High transcriptional activity and clinical correlations in eosinophils of patients with late-onset asthma

doi:10.21203/rs.3.rs-1983333/v1

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High transcriptional activity and clinical correlations in eosinophils of patients with late-onset asthma

https://doi.org/10.21203/rs.3.rs-1983333/v1

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Background

The immunological features of eosinophils in early-onset asthma (EOA) differs from that in late-onset asthma (LOA). Clinical trials of anti-interleukin-5 (IL-5) treatment for severe eosinophilic asthma showed a better response for LOA patients than EOA patients. We wonder if the transcriptional activity of activated eosinophils was different in EOA and LOA.

Methods

Eosinophils obtained from well controlled EOA and LOA patients and normal subjects were compared in terms of the mRNA expression of activation-related genes (integrins α4 and β2, CCR3, CCR5, and IL-5Rα, and IL-17RA) and specific markers (IL-4Rα, prostaglandin D2 receptor 2, high-affinity IgE receptor, TGF-β1, aryl hydrocarbon receptor, and endosomal TLR-3, 7, and 9) in eosinophils activated by IL-5 or IL-17. The correlation between mRNA expression and clinical features and lung function were further analyzed.

Results

The transcriptional expression of most genes was higher in activated eosinophils from LOA patients than in those from EOA patients and normal subjects. After IL-17 stimulation, the expression of certain genes was higher in atopic EOA patients than in non-atopic EOA patients. Similar observation was noted in obese EOA patients. After IL-5 stimulation, the transcriptional expression of most genes in eosinophils from LOA patients was negatively correlated with indicators of lung function. These correlations were less pronounced in EOA patients: After IL-17 stimulation, some genes in EOA patients were negatively correlated with post-bronchodilator changes in lung function. There was no significant correlation between mRNA expression and treatment control levels among LOA or EOA patients

Conclusion

This study describes differences in the transcriptional active patterns of eosinophils and their correlation to atopy and obese by age of onset. High transcriptional activity in activated eosinophils and a negative correlation to lung function indicate the importance of eosinophils in the pathogenesis of LOA.

late-onset asthma

early-onset asthma

eosinophils

Messenger RNA

phenotyping

Airway eosinophilia is associated with poor symptom control, acute exacerbation, and airway remodeling in asthma[1–3]. Recruitment of eosinophils from blood requires eosinophil activation, leading to arrest on the endothelium and extravasation, followed by recruitment to the airway. Surface proteins (e.g., integrins, cytokines, and chemokine receptors) have been used to indicate the activation of circulating eosinophils [4–6]. In addition to the expression of activated markers, the priming of eosinophil circulation in asthmatic patients under allergen challenge or cytokine stimulation has also been shown to induce a functional response stronger than that of the eosinophils of control subjects[7, 8]. Researchers have also identified correlations between the surface proteins of circulating eosinophils and the features of asthma; however, the results have been inconsistent. One possible explanation is the extravasation of activated eosinophils from circulation. It has also been suggested that in vitro experiment methods alter protein expression[6].

Early-onset asthma (EOA) and late-onset asthma (LOA) are recognized as distinct phenotypes, in terms of risk factors, remission rate, co-morbidities, eosinophilic inflammation, and gene signatures[9–12]. One study reported that airway eosinophils in patients with LOA were less strongly associated with type 2 inflammation than were the eosinophils of patients with EOA[11]. In cases of LOA, blood eosinophilia levels were also associated with disease severity[13]. Interestingly, recent clinical trials involving anti-interleukin-5 (IL-5) treatment for severe eosinophilic asthma showed a better response for patients with LOA (onset after the age of forty) than for with patients with EOA[14–16]. These findings suggest that there is a fundamental mechanistic difference between eosinophils in cases of EOA and LOA. This prompted us to consider whether there were differences in the priming response of circulating eosinophils in cases of LOA and EOA.

In the current study, we analyzed the transcriptional levels of target genes in the eosinophils from EOA and LOA following activation using IL-5 or IL-17. We also examined the mRNA expression of target genes associated with activated eosinophils, including integrin α4 for arrest on endothelium[17], integrin β2 for recruitment to airways[18], CCR3 for eotaxin receptors, and CCR5 for MIP-1 receptors[19] as well as cytokine receptors, IL-5Rα, IL-17RA, IL-4Rα, prostaglandin D2 receptor 2 (CRTH2), and FcεRI, which are related to eosinophil activation or recruitment[5, 6, 20, 21]. We also examined a broader range of markers, including TGF-β1 for airway remodeling, aryl hydrocarbon receptor (AHR) for tissue adaptation under environmental triggers, and endosomal TLR-3, 7, and 9, which have been linked to eosinophil-related inflammation[22–24]. Finally, we examined correlations between the transcriptional levels of target genes and patient characteristics to identify patient-related factors associated with the transcriptomes of eosinophils.

Subjects and blood sampling

This study recruited asthma patients (diagnosed by pulmonologists in a medical center), based on episodic respiratory symptoms and a history of one or more of the following: 1. >12% and > 200ml improvement in forced expiratory volume in 1 second (FEV1) following the use of a bronchodilator; 2. PC20 of methacholine test < 8 mg/mL; 3. Diurnal variation of > 10% in peak expiratory flow; and 4. Increase in FEV1 by > 12% and > 200 mL or diurnal variation in peak expiratory flow by > 20% from baseline after four weeks of treatment. Asthma control levels were defined in accordance with guidelines outlined in the 2018 Global Initiative for Asthma (GINA)[25]. Pulmonary function, blood eosinophil count, immunoglobulin E (IgE), and specific IgE (ImmunoCAP, Phadia, Sweden) were recorded. Patients with positive specific IgE to allergens (> 0.35 KU/L) were considered atopic. Note that all subjects were non-smokers and clinically free from respiratory or systemic infection during the two months prior to recruitment. All patients provided written informed consent. This study was approved by the Chang Gung Medical Foundation Institutional Review Board (No. 201900943A3).

Preparation of purified eosinophils

Peripheral blood sampled from patients was subjected to Ficoll-Hypaque centrifugation (GE Healthcare, Bio-science AB, Uppsala, Sweden). The lower layer consisted mainly of granulocytes and erythrocytes and was treated using an erythrocyte lysis solution (Bioman Scientific CO., LTD, New Taipei City, Taiwan). Eosinophils were isolated from the granulocyte part using an eosinophil isolation kit through the depletion of CD16 + neutrophils (Miltenyi Biotec Inc. Auburn, CA, USA) in accordance with the manufacturer's specifications. The purification of eosinophils reached > 95%, as confirmed via cytospin flowcytometry. The purified eosinophils were resuspended in RPMI 1640 (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 100 U/ml penicillin G, 10 µg/ml streptomycin, 3 µg/ml L-glutamine, and 20% heat-inactivated fetal calf serum.

RNA purification, cDNA synthesis, and quantitative real-time polymerase chain reaction

Eosinophils were resuspended at a concentration of 5 x 10⁵ in 500 µl RPMI 1640 in 24-well culture plates. Eosinophils were incubated with IL-5 (25 ng/µl), IL-17A (25 ng/µl) (R&D Systems, Minneapolis, MN, USA), or phosphate-buffered saline (PBS) as a control (Sigma-Aldrich, St. Louis, MO, USA) for 30 min, 1 h, or 3 h. Cells were then harvested for total RNA isolation using the TOOLSmart RNA Extractor (BIOTOOLS CO., LTD., Taipei, Taiwan). Complementary DNA was synthesized from 1-100 µg of purified RNA using ToolsQuant2 Fast RT Kit (BIOTOOLS CO., LTD., Taipei, Taiwan) in accordance with the manufacturer’s instructions. RT-PCR primer sets for target genes were designed manually based on the website of the National Center for Biotechnology Information using Primer-BLAST tools (see Additional file 1). Quantitative real-time PCR (qRT-PCR) analysis was performed using the MyGo Pro real-time PCR System (Azura Genomics Inc., Raynham, MA, U.S.A.) using 2X SuperFast SYBR qPCR Reagent (BIOTOOLS CO., LTD., Taipei, Taiwan). Ct values were derived from fluorescence measurements obtained during 3-step real-time PCR (40 cycles of denaturation at 95°C for 5 sec; annealing at 60°C for 10 sec; extension at 72°C for 15 sec). Relative gene expression was normalized to β-actin and calculated based on Ct values using PBS treatment as a baseline control.

Statistical analysis

Patient characteristics were expressed as mean ± standard deviation (SD) or numbers with percentage. Unpaired two-tailed Student t-tests and the Fisher exact test were used to compare continuous variables and categorical data between EOA and LOA. Comparisons of mRNA expression of target genes between groups were performed using the Kruskal-Wallis test in conjunction with the Dunn-Bonferroni post-hoc test. mRNA expression was also tested in the context of patient atopy, obesity status (body mass index ≥ 30), and gender.

Spearman rank correlation was used to assess the correlations between the mRNA expression of target genes and IgE levels, eosinophil counts, GINA levels or lung function following stimulation using IL-5 or IL-17 30 min or 1 hour. The correlations with lung function were further analyzed using heatmaps derived using the Spearman correlation coefficient ρ. Probability values of < 0.05 were considered significant. All analysis was performed using SPSS, version 19 (IBM; Armonk, NY).

This study recruited patients with EOA (n = 18) or LOA (n = 14) and healthy controls (n = 7) between October 2019 and September 2021 (Table 1). The mean age of onset was as follows: EOA (22.9 ± 9.8 years) and LOA (54.7 ± 12.1 years). LOA patients were older than EOA patients, were less likely to have a family history of asthma, were less prone to atopy, and presented lower FEV1/FVC levels.

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/m², 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 X10⁴/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)	5.8 (13.8)	0.572
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)
Data are presented as mean (standard deviation, SD), median (interquartile range, IQR), or number (%), unless otherwise stated.
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.
#: p < 0.05, *: p < 0.01, ¶: p < 0.001 compared with healthy subjects. §: p < 0.001 compared with EOA.

Transcriptional activity was higher in activated eosinophils of patients with LOA than in those with EOA

Figure 1 illustrates the mRNA expression of integrin α4, integrin β2, CCR3, and CCR5 (Figs. 1A to D) in the eosinophils of LOA patients, EOA patients, and normal controls following stimulation using IL-5 (left panels) or IL-17 (right panels). After IL-5 stimulation for 1 hour, the increases in integrin α4 and CCR5 expression levels were more pronounced in LOA patients than in EOA patients. After IL-5 stimulation for 30 min, the increases in integrin α4, integrin β2, CCR3, and CCR5 expression levels were more pronounced in patients with LOA than in normal subjects. Following IL-5 treatment, only integrin α4 expression levels were higher in EOA patients than in normal subjects. Following IL-17 stimulation for 30 min, the mRNA expression levels of all four genes were higher in LOA patients than in EOA patients. Similar results were obtained from IL-5 only after stimulation for 1 hour. Following IL-17 stimulation for 30 min, the mRNA expression levels of all four genes were higher in LOA patients than in normal subjects. Following IL-17 stimulation, we observed no differences between EOA patients and normal subjects in terms of mRNA expression levels.

Figures 2 and 3 present the mRNA expression levels of the other target genes, the results of which were similar to those in Fig. 1. Table 2 lists the mRNA expression of all target genes in LOA patients, EOA patients, and normal subjects following IL-5 and IL-17 stimulation. We found that after IL-17 stimulation, the mRNA levels of all target genes of eosinophils other than TLR-3 were higher in LOA patients than in EOA patients. We found that after IL-5 stimulation, the mRNA expression of integrin α4, CCR5, IL-5Rα, IL-17RA, CRTH2, FcεRI, TGF-β1, AHR, and TLR-9 was higher in the eosinophils of LOA patients than in those of EOA patients. Note that the effects on the mRNA expression of all genes except FcεRI and TLR-3 manifested more rapidly after IL-17 stimulation (30 min) than after IL-5 stimulation (at 1 hour). IL-17 stimulation and IL-5 stimulation were both shown to increase the expression of all eosinophil target genes in LOA patients beyond that of normal controls. Note that between EOA patients and normal subjects, we observed no significant differences in the mRNA expression levels of any markers other than integrin α4.

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	*	1	**	0.5
	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	*	1	No	-
	LOA vs EOA	No		**	0.5
Integrin β2	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	*	0.5
CCR3	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	**	0.5
CCR5	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	*	0.5
IL-5Rα	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	**	1	**	0.5
IL-17RA	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	**	1
IL-4Rα	LOA vs NC	*	0.5	*	1
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	*	0.5
CRTH2	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	**	1
FcεRI	LOA vs NC	**	0.5	*	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	**	0.5	***	0.5
CRLF2	LOA vs NC	No	-	*	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	0.5	**	0.5
IL-7Rα	LOA vs NC	***	0.5	***	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	**	0.5
TGF-β1	LOA vs NC	*	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	*	0.5
AHR	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	No	-
TLR-3	LOA vs NC	*	1	*	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	No	-	**	0.5
TLR-7	LOA vs NC	**	0.5	**	0.5
	EOA vs NC	No	-	No	-
	LOA vs EOA	*	1	**	0.5
TLR-9	LOA vs NC	***	0.5	***	0.5
	EOA vs NC	No	-	No	-
Abbreviations: EOA: early-onset asthma; LOA: late-onset asthma; NC: normal control.
: p < 0.05, : p < 0.01, **: p < 0.001.

Transcriptional activity in activated eosinophils from EOA and LOA as a function of atopy, obesity, or T2 biomarkers

mRNA expression peaked at 30 min or 1 hour after stimulation. We therefore analyzed the correlation between the mRNA levels of target genes at those points versus patient characteristics, including atopy, obesity, and gender.

In the EOA group after IL-5 or IL-17 stimulation for 1 hour, the mRNA expression of integrin β2 was higher in atopic patients than in non-atopic patients. In the EOA group after IL-17 stimulation for 1 hour, the mRNA expression of CRTH2 and FcεRI was higher in atopic patients than in non-atopic patients (Fig. 4A). In the LOA group, we observed no difference in mRNA expression as a function of atopic status.

In the EOA group after IL-17 stimulation for 30 min, the mRNA expression levels of integrin α4, CCR3, IL-5Rα, IL-17RA, CRTH2, AHR, and TLR-7 were significantly higher in obese patients than in non-obese patients (Fig. 4B). In the LOA group after IL-17 stimulation for 1 hour, the mRNA expression levels of IL-17RA and TGF-β1 were higher in obese patients than in non-obese patients (Fig. 4C). In terms of gender, we observed higher CCR5 expression levels after IL-17 stimulation (1 hour) in female LOA patients than in male LOA patients (see Additional file 2).

Table 3 summarizes the correlation between the mRNA expression of target genes in activated eosinophils and T2 markers in patients with EOA and LOA. Among EOA patients, the mRNA expression levels of IL-17RA, CRTH2, AHR, and TLR-7 were positively correlated with total IgE levels. Among LOA patients, we observed a negative correlation between CCR5 and total IgE levels. Among EOA patients, we observed a negative correlation between the mRNA expression of CCR5 and TGF-β1 and eosinophil counts. Among LOA patients, we observed a positive correlation between the mRNA expression of CCR3, IL-5RA, CRTH2, AHR, and TLR-7 and eosinophil counts.

Table 3

mRNA expression of activated eosinophils correlation with T2 markers in patients with EOA and LOA
mRNA expression correlation with total IgE level in patients with EOA
Gene	IL-17RA		CRTH2		AHR		TLR-7
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.

Negative correlation between mRNA gene expression in activated eosinophils and lung function in LOA patients but inconsistent correlation in EOA patients

Figure 5 presents the Spearman correlation coefficient ρ of mRNA expression levels and lung function parameters, where a higher ρ value indicates a stronger correlation. In the LOA group (Fig. 5A), we observed a significant negative correlation between integrin α4, CCR3, CCR5, IL-5Rα, IL-17RA, CRTH2, AHR, and TLR-3, 7, and 9 mRNA expression levels and lung function in terms of FVC (L) and FEV1 (L). We also observed a significant negative correlation between IL-17RA, TLR-3, and TLR-9 mRNA expression levels and post-BD FEV1 (L). TLR-3 alone was also negatively correlated with FVC (%) and FEV1/FVC. TLR3 expression presented the strongest correlation with lung function (ρ= -0.65 to -0.74). Note that all of the significant correlations between specific genes and lung function were observed only after IL-5 stimulation (i.e., not after IL-17 stimulation).

As shown in Fig. 5B, the correlation between mRNA expression and lung function was less pronounced among EOA patients than among LOA patients. After IL-17 stimulation, we observed a negative correlation between IL-5RA, IL-4Rα, CRTH2, AHR, and TLR-3, 7, and 9 mRNA expression levels and changes in post-BD FEV1 changed. After IL-5 stimulation, we observed a correlation between TLR-9 mRNA expression levels and lung function. We also observed positive correlations between mRNA expression of some genes and lung function.

There was no significant correlation between mRNA expression and GINA control levels among LOA or EOA patients (data not shown).

The present study discovered that the mRNA expression of many target genes associated with eosinophilic functions and activation were higher in patients with LOA than in patients with EOA and normal subjects. After IL-17 stimulation, we observed that atopic and obesity status were correlated with higher transcriptionalexpression levels of specific genes in the eosinophils of EOA patients. We also observed that in the EOA group, the transcriptional expression levels of various genes in activated eosinophils were positively correlated with total IgE levels and negatively correlated with blood eosinophil count. Note that this correlation was the opposite of that observed in the LOA group. After IL-5 stimulation, we found that most of the transcriptional activity in eosinophils from LOA patients was correlated with reduced lung function. The correlation between mRNA expression levels and lung function was less pronounced in EOA patients than in LOA patients. To the best of our knowledge, this was the first study to reveal differences between EOA and LOA in terms of the correlation between mRNA expression patterns of activated eosinophils and clinical outcomes. The high transcriptional activity in activated eosinophils and the negative correlation with lung function suggest that eosinophilic inflammation plays a key role in the pathogenesis of LOA.

There have been relatively few studies on the patterns of eosinophilic inflammation in the context of the age of onset. One early study determined that airway eosinophilia was more common in cases of LOA than in cases of EOA and that T2 inflammatory responses were less pronounced[11]. One sophisticated study based on gene set variation analysis revealed that LOA was characterized by enriched gene signatures of eosinophils[10]. Note that their findings are complementary to those in the current study. Post-hoc analysis in trials of anti-IL-5 biologics for severe eosinophilic asthma revealed variations in responses as a function of the age of onset. The effects of reslizumab on exacerbation were 75% less obvious in LOA patients than in EOA patients[14]. In another study on patients with frequent exacerbation, benralizumab was shown to reduce exacerbation by 70% in LOA patients and 42% in EOA patients[15]. In a study conducted in Australia, the benefits of mepolizumab were more pronounced in cases involving elevated blood eosinophil levels and a later age of onset[16]. These differential responses among subgroups suggest that the relative importance of eosinophilic inflammation varies as a function of age (at the time of asthma diagnosis). In the current study, we identified a correlation between an increase in transcriptional activity in the eosinophils of LOA patients after IL-5 stimulation and poor lung function. This may provide a mechanistic explanation for the superior response of eosinophil-targeting biologics in patients with LOA.

Following IL-17 treatment, we observed increases in the mRNA expression of specific genes in the eosinophils of atopic or obese EOA patients, but not in those of LOA patients (Figs. 3A and B). Note that atopy is a well-known risk factor for EOA[9]. In one previous study, a higher BMI was significantly associated with an earlier age of onset[26]. Researchers have also reported increased eosinophil uptake in the lungs of obese asthmatic patients and eosinophil accumulation in airway submucosa[27, 28]. Our results provide further evidence of an interaction between specific phenotypes and eosinophil activation under IL-17 stimulation in EOA patients. Atopy was shown not to have this effect on transcriptional activity in the eosinophils of LOA patients. Furthermore, we observed a correlation between obesity and only two eosinophil-related genes (Fig. 3C). Further studies will be required to identify the mechanism underlying eosinophil activation in non-atopic LOA patients, such as the role of thymic stromal lymphopoietin or innate lymphoid cells in eosinophil recruitment[29, 30].

In addition to atopy, total IgE levels and eosinophil count have been identified as biomarkers of T2 inflammation. The elevated mRNA expression of various eosinophil-related genes was associated with higher IgE levels in EOA patients. Expression of the CCR5 gene was negatively correlated with total IgE levels in LOA patients (Table 3). Differences in the IgE correlation patterns in EOA and LOA patients revealed that the discrepancy between allergic inflammation and eosinophil activation increased with an increase in the onset of asthma age. This result is consistent with previous findings indicating that LOA is associated with non-atopic eosinophilia[9, 31]. We also determined that among EOA patients, elevated mRNA expression levels in eosinophils were correlated with lower blood eosinophil counts. Note that this relationship is the inverse of that observed among EOA patients. These findings indicate that regulating the recruitment of circulating eosinophils may differ between EOA and LOA. In cases of EOA, transcriptional activity may be related to cell extravasation from circulation. In cases of LOA, it appears that transcriptional activity is related to eosinophilia or recruitment into circulation Further research will be required to obtain mechanistic explanations of these phenomena.

Our results have clinical implications pertaining to the negative correlation between the transcriptional activity of circulating eosinophils and the lung function of LOA patients after IL-5 stimulation (Fig. 5A). Elevated transcriptional activity levels in eosinophils were associated with poor lung function, such as FVC and FEV1. Elevated mRNA expression levels of TLR-3 were also associated with lower FVC (%) and FEV1/FVC ratio. At present, the mechanism underlying the strong correlation between mRNA TLR-3 expression and lung function is unknown. One study on allergic rhinitis revealed that eosinophils activated by TLR3 are able to recruit leukocytes to sites of inflammation[23]. Activated TLR-3 has been shown to augment the extracellular production of lung fibroblasts[32]. Further mechanistic studies will be required to determine whether TLR signaling in eosinophils is associated with impaired lung function. In terms of the correlation between mRNA and lung function, the difference between the EOA and LOA groups can be seen in the column on the right of Fig. 5B (post BD FEV1 change %) and the expression levels of IL-17-stimulated IL-5Ra, IL-4Ra, CRTH2, AHR, and TLR-3,7,9 mRNA. As shown in the black box, there was a significantly negative correlation between mRNA and lung function in the EOA group (Fig. 5B). In a previous report, the co-expression of Th2-Th17 inflammation in asthma was characterized by high IL-17A in bronchioalveolar lavage, poor lung function, and high blood eosinophil count[33]. Data obtained in the current study provide further evidence as to the importance of IL-17 in the treatment of eosinophilic inflammation in cases of EOA.

This study was subject to a number of limitations, which limit the generalizability of our findings. First, we observed links between mRNA expression in eosinophils and various features of asthma. Note however that identifying the mechanism by which these genes affect the characteristics of asthma was beyond the aims of the study. Second, the age of EOA and LOA patients was not balanced, and the older age of LOA patients may have been a confounder. Nonetheless, the distinct patterns of correlation between transcriptional activity in eosinophils and the features of asthma in EOA and LOA cannot be explained entirely by age. Furthermore, one previous study reported a decrease in eosinophil function among older patients[34]. Therefore, our study revealed the increasing transcriptional level in eosinophils of LOA patients is a unique finding. The current study was inspired by observed differences in the response to treatment using anti-IL-5 biologics in cases of severe eosinophilic asthma; however, the patients recruited in the current study were of mixed severity in small sample size, such that applying our results are not necessarily indicative of severe asthma. Fourth, to avoid the possibility of protein expression being skewed by in vitro cell culture conditions (as mentioned in the background), we focused on the mRNA expression of selected genes and specific biomarkers. Nonetheless, the post-translational effects of gene expression are unknown. Further research will be required to validate our observations at the protein level.

This study found that patients with EOA differ considerably from those with LOA in terms of background characteristics and the mRNA expression of genes related to cell activation and cell recruitment. Atopy and obese can impact the transcriptional level of eosinophils of EOA but not LOA. High transcriptional activity in activated eosinophils and negative correlations with lung function suggest that eosinophilic inflammation plays a key role in the pathogenesis of LOA.

EOA: early-onset asthma

LOA: late-onset asthma

CCR: C-C motif chemokine receptor

IL-5Rα: interleukin-5 receptor α

IL-17RA: interleukin-17 receptor A

IL-4Rα: interleukin-4 receptor α

CRTH2: prostaglandin D2 receptor 2,

FcεRI: Fcε receptor type I

TGF-β1: transforming growth factor beta 1

AHR: aryl hydrocarbon receptor

TLR: toll-like receptor

Ethics declarations

All patients provided written informed consent. This study was approved by the Chang Gung Medical Foundation Institutional Review Board (No. 201900943A3).

Consent for publication

Not Applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest statement

No conflict of interest to declare

Funding

This study is supported by Chang-Gung Memorial Hospital (Grant no. CMRPG3J1361, CMRPG3J1362).

Authors' contributions

TYL: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization and writing original draft.

CYL, PJC, YUL: Data curation, Investigation, methodology, and resources.

CSL, CHC and CTY: Investigation, Methodology, and resources-Supporting

JHY: Data curation, Formal analysis, methodology, and project administration.

SML: Data curation, formal analysis, resources, supervision, and Writing – review & editing.

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No competing interests reported.

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High transcriptional activity and clinical correlations in eosinophils of patients with late-onset asthma

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Subjects and blood sampling

Preparation of purified eosinophils

RNA purification, cDNA synthesis, and quantitative real-time polymerase chain reaction

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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