Involvement of Transforming Growth Factor-β-Associated Kinase 1 in Fixed Airway Obstruction in Asthmatic Patients with Longer Disease Duration Independent on Airway Eosinophilia

Figures & data

Table 1 Patient Characteristics and Sputum Cytology

Variables	All Participants (n=103)	nFAO (n=43)	FAO (n=60)	P-value*
Mean age (yr) ±SD	60.3 ± 11.5	54.6 ±10.9	64.5 ±10.3	< 0.001^a
Sex, (%)
Male	33	13.9	46.7	0.35^b
Female	67	86.1	53.3	0.11^b
Duration of asthma, y	28.6 ± 16.2	24.6 ± 13.9	31.5 ± 17.1	0.039^a
Number of 10-y exacerbations	0 [0.0–1.0]	0 [0.0–1.0]	0 [0.0–1.75]	0.15^d
Atopy (%)	39 (37.9)	13 (30.2)	26 (43.3)	0.17^b
Body mass index	25.1 ± 4.7	24.6 ± 4.4	25.4 ± 4.8	0.46^a
Comorbidities
Allergic rhinitis (%)	65	72	60.0	0.204^b
CRSwNP (%)	4.9	4.7	5.0	1.0^b
GERD (%)	10.7	18.6	5.0	0.048^b
HT and dyslipidemia (%)	56.3	30.2	75.0	<0.0001^b
Serum total IgE (IU/mL)	165 [68.9–540.5]	157 [70.0–1005]	165 [94.2–476.8]	0.81^c
Post-BD FEV1/FVC (%)	66.2 ± 12.7	77.2 ± 5.1	56.6 ± 8.9	<0.001^c
% Reversibility	15.5 ± 14.5	10.9 ± 9.7	18.8 ± 16.4	0.0037^c
Pre-BD FEV1, (L)	1.4 ± 0.5	1.75 ± 0.5	1.21 ± 0.4	<0.001^c
FEV1% predicted	69.7 ± 22.0	82.7 ±19.3	60.4 ± 19.1	<0.001^c
Post-BD FEV1, (L)	1.62 ± 0.49	1.92 ± 0.45	1.41 ± 0.42	<0.001^c
FEV1% predicted	78.5 ± 20.0	90.5 ± 16.7	69.9 ± 17.7	<0.001^c
Pre-BD FVC, (L)	2.3 ± 0.6	2.4 ± 0.6	2.24 ± 0.63	0.11^c
Post-BD FVC, (L)	2.5 ± 0.59	2.5 ± 0.6	2.5 ± 0.62	1.0^c
FeNO, (ppb)	34.9 ± 29.3	40.5 ± 35.6	31.5 ± 17.1	0.23^c
ACT score (SD)	20.6 ± 4.1	20.2 ± 4.7	20.8 ± 3.7	0.7^c
Median equivalent beclomethasone daily dose (μg) (IQR)	500 [320–640]	500 [320–640]	640 [400–990]	0.06^c
ICS+LABA (%)	64.1	74.4	88.3	0.04^b
LAMA (%)	9.7	4.7	21.7	0.01^b
LTRA (%)	39.8	60.5	60.0	0.93^b
Total sputum cells (×10^6)	0.96 [0.84–1.08]	0.98 [0.86–1.08]	0.92 [0.84–1.15]	0.66^c
Sputum neutrophil number (×10^6)	0.38 [0.28–0.5]	0.4 [0.27–0.46]	0.35 [0.27–0.52]	0.71^c
Sputum neutrophils (%)	36.8 [30.9–52.3]	38.5 [30.9–50.7]	36.0 [30.1–52.7]	0.94^c
Sputum eosinophil number (×10^6)	0.02 [0.0–0.06]	0.02 [0.0–0.06]	0.02 [0.0–0.06]	0.8^c
Sputum eosinophils (%)	2.0 [0.0–6.6]	2.0 [0.0–6.7]	2.0 [0.0–6.1]	0.8^c
Sputum macrophage number (×10^6)	0.53 [0.4–0.7]	0.53 [0.4–0.6]	0.53 [0.4–0.6]	0.75^c
Sputum macrophages (%)	58.5 [43.8–65.3]	58.0 [45.6–63.4]	58.4 [43.2–67.0]	0.98^c
Sputum lymphocyte number (×10^6)	0.0 [0.0–0.0]	0.0 [0.0–0.0]	0.0 [0.0–0.0]	0.38^c
Sputum lymphocytes (%)	0.0 [0.0–0.0]	0.0 [0.0–0.0]	0.0 [0.0–0.0]	0.46^c
Sputum airway epithelial cell number (×10^6)	0.005 [0.0–0.2]	0.01 [0.0–0.02]	0.005 [0.0–0.02]	0.82^c
Sputum airway epithelial cells (%)	0.74 [0.0–1.9]	0.74 [0.0–2.0]	0.73 [0.0–1.9]	0.63^c
White blood cell count (×10^3/μL)	7.4 [5.8–8.7]	7.3 [5.6–8.5]	7.4 [6.0–9.1]	0.66^c
Neutrophils (%)	57.0 [48.9–63.5]	58.9 [50.3–63.1]	55.5 [48.2–64.0]	0.85^c
Lymphocytes (%)	32.0 [24.3–39.1]	32.2 [25.4–38.0]	31.7 [22.8–39.2]	0.94^c
Monocytes (%)	6.9 [5.4–8.1]	6.3 [5.3–7.5]	7.2 [5.6–9.1]	0.06^c
Eosinophils (%)	3.3 [2.0–5.5]	3.6 [1.8–5.7]	3.2 [2.1–5.2]	0.81^c
Basophils (%)	0.6 [0.4–0.8]	0.6 [0.4–0.7]	0.6 [0.5–0.8]	0.83^c
Neutrophils (×10^3/μL)	4.0 [2.8–5.4]	3.9 [2.9–5.2]	4.2 [2.6–5.4]	0.63^c
Lymphocytes (×10^3/μL)	2.1 [1.8–2.5]	2.1 [1.7–2.7]	2.1 [1.8–2.4]	0.91^c
Monocytes (×10^3/μL)	0.5 [0.39–0.57]	0.44 [0.4–0.5]	0.5 [0.4–0.6]	0.009^c
Eosinophils (×10^3/μL)	0.23 [0.1–0.4]	0.19 [0.1–0.5]	0.24 [0.2–0.3]	0.7^c
Basophils (×10^3/μL)	0.04 [0.03–0.06]	0.04 [0.02–0.06]	0.04 [0.03–0.05]	0.51^c

Notes: Data are presented as median [IQR] or otherwise indicated. FAO was defined as postbronchodilator FEV₁/FVC < LLN with the first lung function data at least 3 months before study entry and confirmed the persistence with the second pulmonary function testing at study enrolment as demonstrated in the table p value* nFAO versus FAO. ^ap value from unpaired t-test. ^bp value from chi-square test for trend test. ^cp value from Mann–Whitney test. ^dp value from chi-square test for linear by linear association.

Abbreviations: ACT, asthma control test; BD, bronchodilator; FAO, fixed airway obstruction; nFAO, no fixed airway obstruction; FeNO, fractional exhaled nitric oxide; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; ICS, inhaled corticosteroid; LABA, long-acting β₂ agonist; LAMA, long-acting muscarinic antagonist; LTRA, leukotriene receptor antagonist.

Table 2 Sputum Signaling Molecules

Variables	All Participants (n=103)	nFAO (n=43)	FAO (n=60)	P-value*	95% CI of Difference
TGF-β1 (pg/mL)	32.1 ± 21.5	7.7 ± 3.6	49.6 ± 6.7	<0.001	39.0 to 42.7^a
Total TAK1 (%)	55.2 ± 28.1	23.8 ± 5.4	77.6 ± 10.7	<0.001	52.3 to 59.6 ^a
Neutrophil TAK1 (%)	15.5 ± 10.3	9.6 ± 2.8	19.7 ± 11.5	<0.001	5.1 to 10.9^a
Eosinophil TAK1 (%)	3.7 ± 3.1	1.3 ± 1.0	5.5 ± 2.8	<0.001	3.0 to 4.9^a
Macrophage TAK1 (%)	34.2 ± 21.1	11.9 ± 3.2	50.2 ± 11.8	<0.001	36.3 to 43.8^b
Lymphocyte TAK1 (%)	0.28 ± 0.7	0.15 ± 0.6	0.37 ± 0.7	0.035	0.0 to 0.0^a
Epithelial TAK1 (%)	1.5 ± 1.3	0.9 ± 1.1	1.8 ± 1.3	<0.001	0.5 to 1.4^a
Total p-TAK1 (%)	52.7 ± 27.9	21.5 ± 5.2	75.1 ± 10.7	<0.001	52.7 to 59.3^a
Neutrophil p-TAK1 (%)	14.6 ± 10.2	8.6 ± 2.7	18.9 ± 11.4	<0.001	5.1 to 11.0^a
Eosinophil p-TAK1 (%)	3.3 ± 2.7	1.0 ± 0.8	4.9 ± 2.4	<0.001	2.7 to 4.4^a
Macrophage p-TAK1 (%)	33.2 ± 20.9	11.0 ± 3.1	49.1 ± 11.7	0.0037	36.1 to 43.9^b
Lymphocyte p-TAK1 (%)	0.2 ± 0.6	0.1 ± 0.5	0.3 ± 0.6	0.039	0.0 to 0.0^a
Epithelial p-TAK1 (%)	1.2 ± 1.1	0.7 ± 0.9	1.5 ± 1.1	<0.001	0.5 to 1.2^a

Notes: Data are presented as mean ± SD. FAO was defined as postbronchodilator FEV₁/FVC < LLN with the first lung function data at least 3 months before study entry and confirmed the persistence with the second pulmonary function testing at study enrolment as demonstrated in the table p value* nFAO versus FAO. ^ap value from Mann–Whitney test. ^bp value from unpaired t-test.

Abbreviations: FAO, fixed airway obstruction; nFAO, no fixed airway obstruction; TAK1, transforming growth factor-β-associated kinase 1; p-TAK1, phosphorylated TAK1; TGF-β₁, transforming growth factor-β_1.

Figure 1 TGF-β₁ levels of asthmatics with FAO and non-FAO (nFAO) in the presence or absence of airway eosinophilia as indicated. TGF-β₁ was quantified by ELISA in sputum fluid of FAO and nFAO asthmatic patients with sputum eosinophilia and without sputum eosinophilia. Results are depicted as bar column, with median values, 25th and 75th quartile and the range of values. Mann–Whitney U-test was used for the statistical analysis, and there was no statistical significance within group and associated P values between groups are indicated. ***P < 0.001.

Figure 2 The levels of TAK1 and p-TAK1 in total sputum cells was analyzed by Western blot analysis of whole cell lysate from asthmatics with FAO and without FAO (non-FAO). GAPDH served as the standard.

Figure 3 Correlations between total TAK1 immunopositive cells in sputum of asthmatics with FAO, expressed as percentage, and the pulmonary function tests, including pre-bronchodilator (A) and post-bronchodilator FEV₁/FVC ratios (B). Spearman rank correlation has shown significant negative correlations between TAK1 levels and FEV₁/FVC ratios.

Figure 4 TAK1 and p-TAK1 immunopositive cells (%) in sputum of asthmatics with FAO and without FAO (nFAO) in the presence or absence of airway eosinophilia as indicated. (A) The difference in TAK1 immunopositive cell numbers within and between groups. (B) The difference in p-TAK1 immunopositive cell numbers between patients with and without airway eosinophilia. Results are depicted as bar, column, with median values, 25th and 75th quartile and the range of values. Mann–Whitney U-test and associated P-values are indicated. ***P < 0.001 for comparison with FAO with eosinophilia. ψ for comparison with FAO without eosinophilia (P < 0.001).