Protein networks in induced sputum from smokers and COPD patients

Figures & data

Table 1 Demographics

	Non	HS	CB	COPD	E&C
n	7	13	11	15	10
Age (years)	68±2	42±6	42±5	70±9	69±8
Pack-years	0	25.0±8.6	36.3±15.0	33.4±25.8	47.7±35.7
% female	29%	15%	27%	27%	18%
FEV^1/FVC (% predicted)	79.5%±6.0%	81.4%±5.0%	75.6%±5.8%	49.5%±14.8%*	37.2%±6.3%*

Notes:

* P=0.0002 by two-tailed, unpaired Student’s t-test after significant analysis of variance results between phenotypes. Data expressed as percentages or means ± standard deviation. HS and CB subjects were the youngest in this male-dominated group. COPD and E&C had significantly worse lung function.

Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction; FEV₁, forced expiratory volume in 1 sec; FVC, forced vital capacity.

Table 2 Proteins with the highest spectral counts in the 56 induced sputum specimens (mean [95% confidence interval])

Gene	Protein	Gene number	Log10-normalized spectral counts	Frequency of detection
IGHA1	Igα1 constant	3493	1.73 [1.61–1.85]	96%
DMBT1	Deleted in malignant brain tumors 1	1755	1.71 [1.60–1.83]	100%
IGJ	Ig J polypeptide (linker protein for Ig α and μ polypeptides)	3512	1.63 [1.60–1.82]	95%
ALB	Albumin	213	1.53 [1.35–1.71]	91%
LYZ	Lysozyme (renal amyloidosis)	4069	1.44 [1.28–1.59]	95%
BPIFB1	Bacterial permeability-increasing fold-containing family B, member 1 (chromosome 20 open reading frame 114, C20orf114; long palate, lung, nasal epithelium, carcinoma-associated 1 [LPLUNC1]; von Ebner’s minor salivary gland protein)	92747	1.33 [1.18–1.47]	91%
IGHG1	Igγ1 constant (G1m marker)	3500	1.28 [1.11–1.44]	88%
IGHA2	Igα2 constant (A2m marker)	3494	1.24 [1.11–1.37]	95%
IGK@	Igκ variable region	50802	1.17 [1.01–1.34]	88%
LTF	Lactotransferrin; lactoferrin	4057	1.17 [1.02–1.33]	86%
MUC5B	Mucin 5B, oligomeric mucus/gel-forming	727897	1.12 [0.96–1.28]	84%
SCGB1A1	Secretoglobin, family 1A, member 1 (uteroglobin; Clara cell-specific 10 kD protein)	7356	1.04 [0.86–1.21]	77%
PIGR	Polymeric Ig receptor	5284	1.02 [0.86–1.18]	79%
SLPI	Secretory leukocyte peptidase inhibitor	6590	0.917 [0.749–1.085]	75%
AZGP1	Alpha-2 glycoprotein 1, zinc-binding	563	0.734 [0.563–0.904]	64%
ACTB	Actin-β	60	0.727 [0.576–0.877]	73%

Figure 1 Patterns for relationships between protein spectral count percentiles and frequencies of detection for each phenotype.

Notes: Trends in spectral count percentiles (A, C, ANOVA probabilities) and frequencies of detection (B, D) are shown for the proteins with the largest ranges of expression and differences between phenotypic groups. AZGP1 (open triangles) and SCGB1A1 (open circles) were highest in Non and trended downward in smokers (A, B). MUC5AC (gray squares) was maximal in CB. DEFA1&3 (black diamonds) increased with disease severity from Non to E&C. Spectral count percentiles (C) and frequencies of detection (D) are shown for BPIFB1 (C20orf114; open triangles), IGHG1 (open circles), IGJ (gray squares), and HIST2H2BE (black diamonds).
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction; ANOVA, analysis of variance.

Figure 2 Secretory IgA-related proteins.

Notes: IGHA1 (red circles, prototype sIgA protein), IGHA2 (yellow diamonds), and IGJ (yellow triangles) were highly expressed, although IGJ was detected less frequently in sputum from COPD subjects. PIGR, the serous cell-binding protein for dimeric IgA, had comparable expression (blue).
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 3 Submucosal gland serous cell proteins.

Notes: LYZ (red circles, prototypical glandular serous cell protein), BPIFB1 (yellow diamonds), LTF (yellow triangles), PIGR (blue), and SLPI (yellow squares) had comparable high levels of expression in each phenotype. PIGR is shown in blue for comparison to sIgA-related proteins. BPIFB2 (yellow circles) was essentially secreted only in Non, and absent in smokers.
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 4 Epithelial proteins.

Notes: Three general patterns were seen: 1) goblet-cell MUC5AC (red circles and line) was increased in HS and CB; 2) SCGB1A1 (yellow diamonds), PRR4 (yellow triangles), and AZGP1 (yellow squares) were highest in Non and tended to decrease between HS and E&C (black lines); and 3) in contrast, DEFA1&3 (yellow squares, blue line) and S100A8 (yellow diamonds, blue line) tended to increase from Non to E&C.
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 5 Submucosal gland mucous cell proteins.

Notes: DMBT1 (orange circles), MUC5B (yellow diamonds), and MSMB (yellow triangles) were detected in similar percentages of each phenotypic group.
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 6 Cellular proteins.

Notes: Three patterns were found: 1) ACTB (red circles) had comparable frequencies of detection in all phenotypes.; 2) CST1&4 (blue diamonds and line), GAPDH (blue triangles and line), and GSN (blue squares and line) were most frequently detected in CB; and 3) proteins that trended upward and were highest in E&C included DEFA1&3 (yellow squares), histones (yellow triangles and orange diamonds), LCN2 (yellow circles), MPO (yellow diamonds), ACTG1 (orange circles), and CTSG (orange triangles).
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 7 Plasma proteins.

Notes: Three patterns were found: 1) albumin was detected in all phenotypes (orange circles); 2) the Ig heavy chains IGHG1 (yellow triangles) and IGHG4 (yellow diamonds) were detected in Non and decreased in HS and CB, but increased to higher detection rates in COPD and E&C; and 3) the opposite pattern was shown for TF (blue diamonds and line) and Igλ light-chain variable regions (IGL@; blue triangles and line), with the highest levels in HS and CB.
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction.

Figure 8 Correlation analysis for Non-group proteins.

Notes: The scattergram-plotted frequencies of detection versus the percentiles of spectral counts for each significantly correlated protein (P≤0.05). The proteins were numbered and colored by origin in secretory IgA (1, black), serous cells of submucosal glands (2, green), epithelial cells (3, yellow), mucous cells of submucosal glands (5, red), cellular cytoplasm (6, blue), and plasma (7, magenta). The ellipses enclose groups of proteins that were significantly correlated with each. Solid lines connect smaller sets of significantly correlated proteins, such as ACTB, IGHG1, and IGHG4. The dashed lines indicate negative correlations for IGJ with IGHG1 and C20orf114 (BPIFB1). Positively correlated proteins suggested similar cellular origins and mechanisms of release into sputum.
Abbreviation: Non, nonsmokers.

Figure 9 Correlation analysis for HS-group proteins.

Notes: Secretory IgA (1, black), serous (2, green), epithelial (3, yellow) goblet (4, red), mucous (5, gold), and plasma (7, magenta) proteins were positively correlated (solid lines). Submucosal gland mucous cell products and epithelial proteins had different trends. MUC5B and DMBT1 were positively correlated with DEFA1&3, but negatively correlated with SCGB1A1 (dashed lines). ALB and IGHG1 had surprisingly low expression in HS.
Abbreviation: HS, healthy smokers.

Figure 10 Correlation analysis for CB-group proteins.

Notes: CB proteins were divided into high (upper half of figure)- and low (lower left corner)-abundance subsets. MUC5AC and C20orf114 (BPIFB1) had the highest expression, but few correlations. Secretory IgA (1, black), serous cell LYZ and LTF (2, green), and the mucous gland products MUC5B and DMBT1 (5, yellow) were correlated. Low-abundance cellular proteins (6, blue) were highly clustered and correlated with each other (blue ellipse). MSMB (5, orange) is in the ellipse, but did not correlate with these cellular proteins. LYS and LTF were negatively correlated with MSMB and MUC5AC (dashed green lines). Plasma ALB, IGHG1, and TF were correlated with each other, which was consistent with their entry into sputum via plasma extravasation.
Abbreviation: CB, chronic bronchitic patients.

Figure 11 Correlation analysis for COPD-group proteins.

Notes: Serous (LYZ, LTF, BPIFB1; 2, green) and mucous (MUC5B, DMBT1; 5, orange) proteins had high abundances and were highly intercorrelated (green ellipse). These proteins also correlated (solid green lines) with PIGR (1, black), DEFA1&3 (3, yellow square), MUC5AC (4, red square), and ACTB (6, blue square). Serous cell LTF and LYZ were negatively correlated (dashed green lines) with the abundant ALB (7, magenta ellipse) and low-abundance IGHG4 (7, magenta). Low-abundance proteins were clumped into the lower left corner. IGHG3 and IGHG4 were correlated with each other, but not the adjacent LCN2 (6, blue) or PLUNC (BPIFA1; 3, yellow). LCN2 was correlated with PRR4 (3, yellow), while PLUNC (BPIFA1) correlated with MSMB (5, orange) and MUC5AC (4, red).

Figure 12 Correlation analysis for E&C-group proteins.

Notes: The black ellipse encloses proteins from the secretory IgA (1), serous (2), and mucous (5) groups that were highly intercorrelated. IGHA1, PIGR, LTF, and MUC5B were negatively correlated with ALB (7, plasma source, dashed magenta lines). IGJ was also negatively correlated with IGHG4. Cellular proteins (6, blue symbols, lines and ellipses) were significantly correlated with each other, and negatively correlated with IGHA2 and MUC5B. The low-frequency cellular proteins at the bottom of the frame were negatively correlated (blue dashed line) with epithelial SCGB1A1 (3, yellow square).
Abbreviation: E&C, patients with significant emphysema and airflow obstruction.

Figure 13 Cytoscape network analysis for nonsmokers.

Notes: Three separate networks were identified between proteomically identified proteins (green circles, gene symbols). Inferred linker proteins (diamonds) were introduced by Cytoscape based on text mining. Previously reported interactions are shown by solid blue lines, and inferred relationships for these proteins by dashed lines.

Figure 14 MUC5AC and glandular serous cell proteins LYZ, LTF, SLPI and PIGR in healthy smokers (HS).

Notes: The transcription factors SP1, SP3, and USF2 were linked to goblet-cell MUC5AC and glandular serous cell proteins in sputum from HS subjects.

Figure 15 Chronic bronchitis: the two protein interaction networks suggest that distinct pathological mechanisms contribute to bronchial pathology.

Notes: The map on the left is dominated by connections of GAPDH, ACTB, and GSN. GAPDH is linked to glycolysis proteins. GSN connects phosphoinositides to mechanisms of actin regulation. through the long tail to histones include S100A8 and S100A9. On the right, CEBPA is linked to DMBT1 in the tail, and LTF and potential neutrophil or serous cell products in the interconnected head.

Figure 16 COPD: protein interaction networks suggest central roles for transcription factors in bronchiole pathology.

Notes: The central core of ETS1, CEBPA, SP1, and NFKB1 transcription factors interacted with proteins from serous (LTF, LYZ, PIGR), mucous (DMBT1, MSMB), and goblet (MUC5AC) exocrine cells, and IgA-producing B lymphocytes. This pattern of interactions suggests hypersecretion from the cluster of submucosal gland serous and mucous cells and their neighboring IgA B cells as a potential pathology, bridging chronic bronchitis and COPD. The link of SP1 with MUC5AC may indicate continued goblet cell hyperplasia as seen in healthy smokers with COPD. These support two avenues for the transition from healthy smokers to the airflow limitation that defines COPD. One would involve mechanisms of airflow limitation with their roots in healthy smokers, and the other a progression of bronchial wall submucosal gland mucous hypersecretion pathology through chronic bronchitis to airflow limitation and COPD.

Figure 17 Emphysema: neutrophil and plasma protein inflammatory cascades were inferred from protein interaction networks.

Notes: ITGAM had a central position. It was connected to MPO, and through PRTN3 to CTSG, coagulation cascade enzymes, and their potential inhibitor, SERPINB13. ITGAM and MMP2 were linked to ACTG and actin-sequestering proteins. MMP2 was linked through CEBPA to defensins and histones. These interactions implicate neutrophil and plasma protease inflammatory cascades such as coagulation in emphysema pathology. Other cascades such as complement may also be involved but were not detected here. Histones, defensins, MPO, ACTG, and the multiple proteases supported neutrophil extracellular nets and proteolysis.

Figure 18 Sputum proteome-derived approach to cigarette smoke-induced lung diseases.

Notes: All smokers were at risk of lung cancer. Approximately half of HS progressed to mucous hypersecretion without airflow obstruction (CB). As smokers aged, 5%–15% developed accelerated obstruction (COPD). A smaller proportion developed alveolar destruction (E&C). Diagnostics and treatments may be significantly improved by directing them at the dynamic alterations in pathogenic mechanisms.
Abbreviations: Non, nonsmokers; HS, healthy smokers; CB, chronic bronchitic patients; E&C, patients with significant emphysema and airflow obstruction; sIgA, secretory IgA; GOLD, Global initiative for chronic Obstructive Lung Disease.