Fig 2: Schematic diagram of tracheal dsDNA activating cGAS-STING pathway of macrophages to promote fibrosis in BAS.Upon injury, dsDNA released from the tracheal epithelium activates the STING pathway in macrophages, leading to the release of the inflammatory cytokine IL-6. IL-6 then activates STAT3 signaling in fibroblasts, contributing to the fibrosis of granulation tissue. Treatment with the cGAS inhibitor RU.521, the STING inhibitor C176, or IL-6 neutralizing antibody alleviates granulation tissue fibrosis. Additionally, depletion of macrophages using bisphosphonate liposomes reduces the extent of fibrosis in the granulation tissue.

Fig 3: cGAS-STING signalling is activated in mouse tracheal upon injury.a Schematic diagram of tracheal lavage of the patient. b Quantification analysis of dsDNA in tracheal lavage fluid in control group (n = 5) and BAS patients’ group (n = 5). c Schematic diagram describing experimental strategies in mice with benign airway stenosis. d H&E staining showing Intratracheal and LP conditions in control and BAS mice at 7th day. Scale bars indicate 200μm and 50μm.e Quantitative analysis of LP thickness in control group and BAS group (n = 8 mice per group). f–j Tracheal tissues were collected from mice with BAS at 24 h after tracheal injury. f Quantitative detection of dsDNA in tracheal lavage fluid of mice in control group and BAS group (n = 5 mice per group). g Representative tracheal immunofluorescence images of dsDNA(green) in the BAS group and control group. Scale bars indicate 100μm and 20 μm (n = 3 mice per group). h Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showing that tracheal injury upregulated genes involved in several biological process. i Heatmap showing that the BAS group upregulated genes involved in cytosolic DNA sensor pathway compare to control group (n = 3 mice per group). j Western blot analysis showing the levels of cGAS, P-STING, STING, P-TBK1, TBK1, P-P65, and P65 in the trachea of BAS group and control group (n = 4 mice per group) at 24 h. k Schematic of Ru.521(5 mg/kg, ip) administration in mice. l H&E staining showing Intratracheal and LP conditions in control and BAS mice at 7th day. Scale bars indicate 200 μm and 50 μm.m Quantitative analysis demonstrated decreased lamina propria thickness in BAS group treated with Ru.521 compared to BAS group treated with PBS (n = 4 mice per group). Data are presented as the mean ± SEM. A two-sided student’s T-test was used in (b, e, f). hypergeometric test was used in (h). One-way ANOVA analysis followed by Tukey post hoc multi-comparison test was used in (m). Source data are provided as a Source Data file.

Fig 4: dsDNA activates the STING pathway in macrophages thereby promoting fibroblast activation via the IL6-STAT3 axis.a Gene ontology (GO) functional enrichment analysis showed that the enriched DNA damage and repair genes in the BAS group at 24 h after tracheal injury compare to control group (n = 3 mice per group). b Representative images of H&E staining and immunofluorescence of γH2AX(green) in mouse trachea(n = 3 mice per group). Scale bars indicate 50 μm in H&E staining images, 50 μm and 10 μm in immunofluorescence images. c Representative tracheal immunofluorescence images of dsDNA(pink), F4/80(red), P-STING (green), cGAS(yellow) in mouse trachea (n = 3 mice per group). Scale bars indicate 200 μm and 50 μm. d Schematic of use DECS to stimulate BMDMs. e Quantification analysis of dsDNA in cell culture supernatant in control group and DECS group (n = 4 independent experiments). f Representative images of AGAR gel electrophoresis of cell supernatants from control and DECS groups (n = 3 independent experiments). g, h Protein levels of phosphorylated STING in BMDM group stimulated with or without DECS, n = 3 independent experiments. i Schematic of BMDMs from WT mice stimulated by transfection of mouse tracheal DNA by lipo3000. j, k Protein levels of phosphorylated STING in the BMDM group stimulated with or without DNA (n = 3 independent experiments). l Representative immunofluorescence images of P-STING(green) in BMDM, Scale bars indicate 50 μm. m Quantitative analysis of relative Mean fluorescence intensity of P-STING (n = 5 independent experiments). o Protein levels of IL6 in the supernatant of BMDM group stimulated with or without DNA (n = 4 independent experiments). p mRNA levels of IL6 in control group, DNA stimulated group and DNA stimulated group treated with C176 (n = 4 independent experiments). q, r Representative tracheal immunofluorescence images of and quantitative analysis of mean fluorescence intensity of P-STAT3(green) and IL6(red) in the different groups (n = 3 mice per group). Scale bars indicate 200 μm and 20 μm. s Schematic diagram depicting in-vitro co-culture model. t, u Representative immunofluorescence images of and quantitative analysis of mean fluorescence intensity of P-STAT3(red) and αSMA(green) in fibroblasts (n = 3 independent experiments). Scale bars indicate 10 μm. Data are presented as the mean ± SEM. A two-sided student’s T-test was used in (e, h, k, o). One-way ANOVA analysis followed by Tukey post hoc multi-comparison test was used in (m, p, r, u). Source data are provided as a Source Data file.

Fig 5: Suppression of the STING attenuates the development of BAS.a Schematic of C176 administration in mice. b Western blot analysis showing the P-STING and STING expression in mouse trachea at 24 h after modeling in control group, BAS group treated with PBS and BAS group treated with C176 (n = 3 mice per group). c H&E staining showing Intratracheal and lamina propria conditions in control group, BAS group treated with PBS and BAS group treated with C176 (n = 4 mice per group). Scale bars indicate 200 μm and 50 μm. d Quantitative analysis demonstrated decreased lamina propria thickness in BAS group treated with C176 compared to BAS group treated with PBS (n = 4 mice per group). e H&E staining showing Intratracheal and lamina propria conditions in WT control group,STING KO control group, WT BAS group, STING KO BAS group (n = 5 mice per group). Scale bars indicate 200 μm and 50 μm. f Quantitative comparison of tracheal LP showed reduced thickness at STING KO BAS group compare to WT BAS group (n = 5 mice per group). g, h Tracheal sections stained with Masson trichromatic staining of different groups of mice, and relative collagen content were reduced in STING KO BAS group compare to WT BAS group (n = 5 mice per group).Scale bars indicate 200 μm and 50 μm. i The CT diagram of the area of tracheal stenosis measured by SYNPASE 3D, and results showed that the area of tracheal stenosis in STING KO BAS group was significantly smaller than that in WT BAS group (n = 5 mice per group). j Micro CT scans of different groups in Horizontal, Coronal and Sagittal plane with red arrows indicating the stenosis site. k SYNPASE 3D reconstruction of mouse trachea with red arrows indicating the stenosis site. Data are presented as the mean ± SEM. Two-sided student’s T-test were used in (b). One-way ANOVA analysis followed by Tukey post hoc multi-comparison test was used in (b, d). Two-way ANOVA analysis followed by Tukey post hoc multi-comparison test was used in (f, h, i). Source data are provided as a Source Data file.