NFAT5 inhibits TLR3-induced IFN-I responses in macrophages and DCs. (A) GSEA of Hallmark database for the IFNα response from microarray data comparing wild-type (Nfat5+/+, WT) and NFAT5-deficient (Nfat5−/−, knockout [KO]) BMDMs unstimulated (middle), stimulated with poly(I:C) (pIC; 1 µg/ml, 6 h; top), or stimulated with LPS (0.3 ng/ml, 6 h; bottom). NES, normalized enrichment score; FDR, false discovery rate. (B) Heat map illustrating the differential expression of a set of ISGs in Nfat5+/+ WT and Nfat5−/− KO BMDMs in basal conditions (Unst) or in response to poly(I:C) stimulation (pIC; 0.2 and 1 µg/ml, 6 h; see Table S1 for an extended list of genes responsive to IFNα and β differentially expressed between poly(I:C)-stimulated wild-type and NFAT5-deficient BMDMs). Log2 FC, fold-change in log2. (C) RT-qPCR analysis of the indicated ISGs and the NFAT5-induced Il1b in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 6 h of stimulation with 1 µg/ml of poly(I:C). (D) RT-qPCR analysis of Ifnb1 and the ISGs Ifit2 and Mx1 in Nfat5+/+ and Nfat5−/− BMDCs differentiated with GM-CSF (GM-BMDCs), and peritoneal macrophages left unstimulated or stimulated with poly(I:C) (1 µg/ml, 6 h). (E) RT-qPCR analysis of IFN-I genes, ISGs Ifit2 and Mx1, and Nfat5 in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 6 h of stimulation with poly(I:C), IFNα or Pam3CSK (P3). (F) mRNA expression of the indicated genes in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after stimulation with IFNβ for 2 and 6 h. (G) RT-qPCR analysis of Ifnb1 mRNA expression in peritoneal macrophages isolated from mice with a conditional deletion of Nfat5 in immune cells (Nfat5fl/fl Vav-Cre) or control littermates (Nfat5+/+ Vav-Cre) after in vivo poly(I:C) injection (10 mg/kg, 6 h). (H) IFNβ production measured by ELISA in the supernatants of Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 24 h of poly(I:C) stimulation. (I) Western blot analysis for the indicated proteins in Nfat5+/+ (W) and Nfat5−/− (K) BMDMs left unstimulated or stimulated with poly(I:C) as indicated. All mRNA values were normalized to L32. Error bars in C–H show the mean ± SEM. Data in A and B are from three independent experiments, each comparing cells from one NFAT5-deficient mouse and one wild-type littermate. Results in C–F are from seven (C), five (D), four to five (E), and three (F) independent experiments, each comparing cells from one NFAT5-deficient mouse and one wild-type littermate. Results in G are from three independent experiments and comprise three untreated wild-type, eight poly(I:C)-treated wild-type, and 10 NFAT5-deficient mice. Results in H are from two independent experiments, each comparing cells from two NFAT5-deficient mice and two wild-type littermates. The Western blot in I is representative of two independent experiments (see Fig. S1 E). Statistical significance in C and D was determined with: a one-sample t test using wild-type cells stimulated with 1 µg/ml poly(I:C) as reference with a value of 1; in E with a one-sample t test for comparisons with the reference sample and an unpaired t test for comparisons between other samples; and in G and H with an unpaired t test. *, P < 0.05; **, P < 0.01.
Figure 1.

NFAT5 inhibits TLR3-induced IFN-I responses in macrophages and DCs. (A) GSEA of Hallmark database for the IFNα response from microarray data comparing wild-type (Nfat5+/+, WT) and NFAT5-deficient (Nfat5−/−, knockout [KO]) BMDMs unstimulated (middle), stimulated with poly(I:C) (pIC; 1 µg/ml, 6 h; top), or stimulated with LPS (0.3 ng/ml, 6 h; bottom). NES, normalized enrichment score; FDR, false discovery rate. (B) Heat map illustrating the differential expression of a set of ISGs in Nfat5+/+ WT and Nfat5−/− KO BMDMs in basal conditions (Unst) or in response to poly(I:C) stimulation (pIC; 0.2 and 1 µg/ml, 6 h; see Table S1 for an extended list of genes responsive to IFNα and β differentially expressed between poly(I:C)-stimulated wild-type and NFAT5-deficient BMDMs). Log2 FC, fold-change in log2. (C) RT-qPCR analysis of the indicated ISGs and the NFAT5-induced Il1b in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 6 h of stimulation with 1 µg/ml of poly(I:C). (D) RT-qPCR analysis of Ifnb1 and the ISGs Ifit2 and Mx1 in Nfat5+/+ and Nfat5−/− BMDCs differentiated with GM-CSF (GM-BMDCs), and peritoneal macrophages left unstimulated or stimulated with poly(I:C) (1 µg/ml, 6 h). (E) RT-qPCR analysis of IFN-I genes, ISGs Ifit2 and Mx1, and Nfat5 in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 6 h of stimulation with poly(I:C), IFNα or Pam3CSK (P3). (F) mRNA expression of the indicated genes in Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after stimulation with IFNβ for 2 and 6 h. (G) RT-qPCR analysis of Ifnb1 mRNA expression in peritoneal macrophages isolated from mice with a conditional deletion of Nfat5 in immune cells (Nfat5fl/fl Vav-Cre) or control littermates (Nfat5+/+ Vav-Cre) after in vivo poly(I:C) injection (10 mg/kg, 6 h). (H) IFNβ production measured by ELISA in the supernatants of Nfat5+/+ and Nfat5−/− BMDMs in basal conditions and after 24 h of poly(I:C) stimulation. (I) Western blot analysis for the indicated proteins in Nfat5+/+ (W) and Nfat5−/− (K) BMDMs left unstimulated or stimulated with poly(I:C) as indicated. All mRNA values were normalized to L32. Error bars in C–H show the mean ± SEM. Data in A and B are from three independent experiments, each comparing cells from one NFAT5-deficient mouse and one wild-type littermate. Results in C–F are from seven (C), five (D), four to five (E), and three (F) independent experiments, each comparing cells from one NFAT5-deficient mouse and one wild-type littermate. Results in G are from three independent experiments and comprise three untreated wild-type, eight poly(I:C)-treated wild-type, and 10 NFAT5-deficient mice. Results in H are from two independent experiments, each comparing cells from two NFAT5-deficient mice and two wild-type littermates. The Western blot in I is representative of two independent experiments (see Fig. S1 E). Statistical significance in C and D was determined with: a one-sample t test using wild-type cells stimulated with 1 µg/ml poly(I:C) as reference with a value of 1; in E with a one-sample t test for comparisons with the reference sample and an unpaired t test for comparisons between other samples; and in G and H with an unpaired t test. *, P < 0.05; **, P < 0.01.

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