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 6m FA, CS+Ig or CS+LTβR-Ig fusion protein therapeutically (n=3 mice/group) and publically available array data from lung tissue (GSE47460-GPL14550) of healthy (n=91) v COPD patients (n=145). b, Representative images of immunohistochemical analysis for Axin2 in lung sections from healthy (n=6) and COPD patients (n=8), brown signal indicated by arrow heads, haematoxylin counter stained, scale bar 50μm. c, mRNA expression levels of Nkd1 and Lgr5 relative to Hprt in primary murine alveolar type 2 epithelial cells (AT2) treated with agonistic antibody to LTβR (LTβR-Ag, 2 μg/ml) for 24h +/− murine rWNT3A (100ng/ml) (n=5 individual experiments). d, mRNA expression levels of Tcf4 relative to Hprt in the murine AT2 like cell line - LA4 stimulated with LTβR-Ag (2 μg/ml) or recombinant murine TNF (1 ng/ml) (n=3, repeated three times). e, mRNA expression levels of Tcf4 relative to Hprt in LA4 cells stimulated with LTβR-Ag (2 μg/ml) plus recombinant murine TNF (1 ng/ml) +/− necrostatin-1 (Nec1, 50 μM), 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 (Veh.), in the human AT2 cell line A549 treated with human LTβR-Ag (0.5 μg/ml) for 24h +/− TPCA-1 (5 μM) (n=3 independent experiments). g. Wnt/β-catenin luciferase reporter activity in the murine AT2 cell line MLE12, activated by GSK-3β inhibitor (CHIR99021, 1 μM) and treated with LTβR-Ag at the concentrations indicated for 24h (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) for 24h +/− murine rWNT3A (100ng/ml) 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 24h with recombinant human TNF (20 ng/ml) or agonistic antibody to human LTβR (LTβR-Ag, 2 μg/ml) for 24h (n=5 slices from individual lungs). j, Western blot analysis for β-catenin in MLE12 cells treated with murine rWNT3A (200ng/ml) and TNFSF14 (200ng/ml) for 30h. 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), porcine pancreatic elastase (PPE, 40 U/kg body weight) (n=7 mice/group) or PPE followed by LTβR-Ig fusion protein (80 μg i.p., weekly) 28d later for 2m and all analysed after 3m (n=8 mice/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 Tcf4 and Axin2 in lung sections from the mice descibed (n=4 mice/group, brown signal indicated by arrow heads, haematoxylin counter stained, scale bar 25μm). m, Quantification of alveolar epithelial cells positive for Tcf4 and Axin2 from (l). Data shown individual lungs (c, i) or mean ± SD (d-h, j-k and m). P values indicated, paired Student’s t test (one-tailed (c), two-tailed (i)), two-tailed Student’s t test (h, j) or one-way ANOVA multiple comparisons Bonferroni test (d-g, g compared to vehicle, k and m).