Phenotypic Variations of Mild-to-Moderate Obstructive Pulmonary Diseases According to Airway Inflammation and Clinical Features

Figures & data

Table 1 Clinical Characteristics of Patients with Asthma, COPD and Control Group

	Asthma (n=30)	COPD (n=30)	Non-Smoking Control (n=10)	Smoking Control (n=10)	p-value
Male/female	12/18	14/16	5/5	5/5	0.910
Age (years)	40.5 (30–56)	66.5 (61–75)	33 (29–36)	28 (25–53)	<0.001	§ $
BMI (kg/m^2)	26.5 (22.9–30)	27 (22–29.7)	25 (22.5–26.2)	28.9 (26.2–32.7)	0.066
Smoking status (never/current/ex-smokers)	18/0/12	0/14/16	10/0/0	0/10/0	<0.001
Smoking history (pack-years)	0 (0–2)	40 (25–50)	0 (0–0)	10.5 (5–32)	<0.001	§ $
Positive skin prick tests, n (%)	22 (79)*	14 (52)**	5 (50)	5 (50)	0.132
Total IgE (IU/mL)	152.9 (47.1–228.7)	57 (25.5–113.8)	35.6 (16.8–98.8)	68.1 (32.3–90.4)	0.047	§
FeNO (ppb)	43.4 (24.4–111)	19 (12.1–23.2)	22.8 (19.7–28.6)	12.4 (9.5–18.3)	<0.001	§ ¶
PC20 (mg/mL)	0.6 (0.2–1.7)	n/a	>16 (16–16)	>16 (9.4–16)	<0.001	¶
FEV1 pre-BD (L)	2.6 (2.1–3.2)	1.4 (1.3–1.8)	4.0 (3.6–4.7)	3.8 (2.5–4.3)	<0.001	§ ¶ $
FEV1 pre-BD (% predicted)	74.5 (70–83)	59 (50–70)	108.5 (98–117)	91 (89–94)	<0.001	§ ¶ $
FEV1/VC ratio pre-BD (%)	69.1 (60.3–75.4)	48.7 (44.8–55.8)	81.5 (80–87)	76.5 (74–82)	<0.001	§ ¶ $
FEV1 post-BD (L)	3.4 (2.3–4.2)	1.7 (1.4–2.0)	n/a	n/a	<0.001	§
FEV1 post-BD (% predicted)	85 (76–92)	69 (57–76)	n/a	n/a	<0.001	§
FEV1/VC ratio post-BD (%)	69.6 (62.9–74.2)	52.9 (47.5–60.0)	n/a	n/a	<0.001	§
ΔFEV1 (mL)	630 (360–800)	210 (90–290)	n/a	n/a	<0.001	§
ΔFEV1 (% predicted)	16.5 (11–21)	7 (4–10)	n/a	n/a	0.002	§

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). For multiple comparisons, we indicate the significance by: § Between asthma and COPD. ¶Between asthma vs non-smoking control. $Between COPD vs smoking control. *Out of 28 tests performed. **Out of 27 tests performed.

Abbreviations: BD, bronchodilator; BMI, body mass index; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in one second; IgE, immunoglobulin E; PC₂₀, methacholine concentration causing a 20% drop in FEV₁; VC, vital capacity.

Table 2 Characteristics and Mediators of Sputum in Asthma, COPD and Control Group

	Asthma (n=30)	COPD (n=30)	Non-Smoking Control (n=10)	Smoking Control (n=10)	p-value
Total sputum cell count (× 10^6 cells/g)	1.0 (0.5–1.4)	1.5 (0.8–2.5)	1.3 (1.1–3.2)	1.4 (0.9–2.0)	0.118
Sputum eosinophils (%)	3 (1–11)	1 (0–2)	0 (0–1)	1 (0–1)	<0.001	§ ¶
Sputum eosinophilia, n (%)	18 (60)	7 (23)	1 (10)	1 (10)	0.001	§ ¶
Sputum neutrophils (%)	44 (41–52)	51.5 (45–68)	31.5 (30–47)	45.5 (33–57)	0.012	§
Sputum neutrophilia, n (%)	4 (13)	11 (37)	0 (0)	1 (10)	0.027	§
IL-1β (pg/mL)	3.4 (0.9–5.8)	3.0 (1.1–6.0)	1.5 (0.6–3.6)	0 (0–1.5)	0.012	$
IL-1RA (pg/mL)	2918.1 (1783.0–7521.0)	3475.0 (1629.7–7578.0)	2804.4 (2005.6–4629.1)	1674.4 (1012.8–2310.6)	0.218
IL-3 (pg/mL)	11.6 (8.9–22.5)	13.7 (9.8–33.2)	11.1 (9.1–12.2)	9.8 (0–15.9)	0.285
IL-6 (pg/mL)	3.4 (0–10.0)	17.1 (4.3–54.0)	0 (0–0)	7.4 (0–8.8)	<0.001	§ ¶
IL-7 (pg/mL)	0 (0–1.6)	1.1 (0–1.5)	0 (0–0)	0 (0–0)	0.003	¶ $
IL-8 (pg/mL)	185.3 (60.8–349.5)	550.2 (254.3–1174.7)	79.2 (47.4–176.1)	75.0 (55.4–235.8)	<0.001	§ $
IL-33 (pg/mL)	2.8 (2.4–3.4)	2.6 (2.5–3.3)	3.3 (2.7–3.6)	2.6 (2.5–3.1)	0.493
CCL2 (pg/mL)	2.9 (0–7.1)	5.5 (1.7–19.3)	0 (0–2.8)	2.9 (0–5.0)	0.037
CCL4 (pg/mL)	1.2 (0–5.7)	7.5 (0–27.0)	0 (0–5.6)	0 (0–0)	0.037	§ $
CCL24 (pg/mL)	4.6 (4.2–5.2)	4.6 (4.0–7.2)	4.4 (4.0–5.4)	4.6 (3.9–24.5)	0.943
CCL26 (pg/mL)	4.2 (3.1–5.4)	4.6 (3.1–5.4)	4.2 (2.3–5.4)	3.1 (2.3–3.8)	0.145	$
CXCL10 (pg/mL)	126.7 (58.8–300.5)	107.3 (57.5–293.2)	45.1 (6.1–118.0)	46.1 (6.1–163.6)	0.167
NE (ng/mL)	18.4 (9.9–38.0)	20.8 (12.7–48.5)	13.6 (9.2–18.2)	12.1 (11.0–30.4)	0.097
MMP-9 (ng/mL)	20.7 (14.4–35.6)	33.6 (16.2–37.0)	25.5 (13.5–27.2)	26.8 (9.6–36.9)	0.218
TSLP (pg/mL)	8.9 (7.8–10.8)	8.2 (7.1–10.5)	8.6 (6.9–11.9)	8.7 (7.2–10.3)	0.789
FGF2 (pg/mL)	3.6 (0–6.6)	0 (0–7.0)	0 (0–2.2)	0 (0–0)	0.042	¶
G-CSF (pg/mL)	0 (0–57.8)	17.9 (0–116.1)	0 (0–33.3)	0 (0–42.0)	0.151
TNF-α (pg/mL)	0 (0–9.6)	2.9 (0–10.1)	0 (0–0)	0 (0–0)	0.075

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). Sputum eosinophilia ≥3%; sputum neutrophilia ≥60%. For multiple comparisons, we indicate the significance by: § Between asthma and COPD. ¶ Between asthma vs non-smoking control. $ Between COPD vs smoking control.

Table 3 Logistic Regression Model of Biochemical and Clinical Features for Discrimination of Asthma and COPD Patients

	Multivariate Analysis (AUC=0.947)		Age-Adjusted Analysis (AUC=0.746)
Variables	Crude OR (95% CI)	p-value	Adjusted OR (95% CI)	p-value
IL-6	1.047 (0.987–1.140)	0.243	1.008 (0.986–1.047)	0.570
IL-8	1.000 (0.998–1.001)	0.931	1.000 (0.999–1.001)	0.505
CCL4	0.985 (0.940–1.042)	0.535	0.996 (0.971–1.020)	0.701
FEV1/VC ratio pre-BD (%)	0.783 (0.661–0.874)	<0.001	0.906 (0.836–0.968)	0.007
Sputum neutrophils (%)	1.041 (0.975–1.124)	0.256	1.003 (0.958–1.051)	0.883

Abbreviations: BD, bronchodilator; CI, confidence interval; FEV₁, forced expiratory volume in one second; OR, odds ratio; VC, vital capacity.

Table 4 Clinical Characteristics of Patients in Two Identified Clusters

	Cluster 1 (n=29)	Cluster 2 (n=31)	p-value
Asthma, n (%)	23 (79)	7 (23)	<0.001
COPD, n (%)	6 (21)	24 (77)	<0.001
Male/female	9/20	17/14	0.063
Age (years)	42 (30–62)	65 (60–75)	<0.001
BMI (kg/m^2)	25.7 (22.7–28.1)	27.5 (23.6–30.0)	0.549
Smoking status (never/current/ex-smokers)	13/4 /12	5/10/16	0.036
Smoking history (pack-years)	1 (0–5)	30 (20–50)	<0.001
Positive skin prick tests, n (%)	19 (68)*	17 (63)**	0.703
Total IgE (IU/mL)	74.7 (28.6–198.1)	73.0 (37.1–190.9)	0.590
FeNO (ppb)	40.2 (20.7–111.0)	20.5 (16.0–37.1)	0.039
PC20 (mg/mL)	0.4 (0.2–1.7)	0.5 (0.1–1.5)	0.540
FEV1 pre-BD (L)	2.6 (1.8–3.2)	1.5 (1.3–2.0)	<0.001
FEV1 pre-BD (% predicted)	74 (71–83)	59 (48–70)	<0.001
FEV1/VC ratio pre-BD (%)	68.4 (60.3–74.7)	48.1 (44.8–55.3)	<0.001
FEV1 post-BD (L)	2.7 (1.7–4.1)	1.7 (1.4–2.1)	0.001
FEV1 post-BD (% predicted)	88.5 (74.5–92.5)	70 (56.5–76.5)	<0.001
FEV1/VC ratio post-BD (%)	70 (61–73)	52 (47–60)	<0.001
ΔFEV1 (mL)	330 (95–770)	250 (120–390)	0.347
ΔFEV1 (% predicted)	10.5 (5–18.5)	9.5 (5–15.5)	0.583

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). *Out of 28 tests performed. **Out of 27 tests performed.

Abbreviations: BD, bronchodilator; BMI, body mass index; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in one second; IgE, immunoglobulin E; PC₂₀, methacholine concentration causing a 20% drop in FEV₁; VC, vital capacity.

Table 5 Characteristics and Mediators of Sputum in Two Main Identified Clusters

	Cluster 1 (n=29)	Cluster 2 (n=31)	p-value
Total sputum cell count (× 10^6 cells/g)	0.9 (0.4–1.3)	1.6 (0.9–2.8)	0.003*
Sputum eosinophils (%)	3 (1–8)	1 (0–4)	0.146
Sputum eosinophilia, n (%)	15 (52)	10 (32)	0.126
Sputum neutrophils (%)	42 (32–48)	56 (47–68)	<0.001*
Sputum neutrophilia, n (%)	2 (7)	13 (42)	0.002*
IL-1β (pg/mL)	2.6 (0.9–5.3)	4.1 (1.7–6.6)	0.333
IL-1RA (pg/mL)	2918.1 (1720.3–4858.0)	3498.8 (1915.3–8726.9)	0.283
IL-3 (pg/mL)	11.9 (9.7–25.8)	12.1 (9.5–20.3)	0.728*
IL-6 (pg/mL)	0 (0–3.7)	17.1 (10.1–48.0)	<0.001
IL-7 (pg/mL)	0 (0–0.5)	1.3 (0–3.5)	<0.001
IL-8 (pg/mL)	158.0 (55.4–281.8)	700.6 (334.6–1371.1)	<0.001
IL-33 (pg/mL)	2.6 (2.3–2.8)	3.2 (2.5–3.4)	0.032*
CCL2 (pg/mL)	1.8 (0–4.4)	6.8 (2.8–26.8)	<0.001
CCL4 (pg/mL)	0 (0–2.7)	11.5 (4.6–29.2)	<0.001*
CCL24 (pg/mL)	4.4 (3.9–4.8)	5.1 (4.4–14.7)	0.003*
CCL26 (pg/mL)	3.9 (3.1–5.4)	4.6 (3.1–6.2)	0.394
CXCL10 (pg/mL)	83.7 (40.9–217.7)	210.8 (85.9–907.4)	0.022*
NE (ng/mL)	12.5 (9.6–30.7)	32.0 (14.8–51.5)	0.004
MMP-9 (ng/mL)	17.3 (14.0–25.5)	37.0 (22.6–37.0)	<0.001*
TSLP (pg/mL)	8.2 (7.5–10.4)	8.9 (7.6–10.7)	0.520
FGF2 (pg/mL)	2.9 (0–5.7)	5.7 (0–7.2)	0.334
G-CSF (pg/mL)	0 (0–17.2)	63.2 (15.1–178.0)	<0.001
TNF-α (pg/mL)	0 (0–0)	9.1 (0–18.8)	< 0.001

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). Sputum eosinophilia ≥3%; sputum neutrophilia ≥60%. *Significant after adjustment for age.

Figure 1 Hierarchical clustering heatmap of asthma and COPD patients based on clinical, sputum cytokine and cellular features. The rows and columns are ordered based on the results of hierarchical clustering with dendrograms for the patients shown on the horizontal axis and clinical and laboratory data shown on the vertical axis. The colour scale codes normalized value of a variable with red corresponding to the highest value.

Table 6 Clinical Characteristics of Patients from the Main Two Clusters Divided According to Asthma or COPD Diagnosis

	Cluster 1		Cluster 2		p-value
	Asthma (n=23); Subgroup 1	COPD (n=6); Subgroup 2	Asthma (n=7); Subgroup 3	COPD (n=24); Subgroup 4
Male/female	9/14	0/6	3/4	14/10	0.073
Age (years)	37 (27–55)	62.5 (53–77)	55 (42–72)	69 (61.5–75)	<0.001	§ ¶ ‖
BMI (kg/m^2)	25.3 (22.6–28.1)	27.6 (23.5–31.1)	29.4 (26.9–30.5)	26.8 (22.0–29.6)	0.283
Smoking status (never/current/ex-smokers)	13/0/10	0/4/2	5/0/2	0/10/14	<0.001
Smoking history (pack-years)	0 (0–5)	35 (17–50)	0 (0–0)	40 (25–50)	<0.001	§ ‖
Positive skin prick tests, n (%)	17 (77*)	2 (33)	5 (83**)	12 (57***)	0.134
Total IgE (IU/mL)	99.6 (47.1–214.1)	20.7 (5.3–25.5)	190.9 (37.1–861)	69.6 (32.2–175.9)	0.006	§ $
FeNO (ppb)	49.4 (23.7–114.7)	16.5 (8.9–22.7)	38.6 (27.8–53.8)	19.0 (13.8–25.1)	0.002	§ ‖
FEV1 pre-BD (L)	2.7 (2.4–3.3)	1.6 (1.3–1.8)	2 (1.7–2.5)	1.4 (1.2–1.9)	<0.001	§ ¶ ‖
FEV1 pre-BD (% predicted)	77 (72–84)	70.5 (61–82)	66 (62–83)	57 (45.5–67)	<0.001	$ ‖
FEV1/VC ratio pre-BD (%)	70 (65–75)	58 (56–60)	58 (54–77)	48 (43.5–52)	<0.001	§ $ ‖
FEV1 post-BD (L)	4.1 (3–4.2)	1.7 (1.4–1.8)	2.2 (2.1–2.8)	1.7 (1.4–2.1)	<0.001	§ ‖
FEV1 post-BD (% predicted)	91.5 (84–96)	75.5 (69–92)	77.5 (74.5–79.5)	66 (55.5–75.5)	<0.001	‖
FEV1/VC ratio post-BD (%)	71 (69–76)	60 (59–62)	62 (59–64)	51 (47–56)	<0.001	§ ¶ $ ‖
ΔFEV1 (mL)	650 (390–880)	95 (50–250)	530 (350–750)	220 (110–295)	0.002	§ ‖
ΔFEV1 (% predicted)	17 (11–21)	5 (3–10)	15.5 (12.5–22.5)	7.5 (4.5–13)	0.014	§

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). For multiple comparisons, we indicate the significance by: § Between subgroups 1 and 2. ¶ Between subgroups 1 and 3. $ Between subgroups 2 and 4. ‖ Between subgroups 3 and 4. *Out of 22 tests performed. **Out of 6 tests performed. ***Out of 21 tests performed.

Abbreviations: BD, bronchodilator; BMI, body mass index; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in one second; IgE, immunoglobulin E; PC₂₀, methacholine concentration causing a 20% drop in FEV₁; VC, vital capacity.

Table 7 Characteristics and Mediators of Sputum in Patients from the Main Two Clusters Divided According to Asthma or COPD Diagnosis

	Cluster 1		Cluster 2		p-value
	Asthma (n=23); Subgroup 1	COPD (n=6); Subgroup 2	Asthma (n=7); Subgroup 3	COPD (n=24); Subgroup 4
Total sputum cell count (× 10^6 cells/g)	0.9 (0.4–1.3)	1.1 (0.5–1.9)	1.3 (0.9–5.1)	1.7 (0.8–2.8)	0.027	¶
Sputum eosinophils (%)	3 (1–11)	1 (0–1)	3 (1–13)	1 (0–2.5)	0.044*	§
Sputum eosinophilia, n	14	1	4	6	0.037
Sputum neutrophils (%)	42 (31–48)	44.5 (35–51)	58 (47–67)	53.5 (45–69)	<0.001	¶
Sputum neutrophilia, n	1	1	3	10	0.017	¶
IL-1β (pg/mL)	2.7 (0.9–5.2)	2.5 (1.1–5.3)	4.9 (3.4–14.7)	3.4 (1.4–6.0)	0.418
IL-1RA (pg/mL)	2449.1 (1708.0–4338.8)	5236.5 (3536.3–6101.0)	8191.4 (2176.7–9486.8)	2883.9 (1629.7–7579.0)	0.238	¶
IL-3 (pg/mL)	11.8 (9.0–22.5)	31.5 (11.8–38.5)	11.1 (0–34.8)	12.8 (9.6–18.5)	0.315*
IL-6 (pg/mL)	0 (0–5.7)	0 (0–0)	11.4 (7.2–16.2)	21.9 (10.7–60.7)	<0.001*	¶ $
IL-7 (pg/mL)	0 (0–0)	0 (0–1.0)	3.6 (1.1–6.3)	1.1 (0–1.8)	0.001*	¶
IL-8 (pg/mL)	149.3 (50.0–283.2)	166.6 (115.1–225.2)	940.5 (344.5–1291.4)	627.5 (324.7–1450.7)	<0.001*	¶ $
IL-33 (pg/mL)	2.6 (2.3–2.9)	2.5 (2.3–2.6)	3.3 (3.1–5.2)	2.7 (2.5–3.4)	0.042*	¶
CCL2 (pg/mL)	1.8 (0–4.8)	1.8 (0–3.8)	8.3 (3.9–31.2)	6.7 (2.6–22.3)	0.008	¶
CCL4 (pg/mL)	0 (0–3.6)	0 (0–0)	10.0 (5.7–22.1)	12.3 (3.5–31.5)	<0.001*	¶ $
CCL24 (pg/mL)	4.5 (4.2–4.9)	3.9 (3.8–4.0)	5.2 (4.6–11.6)	5.0 (4.3–23.6)	0.007*	§ ¶ $
CCL26 (pg/mL)	4.6 (3.1–5.4)	3.9 (3.1–4.6)	3.9 (3.1–6.9)	4.6 (3.5–5.8)	0.735
CXCL10 (pg/mL)	79.0 (42.6–251.3)	88.5 (14.0–113.0)	450.1 (122.6–1085.5)	134.7 (67.5–325.3)	0.047*	¶
NE (ng/mL)	12.5 (9.6–30.7)	11.9 (9.6–32.6)	35.1 (22.7–50.2)	23.4 (14.3–58.0)	0.036	¶
MMP-9 (ng/mL)	17.3 (13.5–26.8)	17.7 (14.0–25.4)	36.9 (34.1–36.9)	36.9 (20.2–36.9)	0.001*	¶
TSLP (pg/mL)	8.8 (7.5–11.1)	7.7 (7.1–7.9)	9.0 (8.0–10.8)	8.5 (7.1–10.7)	0.290
FGF2 (pg/mL)	3.6 (0–5.7)	0 (0–0)	5.7 (3.6–9.0)	3.1 (0–7.0)	0.367
G-CSF (pg/mL)	0 (0–22.3)	0 (0–5.1)	123.5 (79.1–159.2)	44.3 (14.7–196.7)	<0.001*	¶ $
TNF-α (pg/mL)	0 (0–5.9)	0 (0–0)	13.1 (6–20.4)	6.7 (0–11.5)	0.002*	¶ $

Notes: Results are presented as median (25th–75th quartiles) or frequency (percentage). Sputum eosinophilia ≥3%; sputum neutrophilia ≥60%. For multiple comparisons, we indicate the significance by: § Between subgroups 1 and 2. ¶ Between subgroups 1 and 3. $ Between subgroups 2 and 4. *Significant after adjustment for age.