/–Casp1/11–/– (n = 5) was determined by 16S sequencing. (F and G) After infection with 1000 PFU of PR8, lungs were harvested on day 7 and analyzed by qPCR for relative expression levels of (F) 16S rDNA and (G) bacteria from the Pasteurellaceae family (n = 3 to 4 per group). (H) A group of Tlr7–/–Mavs–/– mice infected with 100 PFU was administered antibiotics daily by intragastric gavage on days 3 to 10 after infection. Survival was monitored (n = 4 to 5 per group). Data are representative of two similar experiments. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; one-way ANOVA; log-rank (Mantel-Cox).

Supplementary Materials

  • Mx1 reveals innate pathways to antiviral resistance and lethal influenza disease

    Padmini S. Pillai, Ryan D. Molony, Kimberly Martinod, Huiping Dong, Iris K. Pang, Michal C. Tal, Angel G. Solis, Piotr Bielecki, Subhasis Mohanty, Mark Trentalange, Robert J. Homer, Richard A. Flavell, Denisa D. Wagner, Ruth R. Montgomery, Albert C. Shaw, Peter Staeheli, Akiko Iwasaki

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Table S1
    • Figs. S1 to S11
    • References

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