Supplementary Material
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Supplementary materialERJ-00174-2018.Supplement
Supplementary figure S1: Gating strategy for lung immune cell subsets. a) CD4+T cells, b) γδ T cells and NKT cells and c) ILC3.ERJ-00174-2018.Figure_S1
Supplementary figure S2: IL-22 and receptor mRNA in human peripheral lung tissue is unchanged in mild emphysema. Microarray data from peripheral lung tissue of patients with mild emphysema (Accession: GSE8581). a) IL-22, b) IL-22RA1, c) IL-22RA1 and d) IL-10RB. Data are represented as log2intensity robust multi-array average signals.ERJ-00174-2018.Figure_S2
Supplementary figure S3: No correlation between smoking pack-years and IL-22 or receptor expression. a) IL-22, b) IL-22RA1, c) IL-22RA2.ERJ-00174-2018.Figure_S3
Supplementary figure S4: No change in IL-22 or receptors in bronchial brushings in lung cancer. Microarray data from bronchial brushings in lung cancer (Accession: GSE4115). a) IL-22, b) IL-22RA1, c) IL-10RB. Data are represented as log2intensity robust multi-array average signals.ERJ-00174-2018.Figure_S4
Supplementary figure S5: No change in IL-22 or receptors in lung tissue in lung cancer. Microarray data from lung tissue in lung cancer (Accession: GSE1650). a) IL-22, b) IL-22RA1, c) IL-10RB. Data are represented as log2intensity robust multi-array average signals.ERJ-00174-2018.Figure_S5
Supplementary figure S6: Representative images of IL-22 and IL-22 receptor staining in human lung tissue. The four panels are showing the representative images of IL-22 (upper panel), IL-22RA1 (upper middle panel), IL-22RA2 (lower middle panel) and IL-10RB (lower panel) immunohistochemical staining in human peripheral lung tissue. a) and d) Age- and smoke history-matched control smokers with normal lung function. b) and e) Mild-to-moderate stable COPD. Upper lane images show the bronchiolar epithelium whereas lower lanes the alveolar macrophages. Representative images of positive control tissues (tonsils for IL-22, IL-22RA1 and IL-10RB), normal kidney for IL-22RA2 (kindly provided respectively by Prof Stefano Pelucchi and Prof Carmelita Di Gregorio) were stained with primary antibody (c) or with nonspecific immunoglobulin (Ig)G (negative control, f). Total magnification: 1000× (a, b, d, e; bar = 20 μm) or 200× (c, f; bar = 100 μm).ERJ-00174-2018.Figure_S6
Supplementary figure S7: Increased IL-22RA1 protein intensity in the airway epithelium of smokers with COPD. a) IL-22RA1 protein intensity per micrometre (μm) of basement membrane (BM) in non-smokers, healthy smokers without COPD and COPD with or without current smoking separated into GOLD stage 2 and GOLD stage 3–4. b) IL-22RA1 intensity in airway epithelium of non-smokersversussmokers with COPD. c) Representative images of IL-22RA1 positive staining, with red staining in the airway epithelial cells indicating IL-22RA1 positive staining.ERJ-00174-2018.Figure_S7
Supplementary figure S8: IL-22 protein levels are unaltered in the lungs of mice exposed to CS for 1 week. Wild-type (WT) C57BL/6 mice were exposed to normal air or CS for 1 week. IL-22 protein levels in lung homogenates were assessed by ELISA. Data are presented as mean±SEM, n=6, with another independent experiment showing similar results. Two-tailed Mann–Whitney t-test was used to analyse differences between two groups.ERJ-00174-2018.Figure_S8
Supplementary figure S9: Representative images of IL-22RA1 and IL-22RA2 protein in mouse lung tissue sections. Wild-type (WT) C57BL/6 mice were exposed to normal air or CS for 8 weeks. Representative images of negative control (top row), IL-22RA1 and IL-22RA2 staining in mouse lung tissue sections from normal air- (left) and CS-exposed (right) mice.ERJ-00174-2018.Figure_S9
Supplementary figure S10:Representative fluorescence-activated cell sorting (FACS) plots of IL-17A+and IL-22+CD4+T-cells; IL-17A+and IL-22+γδ T-cells; IL-17A+and IL-22+NKT-cells; and IL-17A+and IL-22+ILC3s.ERJ-00174-2018.Figure_S10
Supplementary figure S11: IL-17A, MPO and neutrophil elastase protein levels are increased in experimental COPD, but not in the absence of IL-22. Wild-type (WT) and IL-22-deficient (Il22-/-) C57BL/6 mice were exposed to normal air or CS for 8 weeks to induce experimental COPD. a) IL-17A, b) MPO and c) neutrophil elastase protein levels in lung homogenates. Data are presented as mean±SEM, n=6, with another independent experiment showing similar results. The one-way analysis of variance with Bonferroni post-test analysed differences between 3 or more groups. *: p<0.05 compared to normal air-exposed controls. ns: not significant.ERJ-00174-2018.Figure_S11
Supplementary figure S12: CS induced non-significant reductions in tissue elastance that was not different in (Il22-/-mice. Wild-type (WT) and IL-22-deficient ((Il22-/-) C57BL/6 mice were exposed to normal air or CS for 8 weeks to induce experimental COPD. Lung function was assessed in terms of tissue elastance. Data are presented as mean±SEM, n=6, with another independent experiment showing similar results. The one-way analysis of variance with Bonferroni post-test analysed differences between 3 or more groups. ns: not significant.ERJ-00174-2018.Figure_S12