Figure 3.

STING promotes progression of AAPV. (A) Cell-free DNA in the BALF of mice treated as indicated. Each dot represents an individual mouse. Data is representative of two experiments and 3–4 mice/group. (B) Cell-free mtDNA (16S or Nd1) to nuclear (n) DNA (HK29) ratio obtained by qRT-PCR in BALF of AAPV mice at the indicated time points after disease induction. Data is representative of two independent experiments (n = 3–5 mice/group). (C) Confocal microscopy images of neutrophils treated as indicated and stained with anti-MPO mAbs. Scale bar, 50 µm. Arrowheads indicate extracellular MPO-decorated DNA. (D) Gel electrophoresis of gDNA, DNA isolated from BALF of mice 3 d after treatment as indicated, or 60mer TREX1-protected phosphorylated DNA treated or not with DNases ex vivo as indicated. Data is representative of two experiments with pooled DNA isolated from three mice per group. (E) cGAS activation assay using recombinant cGAS, ATP, and either dsDNA (lane 2), gDNA (lane 13), or DNA isolated from the BALF of mice treated 3 d earlier with LPS (lanes 3–5), fMLP/LPS (lanes 6–8) or fMLP/LPS + anti-MPO (lanes 9–12). Each lane represents an independent DNA sample. (F) cGAMP quantification in the BALF of mice treated as indicated 3 d earlier. Shown are pooled data from two independent experiments (n = 5-14 mice/group), each dot representing a single mouse. LOD, limit of detection. (G) Schematic of experimental design in wild-type or Sting^gt/gt mice. (H and I) Kinetics of weight change (H) and SpO₂ (%) in peripheral blood (I) of mice treated as in G. Shown are pooled data from two independent experiments (n = 10 [H] or 5 [I] mice/group). (J) Confocal microscopy images of unfixed vibratome lung slices showing alveoli (a) and a medium-sized pulmonary blood vessel (v) stained in vivo with anti-CD31 and ex vivo with propidium iodide. Scale bar = 100 µm. (K) Representative photographs and quantification of pulmonary hemorrhages in BALF. Treatments and time points as in G. Shown are pooled data from three independent experiments (n = 11–13 mice/group). (L) H&E staining of lung sections and lung injury score (0–4) for mice treated as indicated. Each dot is an individual mouse from two pooled independent experiments (n = 5–9 mice/group). Scale bar, 500 µm. Bars and line graphs represent mean ± SEM. Asterisks indicate the significance level of unpaired t test (F and K), one-way ANOVA with Kurskal–Wallis multiple comparisons test (B) or Tukey’s multiple comparisons test (L) and two-way ANOVA with Šídák's multiple comparisons test (A, H, and I). *P < 0.05; **, P < 0.01.***, P < 0.001.