High Transcriptional Activity and Clinical Correlations in Eosinophils of Patients with Late-Onset Asthma

Figures & data

Table 1 Characteristics of Healthy Controls and Asthma Patients (EOA vs LOA)

Variables	Health Subjects (n=7)	EOA (n= 18)	LOA (n =14)	p-value
Age, years, mean (SD)	38.1 (7.0)	39.2 (14.4)	65.0 (10.4)^¶§	<0.001
Male, n (%)	2 (28.6)	9 (50.0)	9 (64.3)	0.301
Body mass index, kg/m^2, mean (SD)	23.7 (2.0)	27.8 (1.2)	26.0 (3.5)	0.109
Age of asthma onset, years, mean (SD)	NA	22.9 (9.8)	54.7 (12.1)	<0.001
Family history of asthma, n (%)	NA	26 (42.6)	48 (21.6)	0.02
Atopy, n (%)	NA	13 (72.2)	6 (46.2)	0.262
IgE level, KU/L, median (IQR)	NA	308.5 (133.5–948.0)	172.0 (72.3–672.0)	0.154
Eosinophil counts ×10^4/mL, median (IQR)	7.9 (4.8–11.6)	14.5 (7.8–21.6)	10.6 (5.0–13.2)	0.328
Pulmonary function, mean (SD)
FVC, liter	3.4 (0.4)	3.1 (1.1)	2.6 (0.8)	0.133
FVC, % of pred.	106.0 (15.8)	84.2 (17.0)	82.2 (18.9)^#	0.038
FEV1, liter	2.4 (1.1)	2.4 (0.9)	1.8 (0.6)	0.114
FEV1, % of pred.	104.2 (14.6)	75.6 (17.8)^#	71.8 (18.8)*	0.005
FEV1/FVC, %	87.0 (13.0)	75.9 (12.1)	69.2 (7.2)*	0.006
Post-BD FEV1, liter	2.9 (0.4)	2.6 (0.9)	1.9 (0.6)	0.030
Change of FEV1% post-BD	3.0 (2.5)	10.1 (11.2)	10.5 (9.4)	0.319
Asthma control level, n (%)				0.224
GINA level 2	NA	4 (22.2)	0
GINA level 3	NA	4 (22.2)	4 (28.6)
GINA level 4	NA	6 (33.3)	4 (28.6)
GINA level 5	NA	4 (22.2)	6 (42.9)

Notes: Data are presented as mean (standard deviation, SD), median (interquartile range, IQR), or number (%), unless otherwise stated. ^#p < 0.05, *: p < 0.01, ^¶p < 0.001 compared with healthy subjects. ^§p < 0.001 compared with EOA.

Abbreviations: EOA, early-onset asthma; LOA, late-onset asthma; NA, not available; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; BD, bronchodilator; GINA, Global Initiative for Asthma.

Figure 1 mRNA expression levels in eosinophils stimulated using IL-5 or IL17 for 30 min, 1 hour, or 3 hours: (A) Integrin α4; (B) Integrin β2; (C) CCR3; and (D) CCR5.

Notes: □: Late-onset asthma (LOA); ∆: Early-onset asthma (EOA); ○: Normal control (NC). Post-hoc analysis *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 2 mRNA expression levels in eosinophils stimulated using IL-5 or IL17 for 30 min, 1 hour, or 3 hours: (A) IL-5Rα; (B) IL-17RA; (C) IL-4Rα; (D) CRTH2; and E. FcεRI.

Notes: □: Late-onset asthma (LOA); ∆: Early-onset asthma (EOA); ○: Normal control (NC). Post-hoc analysis *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 3 mRNA expression levels in eosinophils stimulated using IL-5 or IL17 for 30 min, 1 hour, or 3 hours: (A) TGF-β1; (B) AHR; (C) TLR-3; (D) TLR-7; and (E) TLR-9.

Notes: □: Late-onset asthma (LOA); ∆: Early-onset asthma (EOA); ○: Normal control (NC). Post-hoc analysis *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Table 2 Summary of Effects of IL-5 and IL-17 Stimulation on mRNA Expression in Eosinophils of EOA and LOA Patients and Normal Control Subjects

		IL-5 Stimulation		IL-17 Stimulation
mRNA Expression	Groups Compared	Significance	First Timepoint of Significance (hr)	Significance	First Timepoint of Significance (hr)
Integrin α4	LOA vs EOA	(5.1±1.0 vs 1.8±1.3)*	1	(7.7±2.1 vs 1.6±0.4)**	0.5
	LOA vs NC	(5.9±1.6 vs 0.7±0.1)**	0.5	(7.7±2.1 vs 0.8±0.2)**	0.5
	EOA vs NC	(1.8±1.3 vs 0.7±0.1)*	1	No	-
	LOA vs EOA	No		(5.2±1.1 vs 1.6±0.3)**	0.5
Integrin β2	LOA vs NC	(35.3±6.9 vs 2.9±1.1)**	0.5	(5.2±1.1 vs 1.0±0.3)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	(6.6±1.8 vs 1.6±0.3)*	0.5
CCR3	LOA vs NC	(5.6±1.4 vs 0.9±0.2)**	0.5	(6.6±1.8 vs 0.8±0.2)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(7.9±1.7 vs 2.5±0.6)*	1	(10.2±3.3 vs 2.4±0.7)**	0.5
CCR5	LOA vs NC	(4.0±2.7 vs 1.1±0.4)**	0.5	(10.2±3.3 vs 1.5±0.4)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(6.3±1.5 vs 1.9±0.4)*	1	(10.1±3.0 vs 1.5±0.3)*	0.5
IL-5Rα	LOA vs NC	(8.2±2.8 vs 0.8±0.1)**	0.5	(10.1±3.0 vs 0.8±0.2)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(4.7±0.8 vs 1.7±0.4)**	1	(7.7±1.8 vs 1.6±0.3)**	0.5
IL-17RA	LOA vs NC	(5.4±1.2 vs 0.9±0.1)**	0.5	(7.7±1.8 vs 0.9±0.2)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	(2.4±0.6 vs 0.7±0.1)**	1
IL-4Rα	LOA vs NC	(20.9±5.0 vs 4.4±1.1)*	0.5	(2.4±0.6 vs 0.6±0.1)*	1
	EOA vs NC	No	-	No	-
	LOA vs EOA	(3.9±0.8 vs 1.5±0.4)*	1	(7.7±2.1 vs 1.6±0.3)*	0.5
CRTH2	LOA vs NC	(6.1±1.4 vs 0.9±0.2)**	0.5	(7.7±2.1 vs 0.8±0.2)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(5.7±1.0 vs 2.5±1.0)*	1	(6.3±1.1 vs 1.7±0.3)**	1
FcεRI	LOA vs NC	(7.6±2.1 vs 0.7±0.1)**	0.5	(10.9±3.9 vs 0.9±0.2)*	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(13.7±3.8 vs 2.7±0.6)*	1	(20.6±6.6 vs 3.9±1.8)**	0.5
TGF-β1	LOA vs NC	(11.8±3.6 vs 0.7±0.2)*	0.5	(20.6±6.6 vs 1.4±0.6)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(9.2±2.0 vs 3.0±0.5)*	1	(7.7±1.9 vs 1.5±0.3)*	0.5
AHR	LOA vs NC	(6.9±1.7 vs 0.9±0.2)**	0.5	(7.7±1.9 vs 0.8±0.2)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	No	-
TLR-3	LOA vs NC	(16.5±8.0 vs 1.1±0.4)*	1	(20.4±12.9 vs 0.7±0.2)*	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	(7.6±1.7 vs 1.6±0.3)**	0.5
TLR-7	LOA vs NC	(4.9±1.4 vs 0.6±0.1)**	0.5	(7.6±1.7 vs 1.0±0.3)**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	(6.2±2.2 vs 1.7±0.3)*	1	(6.7±1.2 vs 1.6±0.3)**	0.5
TLR-9	LOA vs NC	(7.3±1.8 vs 0.7±0.2)***	0.5	(6.7±1.2 vs 0.7±0.2)***	0.5
	EOA vs NC	No	-	No	-

Notes: mRNA expression (2-∆∆ct) is presented as mean ± standard error. *: p<0.05, **: p<0.01, ***: p<0.001.

Abbreviations: EOA, early-onset asthma; LOA, late-onset asthma; NC, normal control.

Figure 4 mRNA expression of specific genes in eosinophils as a function of atopy or obesity. (A) The expression of integrin β2 after IL-5 or IL-17 stimulation (1-hour) and CRTH2 and FcεRI after IL-17 stimulation (1-hour) were higher in EOA patients with atopy than in EOA patients without atopy; (B) The expression of integrins α4, CCR3, IL-5Rα, IL-17RA, CRTH2, AHR and TLR-7 after of IL-17 stimulation (30 min) were higher in obese EOA patients than in non-obese EOA patients; (C) The expression of IL-17RA and TGF--β1 after of IL-17 stimulation (1-hour) was higher in obese LOA patients than in non-obese LOA patients.

Table 3 mRNA Expression of Activated Eosinophil Correlation with T2 Markers in Patients with EOA and LOA

Gene	IL-17RA	CRTH2	AHR	TLR-7
mRNA expression correlation with total IgE level in patients with EOA
Stimulation	IL-5, 30 min	IL-5, 30 min	IL-5, 30 min	IL-17, 1 hour
Spearman’s ρ	0.507	0.551	0.498	0.507
P-value	0.027	0.015	0.03	0.027
mRNA expression correlation with total IgE level in patients with LOA
Gene	CCR5
Stimulation	IL-17, 1 hour
Spearman’s ρ	−0.593
P-value	0.033
mRNA expression correlation with eosinophil counts in patients with EOA
Gene	CCR5	TGF-β1	TGF-β1
Stimulation	IL-5, 30 min	IL-5, 30 min	IL-17, 30 min
Spearman’s ρ	−0.713	−0.552	−0.653
P-value	0.004	0.041	0.011
mRNA expression correlation with eosinophil counts in patients with LOA
Gene	CCR3	IL-5RA	CRTH2	AHR	TLR-7
Stimulation	IL-17, 30 min	IL-17, 30 min	IL-17, 30 min	IL-17, 30 min	IL-17, 30 min
Spearman’s ρ	0.548	0.577	0.612	0.496	0.605
P-value	0.019	0.01	0.005	0.031	0.006

Abbreviations: EOA, early-onset asthma; LOA, late-onset asthma.

Figure 5 Continued.

Figure 5 Heatmap showing the Spearman correlation coefficient ρ of mRNA expression levels and lung function parameters (A) LOA group; (B) EOA group. Red and blue color respectively indicate negative and positive correlations. The thick black frame indicates that the correlation is statistic significant.

Data Sharing Statement

The datasets used and/or analyzed during the current study are available in the Google drive repository.

[https://drive.google.com/drive/folders/13DXdwj91b2qG4WL9LLTezFFYSNxdsj7a?usp=sharing].