Exacerbated IFN-I response in NFAT5-deficient mouse models causes enhanced activation of HSCs. (A and B) IFNα levels in serum of untreated (Untr, n = 3) or poly(I:C)-treated (n = 6–7) mice (pIC; 2.5 mg/kg, 12 h) were measured by ELISA in wild-type and Nfat5^fl/fl Vav-Cre (A) or Nfat5^fl/fl LysM-Cre mice (B). Results (mean ± SEM) in A and B are from two independent experiments. N.D., not detected. (C) Number of cells, platelets, and erythrocytes in whole blood and bone marrow cellularity in wild-type and Nfat5^fl/fl LysM-Cre mice left untreated or treated with poly(I:C) (two doses of 2.5 mg/kg of poly(I:C) administered 48 h apart and analyzed 24 h after the second injection). Bone marrow cell numbers were counted from two femurs and two tibiae per mouse. Results (mean ± SEM) are from one experiment with five treated and two untreated mice of each genotype. (D) Panels show representative flow cytometry analysis of hematopoietic progenitor and stem cells within LSK cells in the bone marrow of Nfat5^+/+ LysM-Cre and Nfat5^fl/fl LysM-Cre mice treated with poly(I:C) as in C. The gating strategy is shown in Fig. S3 A. (E) The upper panels show the percentages of LSK and the indicated populations within LSK cells in untreated and poly(I:C)-treated mice, the middle panels show the percentage of ESAM⁺ cells in the different subsets, and the bottom panels show the percentage of annexin V⁺ cells in the indicated subsets. Solid dots represent individual NFAT5-deficient mice, and empty dots are wild-type mice. (F) Representative contour plots for cell cycle analysis in HSCs in G. (G) Percentage of quiescent (Ki67^neg) HSCs and their CD41⁺ and CD41^neg subsets in wild-type and NFAT5-deficient mice treated as in C. (H) Phospho-STAT1 (Y701) levels measured by intracellular staining and flow cytometry in LSK cells of untreated Nfat5^+/+ Vav-Cre (wild-type) mice (n = 3), and poly(I:C)-treated (2.5 mg/kg, 16 h) Nfat5^+/+ Vav-Cre (n = 4) and Nfat5^fl/fl Vav-Cre (n = 4) mice. MFI, median fluorescence intensity. (I) Sca-1 levels measured by flow cytometry in HSCs from top panel in E. Error bars in E and G–I show the mean ± SEM. Results for hematopoietic subsets analysis in E, Ki67 analysis in whole HSCs in G, and Sca-1 MFI in I are from two independent experiments, with a total of 3–4 untreated mice of each genotype, 12 poly(I:C)-treated Nfat5^+/+ LysM-Cre mice, and 11 poly(I:C)-treated Nfat5^fl/fl LysM-Cre mice. Results for ESAM, annexin V, and CD41 analyses in E and G are from one experiment with two untreated mice of each genotype, and five wild-type and six NFAT5-deficient poly(I:C)-treated mice. Results for phospho-STAT1 in H are from two independent experiments. (J) Percentage of HSCs in the indicated cell cycle phases after poly(I:C) treatment as in C in wild-type (three untreated, six treated), NFAT5-deficient (Nfat5^fl/fl Vav-Cre, n = 4), IFNAR-deficient (Nfat5^+/+ Vav-Cre, Ifnar1^−/−, n = 6), or NFAT5- and IFNAR-deficient mice (Nfat5^fl/fl Vav-Cre, Ifnar1^−/−, n = 4). Results (mean ± SEM) are from four independent experiments with one to two mice of each genotype per condition and experiment. Statistical significance in A–J was determined with an unpaired t test. *, P < 0.05; **, P < 0.01. ** in J is for the difference between wild-type and NFAT5-deficient HSCs in the percentage of cells in G0 and G1 upon poly(I:C) treatment in vivo. (K) Schematic diagram of the poly(I:C) conditioning and 5-FU challenge experiment (left), and Kaplan-Meier representation (right) of survival of Nfat5^+/+ LysM-Cre (WT) and Nfat5^fl/fl LysM-Cre (KO) mice under this protocol (13 WT mice treated with 5-FU without poly(I:C) conditioning, 14 WT mice treated with 5-FU after poly(I:C), 12 knockout mice treated with 5-FU without poly(I:C), and 12 knockout mice treated with 5-FU after poly(I:C)). Results are from two independent experiments. Statistical significance in K was determined with a Mantel-Cox test. ***, P < 0.001.