Figures
- FIGURE 1
Phenotypic differences among severe asthma current smokers (CSA), severe asthma ex-smokers (ESA) and severe asthma nonsmokers (NSA) were unveiled using the SomaLogic linear model for microarray (limma) analysis of sputum. a–c) Volcano plots showing differentially expressed proteins (DEPs) in limma of sputum SomaLogic in the following comparisons: a) CSA and healthy nonsmokers (NH); b) ESA and NH; and c) NSA and NH. The proteins whose absolute fold change ≥2.0 at false discovery rate (FDR) <0.05 were regarded as DEPs. The number of DEPs of each comparison is shown in the left and right upper areas of each plot. d) Venn diagram showing the numbers and names of DEPs in each comparison.
- FIGURE 2
Differentially expressed proteins in severe asthma sputum according to smoking status. Dot plots with mean±sd showing signal intensity levels of protein expression of a) colony-stimulating factor (CSF)-2, b) anterior gradient protein (AGR)2, c) azurocidin (AZU)1, d) C-X-C motif chemokine ligand (CXCL)8, e) neutrophil elastase (ELANE), f) complement factor properdin (CFP) and g) catalase (CAT) in sputum by SomaLogic (Boulder, CO, USA) analysis in severe asthma current smokers (CSA), severe asthma ex-smokers (ESA), severe asthma nonsmokers (NSA) and healthy nonsmokers (NH). RFU: relative fluorescence units, *: p<0.05; **: p<0.01; ***: p<0.001.
- FIGURE 3
Differentially expressed genes (DEGs) in current smokers (CSA) and nonsmokers (NSA) with severe asthma. Volcano plots showing DEGs between CSA and NSA in a) sputa, b) bronchial biopsies and c) bronchial brushings. The genes whose absolute fold change (FC) ≥1.5 at a false discovery rate (FDR) <0.05 are shown as coloured dots. The number of DEGs in each sample is shown in the left and right upper areas of each plot. d) Hierarchical clustering for DEGs from bronchial brushings in severe asthma patients. ESA: severe asthma ex-smokers.
- FIGURE 4
Differentially expressed genes (DEGs) associated with metabolism of xenobiotics, oxidative stress and endoplasmic reticulum stress in bronchial brushings. Dot plots showing DEGs in bronchial brushings associated with a) xenobiotic metabolism by cytochrome P450 family 1 subfamily B member 1 (CYP1B1), b) aldehyde dehydrogenase 3 family member A1 (ALDH3A1), c) NAD(P)H quinone dehydrogenase 1 (NQO1), d) aldo-keto reductase family 1 member C1 (AKR1C1) and e) heat shock protein family A (Hsp70) member 5 (HSPA5). RFU: relative fluorescence units; CSA: severe asthma current smokers; ESA: severe asthma ex-smokers; NSA: severe asthma nonsmokers; NH: healthy nonsmokers. **: p<0.01; ***: p<0.001.
- FIGURE 5
Gene set variation analysis of selected stress-related pathways in bronchial brushings according to smoking status. Box-and-whisker plots showing pathway enrichment of a) xenobiotic metabolism by CYP450, b) glutathione metabolism, c) response to oxidative stress, d) endoplasmic reticulum stress, e) unfolded protein response, f) lysosome and g) glycolysis and gluconeogenesis in bronchial brushings of severe asthma current smokers (CSA), severe asthma ex-smokers (ESA), severe asthma nonsmokers (NSA) and healthy nonsmokers (NH). *: p<0.05; **: p<0.01; ***: p<0.001.
- FIGURE 6
Gene set variation analysis of selected stress-related pathways in bronchial biopsies according to smoking status. Box-and-whisker plots showing pathway enrichment of a) xenobiotic metabolism by CYP450, b) glutathione metabolism, c) response to oxidative stress, d) endoplasmic reticulum stress, e) unfolded protein response, f) lysosome and g) glycolysis and gluconeogenesis in bronchial biopsies of severe asthma current smokers (CSA), severe asthma ex-smokers (ESA), severe asthma nonsmokers (NSA) and healthy nonsmokers (NH). *: p<0.05; **: p<0.01; ***: p<0.001.
- FIGURE 7
Protein interaction analysis in STRING (version 10.0; STRING Consortium 2016, www.string-db.org) using combined differentially expressed genes (DEGs). Combined DEGs in limma from bronchial brushings, biopsies and sputa were used for protein interaction analysis by STRING. The large pink-coloured area is filled with proteins related to xenobiotic metabolism and oxidation–reduction which contains the pentose-phosphate pathway. These proteins function with those in charge of redox and connect with protein production or modification. Some proteins are associated with innate immunity. The other proteins function as lysosomal, membranous, mucus productive, Golgi homeostatic or structural proteins. Overall, this reveals the relationship between oxidative stress, endoplasmic reticulum stress, metabolism of xenobiotics and innate immunity.
Tables
- TABLE 1
Patient characteristics for sputum SomaLogic analyses
CSA Missing values ESA Missing values NSA Missing values Healthy
nonsmokersMissing values p-value Subjects 11 22 37 18 Female 5 (45.5) 14 (63.6) 22 (59.5) 6 (33.3) 2.01×10−1¶ Age years 50.0±10.6 55.7±9.7 52.6±13.3 39.9±13.8##,¶¶ 3.95×10−3+ Onset age of asthma years 29.8±19.9 39.5±19.0¶¶ 25.0±18.1 2.45×10−2+ Age at starting smoking years 19.3±4.0 16.2±2.5 4.93×10−2§ Years of smoking cessation 13.7±10.5 Smoking pack-years 29.0±18.2 20.8±16.1 0±0 0±0 1.17×10−1§ BMI kg·m−2 27.7±4.7 31.1±6.7 27.5±5.7 25.3±3.2## 2.79×10−2+ Atopic 8 (88.9) 2 10 (62.5) 6 28 (84.8) 4 5 (45.5) 7 3.60×10−2¶ Blood eosinophils cells·μL−1 259±173 296±246 407±357 2 116±71 3.31×10−1+,ƒ Blood neutrophils ×103 cells·μL−1 5.10±1.95 5.84±3.03 4.97±2.16 2 3.35±1.15 6.03×10−1+,ƒ Sputum eosinophils % 7.2±15.2 14.8±16.8 18.8±24.6 0.36±0.57 2.98×10−1+,ƒ Sputum neutrophils % 53.9±16.1 55.2±20.6 50.8±30.9 41.0±26.5 9.28×10−1+,ƒ IgE IU·mL−1 222±201 2 313±499 305±510 3 105±178 8.84×10−1+,ƒ FeNO ppb 15.2±16.6##,¶¶ 40.5±33.9 1 41.2±36.3 3 19.4±9.7 3 7.55×10−4+,ƒ Periostin ng·mL−1 42.8±9.3 2 53.1±18.9 4 54.9±20.3 9 49.7±5.5 4 2.66×10−1+,ƒ FEV1 post-bronchodilator# % 73.7±18.2 78.8±21.1 68.6±21.1 105.2±11.5 1.82×10−1+,ƒ FEV1/FVC post-bronchodilator# % 61.5±10.1 63.4±12.2 60.2±13.9 79.0±5.9 6.27×10−1+,ƒ Airway reversibility % 15.0±9.5 16.7±12.7 17.3±20.4 1 7.45×10−1+ Airflow limitation (missing data n=92 overall) 7 (63.6) 11 (50.0) 28 (75.7) 1.33×10−1¶ Average ACQ-7 2.87±1.31 1 2.67±0.98 3 2.68±1.19 4 8.30×10−1+ Average AQLQ 4.15±1.57 1 4.62±1.04 5 4.35±1.29 2 5.06×10−1+ Exacerbations in previous year 2.6±3.3 2.1±1.9 2.4±1.9 7.47×10−1+ ER visits due to breathing problems 5 (45.5) 14 (63.6) 25 (62.2) 4.41×10−1¶ Comorbidities Allergic rhinitis 2 (25.0) 3 8 (40.0) 2 16 (55.2) 8 2.85×10−1¶ Nasal polyps 2 (20.0) 1 7 (33.3) 1 12 (34.3) 2 7.20×10−1¶ Sinusitis 2 (25.0) 3 6 (28.6) 1 9 (28.1) 5 1.00¶ Chronic bronchitis 1 (11.1) 1 2 (9.1) 4 (12.1) 4 1.00¶ Psychiatric disease 3 (33.3) 2 3 (14.3) 1 5 (13.9) 1 3.84×10−1¶ GORD 4 (50.0) 3 15 (71.4)¶¶ 1 11 (32.4) 3 1.74×10−2¶ Medications Inhaled corticosteroids 11 (100.0) 22 (100.0) 37 (100.0) 1.00¶ Systemic corticosteroids 3 (30.0) 1 14 (63.6) 16 (45.7) 2 1.93×10−1¶ Oral corticosteroid dose mg·day−1 2.50±4.71 1 7.89±8.01 3 4.18±6.61 2 8.53×10−2+ Anti-IgE therapy 0 (0.0) 1 2 (4.0) 2 0 (0.0) 2 1.13×10−1¶ Long-acting β-agonists 11 (100.0) 21 (95.5) 37 (100.0) 4.71×10−1¶ Leukotriene modifiers 4 (36.4) 11 (52.4) 1 19 (51.4) 6.80×10−1¶ Tiotropium 3 (30.0) 1 4 (22.2) 2 12 (34.3) 2 5.61×10−1¶ Macrolide 2 (18.2) 3 (13.6) 4 (10.8) 7.96×10−1¶ Data are presented as n, n (%) or mean±sd, unless otherwise stated. Bold type represents statistical significance (p<0.05). CSA: severe asthma current smokers; ESA: severe asthma ex-smokers; NSA: severe asthma nonsmokers; BMI: body mass index; Ig: immunoglobulin; FeNO: fractional exhaled nitric oxide; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; ACQ: asthma control questionnaire; AQLQ: asthma quality of life questionnaire; ER: emergency room; GORD: gastro-oesophageal reflux disease. #: spirometry data without bronchodilator were used for healthy subjects; statistical analysis was performed using ¶: Fisher's exact test, +: Kruskal–Wallis test; or §: Mann–Whitney U-test; ƒ: healthy subjects were excluded from statistical analyses of several items; ##: p<0.05 versus ESA; ¶¶: p<0.052 versus NSA.
- TABLE 2
Differentially expressed proteins between severe asthma current smokers (CSA) and healthy nonsmokers (NH) and severe asthma nonsmokers (NSA) and NH by sputum assay
Probe ID Protein target Gene symbol Gene name Function CSA-NH SL001726 CSF2 CSF2 (or GM-CSF) Colony-stimulating factor 2 Granulocyte, monocyte, macrophage expansion SL004925 AGR2 AGR2 Anterior gradient protein 2 Mucin (MUC5AC and MUC5B) overproduction in asthma
Localised in endoplasmic reticulum of bronchial epithelial cellsSL000039 IL-8 CXCL8 (=IL8) C-X-C motif chemokine ligand 8 Acts as one of the major mediators of the inflammatory response by recruiting neutrophils NSA-NH SL000342 Catalase CAT Catalase A key antioxidant enzyme in the body's defence against oxidative stress SL000051 CRP CRP C-reactive protein Host defence based on its ability to recognise foreign pathogens and damaged cells of the host and to initiate their elimination by interacting with humoral and cellular effector systems in the blood SL004153 M-CSF R CSF1R Colony-stimulating factor 1 receptor The receptor for CSF-1, a cytokine which controls the production, differentiation and function of macrophages SL004853 B7-H2 ICOSLG Inducible T-cell costimulatory ligand This protein acts as a costimulatory signal for T-cell proliferation and cytokine secretion and induces B-cell proliferation and differentiation into plasma cells SL006108 CD5L CD5L CD5 molecule like This secreted protein is mainly expressed by macrophages in lymphoid and inflamed tissues and regulates mechanisms in inflammatory responses. Regulation of intracellular lipids mediated by this protein has a direct effect on transcription regulation mediated by nuclear receptors ROR-γ (RORC) SL004068 GZMB GZMB Granzyme B Targets cell lysis in cell-mediated immune responses. Processes cytokines and degrades extracellular matrix proteins; these roles are implicated in chronic inflammation and wound healing CSA-NH and NSA-NH SL017613 FCG2A/B FCGR2A Fc fragment of immunoglobulin-γ receptor IIa The protein is a cell surface receptor found on phagocytic cells such as macrophages and neutrophils, and is involved in the process of phagocytosis and clearing of immune complexes SL003524 Protein disulfide isomerase A3 PDIA3 Protein disulfide isomerase family A member 3 Formation of the final antigen conformation, export from the endoplasmic reticulum to the cell surface and adaptation to oxidative damage Assay performed by SomaLogic (Boulder, CO, USA). GM-CSF: granulocyte-macrophage colony-stimulating factor; IL: interleukin.
- TABLE 3
Differentially expressed key proteins in sputum SomaLogic analysis comparing severe asthma ex-smokers (ESA) and healthy nonsmokers (NH) with severe asthma nonsmokers (NSA) and NH
Probe ID Protein target Gene symbol Gene name Function ESA-NH SL004589 AZU1 AZU1 Azurocidin 1 A pre-proprotein of a mature azurophil granule antibiotic protein with monocyte chemotactic and antimicrobial activity SL000401 ELANE ELANE Neutrophil elastase This protease hydrolyses proteins within specialised neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix SL003192 CFP CFP Complement factor properdin A positive regulator of the alternative pathway of complement system SL000039 IL-8 CXCL8 (=IL-8) C-X-C motif chemokine ligand 8 Acts as one of the major mediators of the inflammatory response by recruiting neutrophils NSA-NH SL003524 Protein disulfide isomerase A3 PDIA3 Protein disulfide isomerase family A member 3 Formation of the final antigen conformation, export from the endoplasmic reticulum to the cell surface and adaptation to oxidative damage SL006108 CD5L CD5L CD5 molecule like This secreted protein is mainly expressed by macrophages in lymphoid and inflamed tissues and regulates mechanisms in inflammatory responses. Regulation of intracellular lipids mediated by this protein has a direct effect on transcription regulation mediated by nuclear receptors ROR-γ (RORC) SL004068 GZMB GZMB Granzyme B Targets cell lysis in cell-mediated immune responses. This protein also processes cytokines and degrades extracellular matrix proteins, and these roles are implicated in chronic inflammation and wound healing ESA-NH and NSA-NH SL017613 FCG2A/B FCGR2A Fc fragment of immunoglobulin-γ receptor IIa Cell surface receptor found on phagocytic cells such as macrophages and neutrophils, is involved in the process of phagocytosis and clearing of immune complexes SL000342 Catalase CAT Catalase A key antioxidant enzyme in the body's defence against oxidative stress SL000051 CRP CRP C-reactive protein Host defence based on its ability to recognise foreign pathogens and damaged cells of the host and to initiate their elimination by interacting with humoral and cellular effector systems in the blood SL004153 M-CSF R CSF1R Colony-stimulating factor 1 receptor The receptor for CSF1, a cytokine which controls the production, differentiation and function of macrophages SL004853 B7-H2 ICOSLG Inducible T-cell costimulatory ligand This protein acts as a costimulatory signal for T-cell proliferation and cytokine secretion and induces B-cell proliferation and differentiation into plasma cells Assay performed by SomaLogic (Boulder, CO, USA). IL: interleukin.
Supplementary Material
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Supplementary material ERJ-02173-2017_Supplement
Supplementary figures ERJ-02173-2017_Supplementary_figures
Supplementary tables ERJ-02173-2017_Supplementary_tables
Supplementary file 1 ERJ-02173-2017_File_1
Supplementary file 2 ERJ-02173-2017_File_2
Supplementary file 3 ERJ-02173-2017_File_3
