Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 and Th9–associated markers are elevated in TS patients. A Representative IHC pictures showing PU.1, IRF4, and IL-9 expression in TS patients’ tracheal tissues. IL-9 staining was single-color IHC, CD4/IL-9 co-localization was not assessed. B Quantitative analysis of IHC-positive regions showing PU.1, IRF4, and IL-9 expression in TS tissues ( n = 3). C qRT-PCR analysis of TS tissues and blood samples ( n = 10) for cytokines and fibrosis markers. D , E WB analysis of TS tissues for IFN-γ, fibrosis indicators, IL-9, and IL-4. F ELISA data showing collagen levels in the blood of TS patients. To determine statistical significance, unpaired t-tests were used. In comparison to controls, * P < 0.05, ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Expressing, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Th1/Th2 cytokine balance is skewed in patients with TS. A Method for gating peripheral blood samples and BALF CD3 + CD4 + T cells. Representative FCM plots showing the populations of IL-9 + , IL-4 + , and IFN-γ + CD4 + T cells in the control and TS groups are shown in ( B , D , and F ). C , E , and G Quantitative evaluation of IL-9, IL-4, and IFN-γ expression in CD4 + T cells ( n = 10). Unpaired t -tests were used to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. controls. TS: traumatic tracheal stenosis; BALF: bronchoalveolar lavage fluid; FCM: flow cytometry

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Expressing, Flow Cytometry

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 promotes fibroblast proliferation and activation via the TGF-β1 pathway in vitro. A Relative growth rate of HTFs treated with IL-9, IL-4 + TGF-β1, anti-IL-9, or SB at 24 h and 48 h, assessed by CCK-8 assay ( n = 5). B and C FCM histograms showing collagen I expression in CD3 − CD4 − HTFs following individual stimulation with IL-9, IL-4 + TGF-β1, anti-IL-9, or SB treatment. D and E ELISA and qRT-PCR analysis of collagen I secretion and COL1A1 mRNA expression in HTFs treated as above ( n = 5). F and G WB analysis of collagen III, collagen I, and α-SMA protein expression in HTFs following the same treatments ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. HTF: human tracheal fibroblast; SB: SB-431,542; FCM: flow cytometry; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; WB: western blot; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Activation Assay, In Vitro, CCK-8 Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 blockade suppresses TGF-β1-induced fibroblast activation and collagen I production in vitro. A FCM histograms depict collagen I expression in CD3 − CD4 − HTFs subjected to individually applied IL-9, IL-4, TGF-β1, anti-IL-9, anti-IL-4, anti-TGF-β1, IL-9 + TGF-β1, or anti-IL-9 + TGF-β1 treatment. B Quantitative FCM analysis of collagen I levels in CD3 − CD4 − HTFs treated as above ( n = 5). C – E qRT-PCR analysis of COL3A1, COL1A1, and α-SMA mRNA expression in HTFs treated with the aforementioned agents ( n = 5). F and G WB analysis of collagen III, collagen I, and α-SMA protein expression in HTFs treated as above ( n = 3). H and I IF analysis of α-SMA and collagen I expression in HTFs following treatment with the conditions described above ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. FCM: flow cytometry; qRT-PCR: quantitative real-time polymerase chain reaction; WB: western blot; IF: immunofluorescence; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates Th1/Th2 cytokine profiles via TGF-β1 signaling in vitro . A , C , E , and G FCM scatter plots illustrate the distribution patterns of IL-9 + CD4 + , IL-4 + CD4 + , TGF-β1 + CD4 + , and IFN-γ + CD4 + T cell subsets across various treatment groups, including control, IL-9 alone, IL-4 + TGF-β1 co-stimulation, anti-IL-9 antibody alone, and SB alone. B , D , F , and H Quantification of FCM data for IL-9 + , IL-4 + , TGF-β1 + , and IFN-γ + CD4 + T cells in the treatment groups mentioned above ( n = 5). I and J ELISA and qRT-PCR analyses assessing protein and mRNA levels of IL-9, IL-4, IL-13, and IFN-γ following treatment with IL-9 or IL-4 + TGF-β1 co-stimulation, anti-IL-9 antibody, or SB ( n = 5). K and L WB analysis of IL-9, IL-4, IL-13, TGF-β1, and IFN-γ protein expression in the different treatment groups ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. FCM: flow cytometry; ELISA: enzyme-linked immunosorbent assay; qRT-PCR: quantitative real-time polymerase chain reaction; SB: SB-431,542; WB: western blot; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Th9 blockers inhibit collagen secretion by fibroblasts via the TGF-β1/SMAD2/3 pathway. A Representative WB results display the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and total SMAD2/3 proteins in samples from the control, IL-9, IL-4 + TGF-β1, anti-IL-9, and SB treatment groups. B Quantification of p-SMAD2/3 and SMAD2/3 protein expression following treatment as described in ( A ) ( n = 3). C Representative WB results show the levels of p-SMAD2/3 and SMAD2/3 proteins in samples from the control, IL-9, IL-4, TGF-β1, anti-IL-9, anti-IL-4, anti-TGF-β1, IL-9 + TGF-β1, and anti-IL-9 + TGF-β1 treatment groups. D and E Quantification of p-SMAD2/3 and SMAD2/3 protein expression following treatments as described in ( C ) ( n = 3). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. IL-9 group. WB: western blot; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Expressing, Western Blot

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates the cytokine microenvironment in TS airways via the TGF-β1/SMAD2/3 pathway. A Tracheal tissues from the control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups were immunohistochemically stained with IL-9, IL-4, IL-13, TGF-β1, and IFN-γ. IL-9 staining was single-color IHC, CD4/IL-9 co-localization was not assessed. Scale bar = 200 μm ( n = 3 per group). B The observed expression patterns were confirmed by quantitative analysis of the cytokine-positive region (%). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. TS: traumatic tracheal stenosis; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Staining, Expressing

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates IL-4, IFN-γ, and TGF-β1 in CD4 + T cells and reduces airway collagen I. A – F Representative FCM plots showing the percentages of CD3 − CD4 − collagen I + , IL-9 + , IL-4 + , IFN-γ + , and TGF-β1 + CD4 + T cells in various experimental groups ( n = 6). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. FCM: flow cytometry; SB: SB-431,542; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Control, Flow Cytometry

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: IL-9 modulates the cytokine microenvironment in TS airways via the TGF-β1/SMAD2/3 pathway. A and B WB analysis was performed to examine the expression of fibrosis-related proteins, including collagen III, collagen I, phosphorylated SMAD2/3, total SMAD2/3, and α-SMA, as well as cytokines TGF-β1, IL-9, IL-4, IL-13, and IFN-γ, in tracheal tissues from control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups ( n = 3). C qRT-PCR (tissue and blood) was used to assess the mRNA expression levels of IL-9, IRF4, PU.1, IL-4, IL-13, IFN-γ, COL3A1, and COL1A1 in the same experimental groups ( n = 6). D ELISA (BALF and blood) was used to assess the protein expression levels of IL-9, IL-4, IL-13, and IFN-γ in the same experimental groups ( n = 6). Statistical comparisons were performed using one-way ANOVA followed by Kruskal–Wallis or Newman–Keuls post hoc analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 vs. TS group. TS: traumatic tracheal stenosis; WB: western blot; SB: SB-431,542; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; bronchoalveolar lavage fluid; ANOVA: analysis of variance

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Expressing, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Respiratory Research

Article Title: Th9/IL-9 axis mediates airway fibrosis in traumatic tracheal stenosis via TGF-β1/SMAD2/3 signaling

doi: 10.1186/s12931-025-03431-2

Figure Lengend Snippet: Proteomic analysis of tracheal tissue from rats in five groups (rat TS model). Heatmap showing the patterns of protein abundance that differ across the five experimental groups. A Control, TS, TS + IL-9, TS + anti-IL-9, and TS + SB groups. B To identify important biological processes and functions, KEGG pathway and gene ontology enrichment analyses were conducted on differentially expressed proteins. C Grouped expression profiles showing patterns of grouped protein expression. TS: traumatic tracheal stenosis

Article Snippet: IL-9 and anti-IL-9 were administered daily (MedChemExpress) [ – ], while SB was administered every other day [ ].

Techniques: Quantitative Proteomics, Control, Expressing