Response to interferons and antibacterial innate immunity in the absence of tyrosine-phosphorylated STAT1

Abstract

Signal transducer and activator of transcription 1 (STAT1) plays a pivotal role in the innate immune system by directing the transcriptional response to interferons (IFNs). STAT1 is activated by Janus kinase (JAK)-mediated phosphorylation of Y701. To determine whether STAT1 contributes to cellular responses without this phosphorylation event, we generated mice with Y701 mutated to a phenylalanine (Stat1(Y701F)). We show that heterozygous mice do not exhibit a dominant-negative phenotype. Homozygous Stat1(Y701F) mice show a profound reduction in Stat1 expression, highlighting an important role for basal IFN-dependent signaling. The rapid transcriptional response to type I IFN (IFN-I) and type II IFN (IFNγ) was absent in Stat1(Y701F) cells. Intriguingly, STAT1Y701F suppresses the delayed expression of IFN-I-stimulated genes (ISG) observed in Stat1(-/-) cells, mediated by the STAT2/IRF9 complex. Thus, Stat1(Y701F) macrophages are more susceptible to Legionella pneumophila infection than Stat1(-/-) macrophages. Listeria monocytogenes grew less robustly in Stat1(Y701F) macrophages and mice compared to Stat1(-/-) counterparts, but STAT1Y701F is not sufficient to rescue the animals. Our studies are consistent with a potential contribution of Y701-unphosphorylated STAT1 to innate antibacterial immunity.

Keywords: STAT1; innate immunity; interferon; pathogen; phosphorylation.

Figure 1. Interferon signaling in mice bearing heterozygous Stat1^Y701F mutation resembles that of human cells with heterozygous Stat1^Y701C mutation

1. Western blot analysis of STAT expression and phosphorylation. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F/+ (WT/YF), or Stat1^−/− (S1) mice were treated with 250 IU/ml of IFNβ or 5 ng/ml of IFNγ for 0.5, 6, 12, and 24 h. Whole‐cell extracts were collected and tested in Western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than three independent experiments.

2. Effect of Stat1^Y701F heterozygosity on the expression of type I IFN‐induced genes (ISGs). BMDMs of wild‐type (WT), Stat1^Y701F/+ (WT/YF), or Stat1^−/− (S1) mice were treated with 250 IU/ml of IFNβ for 4 and 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.

3. Effect of Stat1^Y701F heterozygosity on the expression of IFNβ‐induced genes. BMDMs of wild‐type (WT), Stat1^Y701F/+ (WT/YF), or Stat1^−/− (S1) mice were treated with 5 ng/ml of IFNγ for 4 and 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.

Figure 2. STAT1 expression and interferon signaling in Stat1^Y701F mice

1. Effect of Stat1^Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers, or bone marrow‐derived macrophages (BMDMs) were isolated from wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice. Whole‐cell extracts were collected and tested for levels of total STAT1 in Western blot. The blots are representative of more than three independent experiments.

2. Effect of Stat1^Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice and stimulated for 30 min with 250 IU/ml of IFNβ or 5 ng/ml of IFNγ. Whole‐cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in Western blot. The blots are representative of more than three independent experiments.

3. Effect of Stat1^Y701F homozygosity on the expression of IFNβ‐induced genes. BMDMs of wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice were treated with 5 ng/ml of IFNγ for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, P > 0.05); and the P‐values were calculated using paired ratio t‐test.

4. Effect of Stat1^Y701F homozygosity on the expression of type I IFN‐induced genes (ISGs). BMDMs were isolated from wild‐type (WT), Stat1^Y701F, Stat1^−/−, Stat2^−/−, and Irf9^−/− mice treated with 250 IU/ml of IFNβ for 4, 8, 12, 24, or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in untreated wild‐type cells. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (*P ≤ 0.05; **P ≤ 0.01); and the P‐values were calculated using paired ratio t‐test.

5. STAT1 and STAT2 phosphorylation in Stat1^−/−, Stat1^Y701F, Stat2^−/−, and Irf9^−/− macrophages. BMDMs were isolated from wild‐type (WT), Stat1^Y701F (YF), Stat1^−/− (S1), Stat2^−/− (S2), and Irf9^−/− (IRF9) mice and treated with 250 IU/ml of IFNβ for 30 min or 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than three independent experiments.

Figure EV1. STAT1Y701F inhibits late STAT1‐independent, STAT2/IRF9‐dependent ISG expression in response to IFNβ

1. Expression of Stat1 and Stat2 genes. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were treated with 5 ng/ml of IFNγ (IFNg) for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells.

2. Expression of type I IFN‐induced genes. BMDMs of wild‐type (WT), Stat1^Y701F, Stat1^−/−, Stat2^−/−, and Irf9^−/− mice were treated with 250 IU/ml of IFNβ for 4 or 48 h. Gene expression was measured by qPCR and normalized to Gapdh and to the expression levels in uninduced wild‐type cells.

Data information: The bars in (A) and (B) represent a mean value of three independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote level of statistical significance (ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001); the P‐values were calculated using paired ratio t‐test.

Figure EV2. Deletion of ISGF3 subunits or Stat1^Y701F mutation is without effect on the expression or IFNβ‐stimulated phosphorylation of STAT3 and STAT5

Western blot analysis of STAT3 and STAT5 tyrosine phosphorylation. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F (YF), Stat1^−/− (S1), Stat2^−/− (S2), and Irf9^−/− (IRF9) mice were treated with 250 IU/ml of IFNβ for 0.5, 6, 12, or 24 h. Whole‐cell extracts were collected and tested in Western blot for total STAT3 and STAT5 amounts and for their tyrosine phosphorylation at Y705 and Y694, respectively. The blots are representative of more than three independent experiments.

Figure EV3. STAT1Y701F mutant does not bind to ISRE sequences after IFNβ treatment

Genomic analysis of STAT1 binding. ChIP‐seq analysis was performed on bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice. BMDMs were treated with 250 IU/ml of IFNβ (IFNb) for 2 h. Chromatin immunoprecipitation (ChIP) was performed using STAT1 antibody. Four genes are shown as representative examples. A genome‐wide search did not produce evidence of STAT1Y701F binding to ISREs elsewhere in the genome. ChIP‐Seq data for Stat1^Y701F mutant and Stat1^−/− samples are deposited at ArrayExpress under accession number E‐MTAB‐3597 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3597/) and for wild‐type Stat1 sample are deposited at ArrayExpress under accession number E‐MTAB‐2972 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2972/).

Figure 3. Presence of the STAT1Y701F mutant reduces IFNβ‐stimulated binding of STAT2 to nuclear ISRE sequences

1. IFNβ‐stimulated binding of STAT2 to ISRE sequences of Mx2 and Irf7 promoters. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F (YF), Stat1^−/− (S1), Stat2^−/− (S2), and Irf9^−/− (IRF9) mice were treated with 250 IU/ml of IFNβ for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT2‐specific antibody. The amount of precipitated DNA was measured by qPCR. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**P ≤ 0.01); and the P‐values were calculated using paired ratio t‐test.

2. Impact of STAT2 deficiency on IFNβ‐stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild‐type (WT), Stat1^−/− (S1), and Stat2^−/− (S2) mice were treated with 250 IU/ml of IFNβ for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with STAT1‐specific antibody. The amount of precipitated DNA was measured by qPCR. Bars represent mean values of three independent experiments; error bars represent standard error of the mean (SEM).

3. Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP‐reChIP. BMDMs of wild‐type (WT) mice were treated with 250 IU/ml of IFNβ for 2 or 24 h. Cells were cross‐linked, sonicated, and immunoprecipitated with either STAT1‐specific antibody and re‐immunoprecipitated with STAT2‐specific antibody or vice versa. The amount of precipitated DNA was measured by qPCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).

4. Impact of STAT1Y701F on delayed, STAT2‐mediated expression of IFN‐induced genes. Stat1^−/− fibroblasts were transfected with plasmids driving expression of the indicated proteins. Twenty‐four hours after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by qPCR after 48 h of cytokine treatment. Gene expression was measured by qPCR and normalized to Gapdh. Bars represent a mean value of three independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*P ≤ 0.05); the P‐values were calculated using paired ratio t‐test.

Figure 4. STAT1Y701F mutant reduces IFNβ‐stimulated nuclear translocation of STAT2

1. Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F (YF), Stat1^−/− (S1), Stat2^−/− (S2), and Irf9^−/− (IRF9) mice were seeded on cover slips and stimulated with 250 IU/ml of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2‐specific antibody followed by Alexa Fluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.

2. Quantitative evaluation of STAT2 nuclear translocation. The intensity of STAT2‐dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***P ≤ 0.001); P‐value was calculated using unpaired t‐test.

Figure 5. STAT1Y701F mutant counteracts the inhibition of Legionella pneumophila replication by delayed, STAT2/IRF9‐dependent IFN signaling

1. Legionella pneumophila growth in unstimulated macrophages. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were seeded in 24‐well plates and infected with Le. pneumophila (JR32 Fla−, MOI 0.25). The numbers of colony‐forming units (CFUs) were determined 24, 48, and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.

2. Legionella pneumophila growth in IFNβ‐treated macrophages. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were seeded in 24‐well plates, treated with 500 U/ml of IFNβ, and then infected with Le. pneumophila (JR32 Fla−, MOI 0.25). The numbers of colony‐forming units (CFU) were determined 24, 48, and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.

Data information: The results in (A) and (B) represent six biological repeats and the data are presented as mean values. Asterisks denote statistically significant differences between CFU numbers from Stat1^Y701F and Stat1^−/− cells (***P ≤ 0.001); P‐values were calculated using unpaired t‐test.

Figure 6. Interferon signaling in Listeria monocytogenes‐infected Stat1^Y701F bone marrow‐derived macrophages

1. Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole‐cell extracts were collected and tested in Western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine 701 (Y701). The blots are representative of more than three independent experiments.

2. Impact of Stat1^Y701F mutation, or of deletion of ISGF3 subunits on the expression of the Ifnb gene. BMDMs of wild‐type (WT), Stat1^Y701F, Stat1^−/−, Stat2^−/−, and Irf9^−/− mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24, or 48 h. Levels of Ifnb gene expression were determined by qPCR. Bars represent mean values of three independent experiments. Error bars represent standard error of the mean (SEM).

Figure 7. STAT1Y701F contributes to clearance of L. monocytogenes infection

1. Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages. Bone marrow‐derived macrophages (BMDMs) of wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were infected with L. monocytogenes (LO28, MOI 10). Colony‐forming unit (CFU) numbers were determined 1, 2, 4, 6, or 8 h after infection by plating on brain–heart infusion (BHI) agar plates. The graph represents biological triplicates and the data are represented as mean values. Error bars represent standard deviation (SD) and asterisks denote statistically significant differences (ns, P > 0.05; **P ≤ 0.01; ***P ≤ 0.001); P‐values were calculated using unpaired t‐test.

2. Survival of mice infected with L. monocytogenes. 10 wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice per group were infected by intraperitoneal injection of 1 × 10² viable L. monocytogenes. Survival was monitored over 10 days. The study is representative of more than three independent experiments.

3. Organ pathogen burdens of mice infected with L. monocytogenes. Wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice were infected by intraperitoneal injection of 1 × 10² viable L. monocytogenes. Number of colony‐forming units (CFU) in organs was determined at 48, 72 h, or at the terminal stage of infection by plating homogenates on brain–heart infusion (BHI) agar plates or Oxford agar plates (for lungs). Dots represent pooled data of three independent experiments. Lines represent the median and asterisks denote statistically significant differences (ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001); P‐values were calculated using Mann–Whitney U‐test.

Figure 8. Liver inflammation in Stat1^Y701F and Stat1^−/− mice after infection with Listeria monocytogenes

1. Immunohistochemical analysis of infection and inflammatory infiltrates. Wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were infected by intraperitoneal injection of 1 × 10² viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G‐specific antibody. The scale bars on 20× magnification images represent 100 and 50 µm on the 63× magnification images.

2. Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size.

3. Liver pathology in infected mice. Wild‐type (WT), Stat1^Y701F (YF), and Stat1^−/− (S1) mice were infected by intraperitoneal injection of 1 × 10⁵ viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*P ≤ 0.05; ***P ≤ 0.001); P‐values were calculated using unpaired t‐test.

Figure EV4. Immune cell infiltrates in Stat1^Y701F mice livers are smaller in numbers and size compared to Stat1^−/− livers, and are mostly composed of F4/80‐negative cells

Wild‐type (WT), Stat1^Y701F, and Stat1^−/− mice were infected by intraperitoneal injection of 1 × 10² L. monocytogenes for 48 h. Immunohistochemistry was performed on liver sections using F4/80‐specific antibody. The scale bars on 20× magnification images represent 100 and 50 µm on the 63× magnification images.