Extended Data Fig. 5: Inhibition of LTβR signalling strongly reduces non-canonical but not canonical NF-κB signalling in lung.

a, Principal component analysis of microarray data, using Mouse Ref-8 v2.0 Expression BeadChips (Illumina), performed on lung tissue from mice exposed to filtered air or cigarette smoke for 6 months, plus LTβR–Ig fusion or control Ig (80 μg intraperitoneally, weekly) therapeutically from 4 to 6 months (n = 3 mice per group). b, Principal component analysis of normalized z-scored mass spectrometry intensities from proteomics 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. c, Heat map depicting the top 20 up and down LTβR–Ig regulated genes presented as fold change (FDR < 10%) from the microarray data described in a. Left, expression in mice exposed to cigarette smoke plus Ig relative to mice exposed to filtered air. Right, expression in mice exposed to cigarette smoke plus LTβR–Ig relative to mice exposed to cigarette smoke plus Ig. d, GSEA of the NIK (non-canonical) NF-κB signalling (GO:0038061) pathway of the microarray data from a. e, Heat map of significantly regulated proteins from the NIK (non-canonical) NF-κB signalling (GO:0038061) pathway as determined by Student’s t-test statistic from the proteomics data described in b. f, GSEA of the NIK (non-canonical) NF-κB signalling (GO:0038061) pathway of the normalized proteome data described in b. g, Representative images of two independent experiments of immunohistochemical analysis for RELB in lung sections from B6 mice exposed to filtered air or cigarette smoke for 4 or 6 months, plus LTβR–Ig fusion 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 (brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 25 μm. h, Quantification of RELB-positive alveolar epithelial nuclei from the immunohistochemistry sections in g (n = 3 mice per group). i, Representative images of two independent experiments of immunohistochemical analysis for RELA in lung sections from the mice described in g (brown signal indicated by arrowheads, nuclei counterstained with haematoxylin). Scale bar, 25 μm. j, Quantification of RELA-positive alveolar epithelial nuclei from the immunohistochemistry sections in i (n = 3 mice per group). k, mRNA expression levels of Ccl2, Ccl3, Cxcl1 and Tnf determined by qPCR in whole lung from the mice described in g (n = 4 mice per group, repeated twice, pooled data shown). l, mRNA expression levels of LTA, CXCL13 and TNF determined by qPCR in ex vivo human precision-cut lung slices stimulated for 24 h with LPS (10 μg ml⁻¹) in the presence or absence of human LTβR–Ig fusion protein (1 μg ml⁻¹) (n = 3 independent experiments from three separate lungs). Left image shows a representative picture of preparing a lung slice from the three independent experiments. Data are mean ± s.d. P values determined by one-way ANOVA multiple comparisons Bonferroni test.

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