Extended Data Fig. 9: LTβR stimulation regulates WNT/β-catenin-signalling.

a, GSEA of canonical WNT signalling (GO: 0060070) and β-catenin/TCF transcription factor complex assembly (GO:1904837) in transcriptomic array data from the lungs of B6 mice after 6 months exposure to filtered air, cigarette smoke plus Ig, or cigarette smoke plus LTβR–Ig fusion protein therapeutically (n = 3 mice per group) and publicly available array data from lung tissue (GSE47460–GPL14550) of healthy participants (n = 91) versus patients with COPD (n = 145). b, Representative images of immunohistochemical analysis for AXIN2 in lung sections from healthy participants (n = 6) and patients with COPD (n = 8) (brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 50 μm. c, mRNA expression levels of Nkd1 and Lgr5 relative to Hprt in primary mouse AT2 cells treated with LTβR-Ag (2 μg ml⁻¹) for 24 h, with or without mouse rWNT3A (100 ng ml⁻¹) (n = 5 individual experiments). d, mRNA expression levels of Tcf4 relative to Hprt in the LA4 cells stimulated with LTβR-Ag (2 μg ml⁻¹) or recombinant mouse TNF (1 ng ml⁻¹) (n = 3, repeated three times). e, mRNA expression levels of Tcf4 relative to Hprt in LA4 cells stimulated with LTβR-Ag (2 μg ml⁻¹) plus recombinant mouse TNF (1 ng ml⁻¹) with or without necrostatin-1 (50 μM), and the IKK kinase inhibitors TPCA-1 (10 μM) or BAY 11-7082 (10 μM) (n = 2, repeated twice). f, mRNA expression levels of AXIN2 relative to HPRT and normalized to vehicle, in human A549 cells treated with human LTβR-Ag (0.5 μg ml⁻¹) for 24 h with or without TPCA-1 (5 μM) (n = 3 independent experiments). g, WNT/β-catenin luciferase reporter activity in mouse MLE12 cells, activated by GSK-3β inhibitor (CHIR99021, 1 μM) and treated with LTβR-Ag at the concentrations indicated for 24 h (activity relative to CHIR alone, n = 2–9). h, Western blot analysis for β-catenin in MLE12 cells treated with LTβR-Ag (2 μg ml⁻¹) for 24 h with or without mouse rWNT3A (100 ng ml⁻¹) plus bortezomib (10 nM). Quantification relative to actin shown (n = 3 independent experiments). For gel source data, see Supplementary Fig. 1. i, mRNA expression levels of TCF4 relative to HPRT in ex vivo human precision-cut lung slices stimulated for 24 h with recombinant human TNF (20 ng ml⁻¹) or LTβR-Ag (2 μg ml⁻¹) for 24 h (n = 5 slices from individual lungs). j, Western blot analysis for β-catenin in MLE12 cells treated with mouse rWNT3A (200 ng ml⁻¹) and TNFSF14 (200 ng ml⁻¹) for 30 h. Quantification relative to vinculin shown (n = 3 independent experiments). For gel source data, see Supplementary Fig. 1. k–m, B6 mice were treated with a single oropharyngeal application of PBS (n = 8), PPE (40 U kg⁻¹ body weight) (n = 7 mice per group) or PPE followed by LTβR–Ig fusion protein (80 μg intraperitoneally, weekly) 28 days later for 2 months and all analysed after 3 months (n = 8 mice per group); see Extended Data Fig. 7a. k, mRNA expression levels of Axin2, Bcl9l, Cdh1, Dvl1, Gsk3b, Rab5a, Tcf4, Wif1, Wnt2 and Wnt4 relative to Hprt, determined by qPCR in whole lung. l, Representative images of immunohistochemical analysis for TCF and AXIN2 in lung sections from the mice described (n = 4 mice per group, brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 25 μm. m, Quantification of alveolar epithelial cells positive for TCF4 and AXIN2 from l. Data shown as individual lungs (c, i) or mean ± s.d. (d–h, j, k and m). P values determined by one-tailed (c) or two-tailed (i) paired Student’s t-test, two-tailed unpaired Student’s t-test (h, j) or one-way ANOVA multiple comparisons Bonferroni test (d–g (compared to vehicle in g), k, m).

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