Extended Data Fig. 8: Inhibiting LTβR-signalling suppresses cigarette-smoke-induced apoptosis.

a, Representative images of immunohistochemical analysis for cleaved caspase-3 in lung sections from healthy participants and patients with COPD (n = 5, brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 50 μm. b, Quantification of alveolar epithelial cells positive for cleaved caspase-3 from the lung sections stained in a. Data are mean ± s.d. (n = 5 patients per group). P = 0.0079, Mann–Whitney two-sided test. c, d, GSEA of apoptosis (Hallmark collection) in transcriptomic array data from publicly available array data of lung tissue (GSE47460–GPL14550) from healthy participants (n = 91) versus patients with COPD (n = 145) (c) and the lungs of B6 mice after exposure for 6 months to filtered air, cigarette smoke plus Ig, or cigarette smoke plus LTβR–Ig fusion protein therapeutically (n = 3 mice per group) (d). e, Box and whiskers plot (box representing 25th–75th percentile, median line indicated and Tukey whiskers representing ± 1.5 × IQR) showing the relative score for apoptosis (Hallmark collection) in AT2 cells after scRNA-seq analysis of lungs from B6 mice after exposure for 6 months to filtered air (n = 3 mice per group), cigarette smoke plus Ig (n = 5 mice per group) or cigarette smoke plus LTβR–Ig fusion protein (n = 5 mice per group) therapeutically. Statistical significance was determined by Wilcoxon rank-sum two-sided test on normalized, log-transformed count values and corrected with Benjamini–Hochberg. f, g, Proteome analysis of whole lung lysates from mice exposed to filtered air (n = 6) or cigarette smoke for 6 months, plus LTβR–Ig fusion (n = 7) or control Ig (n = 4) (80 μg intraperitoneally, weekly) from 4 to 6 months was undertaken. f, Heat map of the significantly regulated proteins from the Hallmark apoptosis list as determined by Student’s two-sided t-test. g, GSEA of the Hallmark apoptosis list on the normalized proteome data. h, Representative images of immunohistochemical analysis for cleaved caspase-3 in lung sections from B6 mice exposed to filtered air or cigarette smoke for 4 and 6 months, plus LTβR–Ig fusion protein or control Ig (80 μg intraperitoneally, weekly) prophylactically from 2 to 4 months and analysed at 4 months, and therapeutically from 4 to 6 months and analysed at 6 months (n = 4 mice per group, brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 50 μm. Quantification of cleaved caspase-3-positive alveolar epithelial cells from the immunohistochemistry sections also shown. i, Western blot analysis for cleaved caspase-3 (c-Cas-3) in total lung homogenate from mice described in h, quantification relative to β-actin (prophylactic groups: FA n = 7, CS+Ig n = 7, CS+LTβR-Ig n = 6 mice per group, therapeutic groups: FA n = 6, CS+Ig n = 5, CS+LTβR-Ig n = 6 mice per group, pooled from two independent experiments), individual mice shown. For gel source data see Supplementary Fig. 1. j–l, The mouse AT2-like cell line LA4 was stimulated with LTβR-Ag (2 μg ml⁻¹), recombinant mouse TNF (1 ng ml⁻¹) or a combination of both, in the presence or absence of necrostatin-1 (Nec1, 50 μM) (j) and (k) or Z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD, 20 μM) (l). Apoptosis was assessed at 6 h (j–l) and 24 h (k, l) by flow cytometric analysis of annexin V and propidium iodide (PI) staining (n = 2–3, repeated twice, pooled data shown in k). m, n, Wound-healing assay in LA4 cells grown to confluence, scratched and then incubated with LTβR-Ag (2 μg ml⁻¹), recombinant mouse TNF (1 ng ml⁻¹) or a combination of both, in the presence or absence of necrostatin-1 (50 μM). m, Representative images at 0 h and 56 h after scratch are shown (n = 4 from one experiment). Scale bar, 200 μm. n, Degree of wound closure (100% representing fully closed) at 56 h (n = 4). Data are mean ± s.d. P values determined by one-way ANOVA multiple comparisons Bonferroni test (h, i, k, l, n).

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