Subjects and study protocol

This was a single center cross-sectional study, involving patients with uncontrolled asthma. Patients referred to Beijing Tongren Hospital for complaints of recurrent and intermittent wheeze, shortness of breath, chest tightness, and cough were consecutively enrolled into the study from Sept 2017 to Sept 2019.

Asthma and uncontrolled asthma were defined according to the criteria of Global Initiative for Asthma (GINA) [11]. Briefly, asthma was diagnosed based on history of variable respiratory symptoms (wheeze, shortness of breath, chest tightness and cough), variable expiratory airflow limitation confirmed by spirometry (positive bronchodilator (BD) reversibility test/positive bronchial challenge test/significant increase in lung function after 4 weeks of anti-inflammatory treatment), and exclusion of other conditions that mimic asthma. The variable airflow limitation was determined based on the results of both lung function measurement on enrollment and previous results, as most of the patients were previously diagnosed as asthmatic. In this study, BD reversibility test was positive at least once in 121 patients, bronchial challenge test was positive at least once time in 15 patients, and significant increase in lung function after 4 weeks of anti-inflammatory treatment was found in 12 patients. At enrollment, all but five patients were found to have been diagnosed as asthma patients for at least 3 months; with the five patients being newly diagnosed as asthmatic, based on typical symptoms manifestation and results of lung function tests. Moreover, the diagnosis was further confirmed during follow up. Asthma control was evaluated based on the following 4 items: (1) Daytime asthma symptoms more than twice/week? (2) Any night waking due to asthma? (3) Reliever needed for symptoms more than twice/week? (excluding reliever taken before exercise) (4) Any activity limitation due to asthma? Uncontrolled asthma was defined if at least 3 of these 4 items were prevailing during the week before enrollment, regardless of the medication status [11, 12]. Chronic rhinosinusitis (CRS) was diagnosed based on the European Position Paper on Rhinosinusitis and Nasal Polyps (EP3OS) criteria by clinical symptoms and features of sinus computed tomography (CT) [13]: (1) For the clinical diagnosis of sinusitis, major and minor criteria were adopted. Major symptoms included nasal obstruction/blockage and nasal or post-nasal discharge/purulence. Minor symptoms were facial pain/pressure/fullness, headache, and hyposmia/anosmia. Patients were classified as having CRS if they reported usual presence of at least two of these symptoms for 12 consecutive weeks, and at least one of the two major symptoms was present. (2) CT features included mucosal changes of sinuses and/or ostiomeatal complex. The presence/absence of nasal polyps (NP) was ascertained by nasal endoscopy performed by two experienced otolaryngologists specifically assigned to do this, and any differences in the diagnoses by the otolaryngologists were resolved by discussion and their final consensus. All patients were aged ≥ 18 years, and patients were excluded from the study if they met any of the following criteria: (i) smoking index > 10 pack·year, (ii) received systemic steroid therapy in the proceeding 4 weeks, (iii) received any other immunosuppressive therapy or biologics treatment, (iv) diagnosed with other lung disease such as chronic obstructive pulmonary disease, pneumonia, lung cancer, allergic bronchopulmonary aspergillosis, active pulmonary tuberculosis and interstitial lung disease; (v) diagnosed with immunodeficiency or autoimmune disease, (vi) diagnosed with gastroesophageal reflux disease or parasite infection, and vii) with severe heart failure or significant dysfunction of other organs. Similarly, patients with fungal sinusitis, inverted papilloma, or other nasal diseases that could affect results of the study were also excluded. Additionally, patients with a post-bronchodilator FEV1/FVC less than 0.7 were required to have no history of smoking, no exposure to burning of wood and other biomass fuels, no occupational exposure to dusts and chemical agents, and no definite lung hyperinflation on CT images, in order to exclude COPD comorbidity as much as possible.

On enrollment, demographic and clinical data were collected for each patient using standard questionnaires, medical histories, and other records; including general demographics, age at first symptoms of asthma, nasal disease and other underlying disease, and medication use for asthma. Blood and sputum were collected for further analyses, and the patients were examined for various clinical parameters as detailed below. 22 parameters associated with demographic characteristics (gender, age, body mass index [BMI], asthma onset age), comorbid CRS (comorbidity of CRS/NP, Lund-Mackay score [LMS]), severity of airflow obstruction and airway inflammation (pulmonary function parameters, blood gas analysis, thickness of bronchial wall, comorbidity of bronchiectasis), and inflammation type of asthma (atopy, immunoglobulin E [IgE] level, blood/sputum eosinophil [EOS] level, fractional exhaled nitric oxide [FeNO]), were collected and clustered using two-step cluster analysis. Sputum samples from a number of patients in each cluster were subsequently analyzed for several cytokines to investigate the possible mechanism/s of airway inflammation. The study flow chart is shown as Fig. 1.

Fig. 1
figure 1

Study flowchart. GINA global initiative for asthma, CRS chronic rhinosinusitis

The study was approved by the local ethics committee of Beijing Tongren Hospital, Capital Medical University, and written informed consent was obtained from all patients prior to entry into the study.

Detection of serum total (T)-IgE and allergen specific (s)-IgE

An automatic immunoassay system (ImmunoCap TM100, Pharmacia Company, Sweden) was used to assess serum T-IgE and s-IgE, according to the manufacturer′s directions. The lower limit of detection of serum T-IgE and allergen s-IgE were 2 kU/L and 0.01 kUA/L, respectively. Serum T-IgE > 60 kU/L and allergen specific IgE > 0.35 kUA/L were considered abnormally elevated [14, 15].

Determination of atopic status

Atopy was assessed by serum levels of specific IgE (s-IgE) generated to a panel of common aeroallergens (dust mites, pollen, cat, dog, cockroach, molds) and food allergens (albumen, milk, peanut, meat, seafood). Atopy was defined when any of the serum s-IgE tested was > 0.35 kUA/L [14].

Fractional exhaled nitric oxide test

Fractional exhaled nitric oxide (FeNO) was measured using a nitric oxide analyzer (Niox; Aerocrine, Solna, Sweden) at a flow rate of 50 mL/s via the oral cavity. The exhaled gas was continuously routed into the analyzer via a side port. The procedure was repeated three times and a mean concentration of FeNO was calculated [15].

Pulmonary function test

Spirometry (JAEGER, MasterScreen-body + diffusion + APS, Germany) was performed to determine the lung function measurements and bronchodilator reversibility. Pre- and Post-bronchodilator forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC)%, and FEV1%predicted (FEV1%pred) were measured before bronchodilator (BD) treatment and 15 min after inhalation of 400 μg salbutamol, respectively.

Blood gas analysis

Samples of arterial blood (2 ml) were withdrawn during normal air breathing and assessed for partial pressure of oxygen (PaO2) and partial pressure of carbon dioxide (PaCO2) using a blood gas analyzer [GEM Premier 3000, United States]. Blood gas analysis was completed within 5 min after collection of the blood sample.

Peripheral eosinophil count

Blood samples were drawn in vacutainer tubes with EDTA as anticoagulant (Ref. 368274, Becton Dickinson, USA). Leukocyte count and eosinophil (EOS) differentiation in anticoagulated blood samples were performed on an automatic blood analyzer (sysmex XN10B3, Japan).

Chest CT, determination of bronchiectasis and bronchial wall evaluation

High resolution computed tomography (HRCT) of the chest was performed using a 64-row, multiple-detector CT scanner (Philips Company, Netherlands). An experienced radiologist blinded to the clinical and laboratory data reviewed the CT scans. A diagnosis of bronchiectasis on chest HRCT was made if bronchial wall thickening was present and the ratio of the diameter of bronchus to that of the accompanying pulmonary artery was greater than 1.1 (signet ring sign), or there was lack of tapering of bronchi (tramline sign). Mild bronchiectasis only visible in a single pulmonary segment was not taken into consideration, as this may exist in a significant percentage of healthy population [16]. Bronchial wall thickness was evaluated based on chest HRCT according to the method described by Awadh and colleagues [17]. Both the ratio of airway wall thickness to total diameter (the T/D ratio) and the percentage wall area (WA%) were used to evaluate airway wall thickening.

Sinus CT and evaluation of CRS

Sinus CT was performed using a 64-row, multiple-detector CT scanner (Philips Company, Netherlands). The radiographic severity of CRS was assessed, by an experienced radiologist blinded to the clinical and laboratory data, using the Lund-Mackay CT staging system (0–24) [18]. The Lund-Mackay score (LMS) of “0” was considered as normal or negative, whereas a score ≥ 1 was considered as abnormal or positive.

Sputum induction, eosinophil count and cytokine measurement

Sputum induction was performed in all the patients and the samples were prepared for routine clinical examination, according to the method described before [19]. Briefly, the sputum samples were kept on ice and processed within 1 h. Sputum was weighed and mixed with 0.1% dithiothreitol in a ratio of 4:1. The sample was then filtered through 48-μm gauze to increase homogenization and centrifuged at 1,500 × g for 15 min at 4 °C. The supernatants from the sputum samples of the final 23 patients enrolled in this study (5 from cluster 1, 9 from cluster 2, and 9 from cluster 3) were harvested and stored at -80 °C until analysis for cytokines; including IL-4, IL-5, IL-13, IL-25, IL-33, IFN-γ, TNF-α, IL-10 and IL-17; using the Luminex assay (Luminex, Austin, Tex). The cell pellets were used to prepare cytospin slides according to Brightling and colleagues [20], and then stained with Wright Giemsa stain for assessment of eosinophil counts. The stained slides were examined by an experienced observer, who was blinded to the patient’s clinical data, to obtain a differential cell count by counting 400 non-squamous cells.

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