NFAT5 represses IFN-I responses in macrophages and DCs infected with different viruses. (A and B) Expression of the mRNA of Ifnb1 and ISGs Ifit2 and Mx1 in NFAT5-deficient (Nfat5−/−) and wild-type (Nfat5+/+) BMDMs (A) and BMDCs differentiated with GM-CSF (GM-BMDCs; B) at different times after infection with VSV (top) or MCMV (bottom), using a multiplicity of infection (MOI) of 1. mRNA values for each gene were normalized to L32 RNA and are represented as relative to the 12-h infection time point in wild-type cells. Expression of Nfat5 mRNA in wild-type BMDMs is also shown (n = 4). (C) Wild-type (Nfat5+/+) and NFAT5-deficient (Nfat5−/−) BMDCs differentiated with Flt3L and sorted as pDCs (CD11c+ B220+) were infected with VSV (for 6 h; left) or LCMV (for 12 h; right) to analyze mRNA levels of Ifnb1, Ifna1, and Ifna7, and the ISG Ifit2. mRNA values for each gene were normalized to L32 RNA and are represented as relative to infected wild-type cells. (D) Expression levels of the mRNA of Ifnb1, Ifna1, and Ifna7 in wild-type, NFAT5-deficient (Nfat5−/− Vav-Cre), IFNAR-deficient (Nfat5+/+ Vav-Cre Ifnar1−/−), or NFAT5- and IFNAR-deficient (Nfat5−/− Vav-Cre, Ifnar1−/−) BMDMs at the indicated times after VSV infection at MOI 1. mRNA values for each gene were normalized to L32 RNA and are represented as relative to VSV-infected wild-type cells. (E) Expression of Ifnb1 and Ifit2 mRNA in Nfat5+/+ and Nfat5−/− BMDMs 12 h after infection with UV-inactivated VSV. Error bars in A–E show the mean ± SEM. Results 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 comprise six BMDC cultures of each genotype for VSV and four for LCMV analyzed in four independent experiments. Results in D comprise six BMDM cultures of each genotype from three independent experiments. Results in E show the mean ± SEM of five BMDM cultures of wild-type and NFAT5-deficient from three independent experiments. Statistical significance in A and B was determined with a one-sample t test for comparisons with the reference sample (wild-type cells infected for 12 h) or with an unpaired t test for comparisons between the other samples. Statistical significance in C was determined with a one-sample t test using infected wild-type cells as reference. Statistical significance in D and E was determined with an unpaired t test. *, P < 0.05; **, P < 0.01.
Figure 3.

NFAT5 represses IFN-I responses in macrophages and DCs infected with different viruses. (A and B) Expression of the mRNA of Ifnb1 and ISGs Ifit2 and Mx1 in NFAT5-deficient (Nfat5−/−) and wild-type (Nfat5+/+) BMDMs (A) and BMDCs differentiated with GM-CSF (GM-BMDCs; B) at different times after infection with VSV (top) or MCMV (bottom), using a multiplicity of infection (MOI) of 1. mRNA values for each gene were normalized to L32 RNA and are represented as relative to the 12-h infection time point in wild-type cells. Expression of Nfat5 mRNA in wild-type BMDMs is also shown (n = 4). (C) Wild-type (Nfat5+/+) and NFAT5-deficient (Nfat5−/−) BMDCs differentiated with Flt3L and sorted as pDCs (CD11c+ B220+) were infected with VSV (for 6 h; left) or LCMV (for 12 h; right) to analyze mRNA levels of Ifnb1, Ifna1, and Ifna7, and the ISG Ifit2. mRNA values for each gene were normalized to L32 RNA and are represented as relative to infected wild-type cells. (D) Expression levels of the mRNA of Ifnb1, Ifna1, and Ifna7 in wild-type, NFAT5-deficient (Nfat5−/− Vav-Cre), IFNAR-deficient (Nfat5+/+ Vav-Cre Ifnar1−/−), or NFAT5- and IFNAR-deficient (Nfat5−/− Vav-Cre, Ifnar1−/−) BMDMs at the indicated times after VSV infection at MOI 1. mRNA values for each gene were normalized to L32 RNA and are represented as relative to VSV-infected wild-type cells. (E) Expression of Ifnb1 and Ifit2 mRNA in Nfat5+/+ and Nfat5−/− BMDMs 12 h after infection with UV-inactivated VSV. Error bars in A–E show the mean ± SEM. Results 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 comprise six BMDC cultures of each genotype for VSV and four for LCMV analyzed in four independent experiments. Results in D comprise six BMDM cultures of each genotype from three independent experiments. Results in E show the mean ± SEM of five BMDM cultures of wild-type and NFAT5-deficient from three independent experiments. Statistical significance in A and B was determined with a one-sample t test for comparisons with the reference sample (wild-type cells infected for 12 h) or with an unpaired t test for comparisons between the other samples. Statistical significance in C was determined with a one-sample t test using infected wild-type cells as reference. Statistical significance in D and E was determined with an unpaired t test. *, P < 0.05; **, P < 0.01.

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