Research ArticleINNATE IMMUNITY

Selective Janus kinase inhibition preserves interferon-λ–mediated antiviral responses

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Science Immunology  14 May 2021:
Vol. 6, Issue 59, eabd5318
DOI: 10.1126/sciimmunol.abd5318
  • Fig. 1 BMS-986165 preferentially inhibits type I compared with type III IFN–mediated gene expression in human A549.

    Human A549 cells were treated with the indicated concentrations of the JAK1/2 inhibitor baricitinib (left) or the selective TYK2 inhibitor BMS-986165 (right) for 1.5 hours before IFN-αB/D (0.03 ng/ml) or hIFN-λ1 (10 ng/ml) was added to the medium. After 4 hours, gene expression levels of the representative ISGs ISG15 and MX1 were quantified relative to the housekeeping gene HPRT. Inhibition of IFN-mediated gene expression is indicated as percentages of maximum gene expression levels without inhibitor. Symbols represent means ± SEM; data are pooled from two independent experiments with a total n = 4 to 7. ****P < 0.0001, **P < 0.01, and * P < 0.05, by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test.

  • Fig. 2 IFN-α– but not IFN-λ–mediated expression of ISGs depends on TYK2 in primary mouse epithelial cells.

    (A) Primary AECs derived from Tyk2+/− or Tyk2−/− mice were treated with increasing concentrations of IFN-αB/D or IFN-λ2 for 4 hours. Expression of Isg15 was quantified relative to Hprt. Symbols represent means ± SD; data are representative of two independent experiments with n = 2 per experiment. ***P < 0.001, **P < 0.01, and *P < 0.05, by two-way ANOVA with Tukey’s multiple comparisons test. (B) AECs derived from Tyk2+/− or Tyk2−/− mice were treated with IFN-αB/D or IFN-λ2 (10 ng/ml) for 1 hour and analyzed at the indicated time points. Expression of Isg15 was quantified relative to Hprt. Symbols represent means ± SD (n = 2). ****P < 0.0001, ***P < 0.001 and **P < 0.01, by two-way ANOVA with Sidak’s multiple comparisons test. (C) Heat map of genes differentially expressed relative to mock (two-way ANOVA, fold change >2; P < 0.01) in WT or Tyk2−/− primary mouse AECs (n = 3) treated with IFN-αB/D or IFN-λ2 (0.3 ng/ml) for 4 hours. Color range is depicted as log2 scale. (D) Scatterplot of IFN-induced genes (fold change >2 over mock in any IFN treatment at any concentration, n = 3). Data used for plot correspond to the Wald statistic value [log2(fold change) divided by SE], obtained from DESeq2. Lines indicate the x = y diagonal. (E) Mini-gut organoids derived from WT or Tyk2−/− mice were treated with increasing concentrations of IFN-αB/D or IFN-λ2 for 4 hours. Expression of Isg15 was quantified relative to Hprt. Symbols represent means ± SD; data are pooled from three independent experiments with a total n = 3. ****P < 0.0001, **P < 0.01, and *P < 0.05, by two-way ANOVA with Sidak’s multiple comparisons test.

  • Fig. 3 Loss of TYK2 moderately influences IFN-λ–mediated gene expression in mouse neutrophils.

    (A and B) BM-derived neutrophils from three individual mice were treated with IFN-αB/D or IFN-λ2 (3 ng/ml) for 4 hours and then processed for RNA-seq analysis. Heat map shows genes regulated (one-way ANOVA, P < 0.01; fold change >4 relative to mock) by both types of IFN (A) or exclusively by IFN-αB/D (B). Color range is depicted as log2 scale. (C) WT BM-derived neutrophils from four individual mice were treated with IFN-αB/D or IFN-λ2 (1 μg/ml) for 4 hours. Expression levels of Isg15 and Mx1 were quantified relative to Ubc and are shown normalized to untreated controls. ****P < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test. (D) Differentiated WT or Tyk2−/− Hoxb8 neutrophils were treated (n = 3) with increasing concentrations of IFN-αB/D or IFN-λ2 for 4 hours. Expression levels of Isg15 were quantified relative to Ubc. Symbols represent means ± SD; data are representative of two independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, by two-way ANOVA with Sidak’s multiple comparisons test. (E and F) WT, Tyk2−/−, or Ifnar1−/− BM-derived neutrophils were treated with increasing concentrations of IFN-αB/D or IFN-λ2 for 4 hours (mock, n = 3 to 4; 0.03 to 0.3 ng/ml, n = 2 to 3; 3 ng/ml, n = 1 to 2). Expression levels of Isg15 (E) and Oasl2 (F) were quantified relative to Ubc. Symbols represent means ± SD; data are representative of three independent experiments. ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, by two-way ANOVA with Sidak’s multiple comparisons test. Asterisks indicate significant differences between WT and Tyk2−/−. (G) BM-derived neutrophils from three individual mice were treated with IFN-αB/D or IFN-λ2 (0.1 ng/ml) for 4 hours and then processed for RNA-seq analysis. Heat map of IFN-λ–regulated genes (two-way ANOVA, P < 0.01; fold change >4 relative to each mock). Color range is depicted as log2 scale. (H) Scatterplot of IFN-induced genes (fold change >2 over mock in any IFN treatment at any concentration, n = 3). Data used for plot correspond to the Wald statistic value [log2(fold change) divided by SE], obtained from DESeq2. Lines indicate the x = y diagonal.

  • Fig. 4 TYK2 inhibition preferentially inhibits type I IFN–mediated gene expression in human HAP1.

    (A) HAP1-WT or HAP1-TYK2ko cells were treated with increasing concentrations of IFN-αB/D or hIFN-λ1 for 4 hours. Expression levels of ISG15 were quantified relative to HPRT. Symbols represent means ± SD; data are representative of two independent experiments with n = 2 per experiment. ****P < 0.0001, ***P < 0.001, and **P < 0.01, by two-way ANOVA with Sidak’s multiple comparisons test. (B) HAP1-WT or HAP1- TYK2ko cells were treated with increasing concentrations of IFN-αB/D or hIFN-λ1 for 16 hours before infection with VSV-GFP [multiplicity of infection (MOI) ~ 1] for 6 hours. Data are shown as percentage of GFP-positive cells normalized to mock-treated controls infected with VSV-GFP. Bars represent means ± SD; data are representative of two independent experiments with n = 2 per experiment. ****P < 0.0001, by two-way ANOVA with Sidak’s multiple comparisons test. (C) HAP1-WT cells were treated with the indicated concentrations of the JAK1/2 inhibitor baricitinib (left) or the selective TYK2 inhibitor BMS-986165 (right) for 1.5 hours before IFN-αB/D (0.1 ng/ml) or hIFN-λ1 (10 ng/ml) was added to the medium. After 4 hours, gene expression levels of the representative ISGs ISG15 and RSAD2 were quantified relative to the housekeeping gene HPRT. JAK inhibitor–mediated inhibition of IFN-mediated gene expression is indicated as percentages of maximum gene expression levels without inhibitor. Symbols represent means ± SEM; data are pooled from three independent experiments with a total n = 6 to 10. ****P < 0.0001 and ***P < 0.001, by two-way ANOVA with Sidak’s multiple comparisons test.

  • Fig. 5 IFN-λ–mediated gene expression is independent of TYK2 and JAK2 in human A549 cells.

    (A) A549 control and TYK2ko cells were treated with increasing concentrations of IFN-αB/D or hIFN-λ1 for 4 hours. Expression of ISG15 and MX1 is shown relative to HPRT. Symbols represent means ± SD. ****P < 0.0001 and *P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test. Data are representative of two independent experiments with n = 2 per experiment. (B) A549 control and TYK2ko cells were treated with either IFN-αB/D or hIFN-λ1 (1 or 10 ng/ml) for 16 hours before infection with VSV-GFP (MOI ~ 1) for 6 hours. Data are shown as percentage of GFP-positive cells normalized to mock-treated controls infected with VSV-GFP. Bars represent means ± SD; data are pooled from two independent experiments with a total n = 4. ***P < 0.001, **P < 0.01, ns = not significant, unpaired t test. (C and D) A549 control and JAK2ko cells (C) or JAK2koTYK2ko cells (D) were treated with increasing concentrations of IFN-αB/D or hIFN-λ1 for 4 hours. Expression of ISG15 and MX1 is shown relative to HPRT. Symbols represent means ± SD. ****P < 0.0001 and *P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test. Data are representative of two independent experiments with n = 2 per experiment.

  • Fig. 6 IFN-λ–mediated protection of mice against influenza A virus–induced disease is not dependent on TYK2.

    (A) WT (n = 13), Tyk2−/− (n = 14), or Ifnar1−/−Ifnlr1−/− (n = 13) mice were infected with 200 PFU of the influenza A virus strain Udorn (H3N2) in a 10-μl volume. Viral load in the upper airways was determined 5 days post-infection (d p.i.) by plaque assay. All mice carried two functional alleles of the IFN-regulated influenza virus resistance gene Mx1. Symbols represent individual mice. Left: ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test; right: *P < 0.05, Fisher’s exact test. (B and C) Tyk2+/− or Tyk2−/− mice were treated intranasally with PBS or 0.66 μg of either IFN-αB/D or IFN-λ2 in 30 μl. After 18 hours, mice were infected with 160 PFU of a highly virulent variant of the influenza A virus strain PR8 (hvPR8; H1N1) in a 40-μl volume. All mice used for these experiments carried one functional allele of the IFN-regulated influenza virus resistance gene Mx1. Weight loss (B) and survival (C) were monitored for 14 days. Symbols in (B) represent means ± SEM; group sizes are indicated in (C). Pooled data from three experiments are shown.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/6/59/eabd5318/DC1

    Fig. S1. Both IFN-α– and IFN-λ–mediated expression of ISGs depends on TYK2 in mouse neutrophils.

    Fig. S2. IFN-λ stimulation of naïve human neutrophils does not induce expression of ISGs.

    Fig. S3. JAK2 does not affect IFN-λ–mediated gene expression.

    Data file S1. Raw data.

  • The PDF file includes:

    • Fig. S1. Both IFN-α– and IFN-λ–mediated expression of ISGs depends on TYK2 in mouse neutrophils.
    • Fig. S2. IFN-λ stimulation of naïve human neutrophils does not induce expression of ISGs.
    • Fig. S3. JAK2 does not affect IFN-λ–mediated gene expression.
    • Legend for data file S1

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    Other Supplementary Material for this manuscript includes the following:

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