Fig 1: Stat1 signaling transactivates Irf1 in lung endothelial cells(A) Representative immunoblots and densitometric quantification of Stat1Ser727 phosphorylation levels in MLECs isolated from Ifr1fl/fl control mice pre- and post-LPS. (B) Representative immunoblots and densitometric quantification of Stat1Ser727 phosphorylation levels in MLECs in Ifr1fl/fl control mice and endothelial cell-specific Irf1 knockout mice at baseline. (C) Representative immunoblots and densitometric quantification of Stat1Ser727 phosphorylation and Irf1 protein levels in the indicated MLEC cell lines following LPS (10 μg/ml) treatment. (D) Representative immunoblots and densitometric quantification of STAT1Ser727 phosphorylation and IRF1 protein levels in the indicated HLMVEC cell lines following LPS (10 μg/ml) treatment. (E) Schematic of the human IRF1 promoter region depicting the two highly-conserved STAT1 binding site at −44 ∼ −56 bp and −177 ∼ −188 bp. The WT and MUT GAS1 sequences used in panel (G) are provided. (F) HLMVECs subjected to vehicle or LPS conditions for 8 hours, followed by ChIP-qPCR assays for detection of STAT1 binding to the two binding sites within the IRF1 promoter region. (G) HLMVECs co-transfected with control or STAT1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or 6 independent biological replicates per group. Data represented as means ± SDs. *P<0.05, **P<0.01 [(A, C, D) one-way ANOVA with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests; (B) two-tailed Student’s t-tests; (E, H, I) two-way ANOVA with Bonferroni post-hoc tests].
Fig 2: Irf1 overexpression inhibits lung endothelial regeneration in a Lif-dependent manner(A) Confocal microscopy illustrating FLAG staining along with CD31+ and DAPI staining in lung cryosections obtained from mice receiving either a scrambled control (Ctrl), FLAG-Irf1, or FLAG-Irf1/shLif construct. Scale bar = 50 and 20 μm (enlarged panel). Co-localization coefficient for FLAG-Irf1 staining in CD31+ MLECs. (B) FLAG-Irf1 and Lif protein levels in MLECs derived from mice receiving either a Ctrl, FLAG-Irf1, or FLAG-Irf1/shLif construct. (C) Lung vessel permeability pre- and post-LPS in mice receiving either a Ctrl, FLAG-Irf1, or FLAG-Irf1/shLif construct. (D) Flow cytometric analysis of CD31+CD45− endothelial cells pre- and post-LPS in mice receiving either a Ctrl, FLAG-Irf1, or FLAG-Irf1/shLif construct. (E) BrdU+ MLEC quantification in lung cryosections obtained from mice receiving either a Ctrl, FLAG-Irf1, or FLAG-Irf1/shLif construct (n = 6 cryosections per mouse). (F) Survival curves following LPS challenge in mice receiving either a Ctrl, FLAG-Irf1, or FLAG-Irf1/shLif construct (n = 30 mice per group). All experiments except (F): n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. *P<0.05, **P<0.01 [(A–E) two-way ANOVA with Bonferroni post-hoc test; (F) Log-rank test].
Fig 3: LPS induces Irf1-mediated Lif transactivation in lung endothelial cells(A) Venn analysis identifying profoundly up-regulated Irf1 target genes in murine lung endothelial cells (MLECs). (B) Web-based term association analysis to identify which Irf1 target gene(s) have the strongest association with endothelial regeneration-associated search terms. (C) qPCR analysis of Irf1 and Lif gene expression in MLECs isolated from mice pre- and post-LPS. (D) Representative immunoblots and densitometric quantification of Irf1 and Lif protein levels in MLECs pre- and post-LPS challenge. (E) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) isolated from mice pre- and post-LPS. (F) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs pre- and post-LPS challenge. (G) qPCR analysis of IRF1 and LIF gene expression in human lung microvascular endothelial cells (HLMVECs) transfected with control or IRF1 vector. (H) Representative immunoblots and densitometric quantification of IRF1 and LIF protein levels in HLMVECs with control vector or IRF1 overexpression. (I) Schematic of the human LIF promoter region depicting the highly-conserved IRF1 ISRE binding site at −102 ∼ −127 bp. The WT and MUT ISRE sequences used in panel (K) are provided. (J) HLMVECs transduced with either control or IRF1 plasmids, subjected to vehicle or LPS conditions for 8 h, followed by ChIP-qPCR assays for detection of IRF1 binding to the −102 bp binding site within the LIF promoter region. (K) HLMVECs co-transfected with control or IRF1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. **P<0.01 [one-way ANOVA (C) and two-way ANOVA (E–H) with Bonferroni post-hoc tests].
Fig 4: Endothelial Irf1 knockout in mice inhibits lung endothelial cell regeneration(A) Schematic illustration depicting Irf1fl/fl × Cdh5-CreERT2 mice crosses to construct endothelial cell-specific Irf1 knockout mice (Irf1EC−/−). Tamoxifen was administered for five consecutive days, left to rest for 4 weeks prior to a sub-lethal (8 mg/kg) LPS challenge. (B) Irf1 protein levels in endothelial cells isolated from flushed lungs from control and Irf1EC−/− mice. (C) Quantification of protein levels. (D) Time course illustrating lung vessel permeability post-LPS in control and Irf1EC−/− mice. (E) Flow cytometric analysis of CD31+CD45− endothelial cells in mice post-LPS injury. (F) Representative images of BrdU-APC, CD31-AF488, and DAPI co-staining in lung cryosections obtained from control Irf1fl/fl mice and Irf1EC−/− mice 3 days post-LPS. BrdU+ MLEC quantification in lung cryosections obtained from mice post-LPS (n = 10 cryosections per mouse). (G) Re-introduction of Irf1 through liposome vector plasmids (50 μg) in Irf1EC−/− mice post-LPS. (H) Lung vessel permeability at day 3 following LPS challenge in Irf1EC−/− mice with re-introduced Irf1. (I) Flow cytometric analysis of CD31+CD45− endothelial cells at day 3 following LPS challenge in Irf1EC−/− mice with re-introduced Irf1. (J) BrdU+ MLEC quantification in lung cryosections obtained at day 3 following LPS challenge in Irf1EC−/− mice with re-introduced Irf1 (n = 10 cryosections per mouse). All experiments: n = 6 mice or six independent biological replicates per group. Data represented as means ± SDs. *P<0.05, **P<0.01 [two-tailed Student’s t-tests (C, J) and two-way ANOVA (D, E–H) with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests].
Fig 5: R-based bioinformatics analysis identifies Irf1 as a key up-regulated gene in LPS-exposed murine lung endothelial cellsR-based bioinformatics analysis of published microarray data (GEO acc no. GSE5883) in which cultured human lung microvascular endothelial cells (HLMVECs) were left untreated (Ctrl) or exposed to LPS (10 ng) for 4 h (n = 4 biological replicates per group). (A) Gene set enrichment analysis (GSEA)-based identification of two discreet gene co-expression modules associated with LPS exposure: M1 and M2. Red coloring denotes a positive NES score, while blue coloring denotes a negative NES score. (B) Network plots for the two gene co-expression modules M1 and M2. (C) Reactome enrichment analysis for the two gene co-expression modules M1 and M2. The color saturation denotes the prediction confidence. (D) Volcano plots of differentially expressed genes (DEGs) with staining for M1 module membership (blue, top panel) or M2 module membership (pink, bottom panel). (E) Heatmap of the top 50 M2 module up-regulated DEGs ordered by descending log2 fold-change. Up-regulation is denoted by green coloring, while down-regulation is denoted by red coloring. IRF1 is marked by a red rectangle.
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