Clinical, functional and inflammatory evaluation in asthmatic patients after a simple short-term educational program: a randomized trial

This study aimed to evaluate the clinical evolution, functional parameters and inflammatory activity of asthma in patients who submitted to an educational intervention. 58 adult patients over 18 years of age with partly controlled and uncontrolled asthma were randomized into an intervention group (IG) (N = 32) and a control group (CG) (N = 26) and evaluated for 12 weeks. The Asthma Control Test (ACT), Asthma Control Questionnaire (ACQ), Asthma Quality Life Questionnaire (AQLQ) and Beck Depression Inventory (BDI) questionnaires were applied. Spirometry, exhaled nitric oxide (NO), exhaled breath condensate (EBC) and induced sputum (IS), measurement of the peak flow and symptoms were performed. The IG patients received an educational activity for 30 min applied by a nurse. Statistical analysis: analysis of variance with repeated intragroup measures. IG presented a decreased number of eosinophils in IS and IL-17A in EBC, an increase in the percentage of FEV1 before and after bronchodilator and an improvement in quality of life compared to the CG. There was an improvement in depression levels and a decrease in IL-4 and IL-5 in the IS and in the EBC in both groups. Our results suggest that an educational intervention can bring benefits concerning the control of inflammation, lung function alterations, quality of life and levels of depression in asthmatic patients. Registration: ClinicalTrials.gov; NCT03655392.

It is estimated that approximately 358 million individuals worldwide are affected by asthma 1 , and the global prevalence can vary from 1 to 18% 2 . Chronic inflammation in asthma is a consequence of the participation of several mediators that lead to influx of inflammatory cells and airway remodeling as a consequence of goblet cell metaplasia, excessive subepithelial collagen deposition, airway smooth muscle hyperplasia, and increased vascularity. These characteristic features are orchestrated mainly by Type 2 (Th2) cells and cytokines [3][4][5] .
Thus, the main goal of asthma treatment is to reduce inflammation in the airways and consequently control the disease and its symptoms. Asthma treatment includes inhaled corticosteroids (ICS) and long-acting bronchodilators (LABA), and, more recently, for severe asthma, biologicals that target Th2 mediators. Factors contributing to an uncontrolled asthma are poor adherence and inappropriate inhaler technique. Studies have found that most patients do not use their inhalation device correctly 2,6-8 . The correct use of these drugs is associated with better efficacy and fewer collateral effects related to therapy 9 .
Inhaled corticosteroids are the basis of treatment for most cases of asthma. Their constant use is associated with an improvement of symptoms and a reduction of morbidity and mortality related to disease 2,10,11 .
Several studies showed that poor adherence to asthma medications and inadequate inhaler technique were associated to difficult-to-control asthma. The asthma education program is considered one of the fundamental pillars for adherence and proper treatment of the disease. It is estimated that more than half of patients treated for asthma do not have adequate adherence to the prescribed medication or do not perform the treatment properly 2,12,13 .
A review of interventions for inhaler techniques concluded that most of the studies showed that an intervention improved inhaler technique when assessed by a checklist or dichotomously; and that it was true for both children and adults with asthma 14 . Melani et al. observed that critical errors in the inhalation technique were observed in 36% of the patients with chronic airflow obstruction 15 .
We believe that an educational intervention aims at better comprehension on asthma care, including environmental control, adherence to medication use, and correct use of the inhalation device. These interventions can improve clinical, functional and inflammatory parameters of asthma. Thus, the objectives of this study were to assess asthma control, quality of life, levels of depression, lung function and inflammatory parameters of patients with asthma before and after an educational intervention.
Individuals with a clinical asthma diagnosis were recruited from an outpatient allergy and immunology clinic at a university hospital. Written informed consent was obtained from all participants included in the study. All participants received explanations, signed and received a copy of the written consent form. Participation was voluntary and participants were free to withdraw from the study at any stage. Participants were recruited between July 2015 and December 2017.
Eligibility criteria. Adults between 18 and 69 years of age with clinical diagnosis of asthma 2 ; asthma with partly controlled or uncontrolled symptoms 2 ; continuous medical follow-up and patients using ICS with or without LABA (long-acting beta-agonist) for at least 1 year; increase in FEV 1 of > 12% and > 200 ml from baseline 10-15 min after 400 mcg Salbutamol; dose of ICS stable in the last 8 weeks before recruitment; and nonsmokers or ex-smokers less than or equal to 10 pack-years. Subject characteristics are summarized in Table 1.
Exclusion criteria: clinical asthma diagnosis and upper respiratory tract infection less than 30 days before; systemic steroids within 4 weeks of enrollment; pregnancy; other lung or uncontrolled chronic disease and chronic obstructive pulmonary disease (COPD) according to the Global Initiative for COPD (GOLD) 16 .
Outcome measures. Individuals were recruited after regular medical visits in outpatient clinics, by the researchers and physicians, after routine outpatient medical care. The study protocol was explained to the possible participant, and signed informed consent was requested within 28 days (T0). Subjects were followed for 56 days at three visits every 4 weeks (day 0 = T1 (baseline), day 28 = T2 and day 56 = T3) ( Fig. 1).
At the first visit (T1), the patients were randomized in the order of inclusion in a 1:1 ratio for the IG (intervention group) or CG (control group) groups. The symptom diary and a manual and portable peak flow meter (Mini-Wright, Clement Clark International) were delivered to all patients. Then, they answered the following questionnaires administered by a blind investigator: Asthma Control Test (ACT) 17 , Asthma Control Questionnaire 18 , Asthma Quality Life Questionnaire (AQLQ) 19 and Beck Depression Inventory II (BDI II) 20 . After, individuals underwent spirometry for the collection of exhaled breath condensate (EBC) and induced sputum (IS). All these data were collected from both groups at the three study visits (T1, T2 and T3).
Additionally, IGs were submitted to educational intervention at all three visits (T1, T2 and T3) and received phone calls every 2 weeks, reinforcing the educational content and the date of return. CGs were only advised as to the date of return and received phone calls 2 days before to confirm attendance.
Educational intervention was applied by a trained nurse in a 30-min session. Its main objective was to verify whether the patient was using the ICS properly and, if necessary, to teach the correct inhalation technique. It included the asthma education components recommended in the Global Initiative for Asthma (GINA) main report 2 and a simplified explanation of this disease and its main characteristics, guidance on inhalation technique using illustrations, a script and an explanatory leaflet.
Skills for correct medication inhalation from a metered-dose inhaler and for peak flow measurement were taught and practiced. The inhalation technique was explained and demonstrated to the patients. As a complementary material, a four-minute explanatory video addressing the correct inhalation technique was shown (available at http:// www. incor. usp. br/ sites/ incor 2013/ videos/ asma-dpoc/). Environmental control was also briefly addressed, and IG subjects were guided on measures to avoid exposure to allergens and irritants.
Finally, patients were invited to demonstrate the inhalation technique. When necessary, the technical errors were corrected by the nurse until the inhalation technique was performed correctly. This activity was performed considering the particularities of each patient.
The symptoms of asthma and peak flow measurement were recorded in a diary of symptoms already used by patients in hospital outpatient clinics 21 . Patients registered these data twice a day in the morning and evening before using ICS. Symptom-free days were accounted for by each asthma symptom individually (cough, wheezing, shortness of breath, waking up at night for asthma and use of rescue medication). At each new visit (T2 and T3), patients returned the symptom diary and the annotation of the peak flow values. Inflammatory parameters. Sputum was induced by standard methodology 26,27 . Patients were asked to inhale 400 μg Salbutamol via a metered-dose inhaler 20 min before induction and then inhaled hypertonic saline Table 1. Baseline data. Data are expressed as the numbers and percentages-N (%) or means ± SD (standard deviation). Statistical analysis: Chi-square (qualitative variables) and T-test (numerical variables). CG control group, IG intervention group. T1 = day 0 (baseline). T2 = day 28. T3 = day 56. *p < 0.05 compared to T1. **Values are expressed as the percentages of patients with sputum samples sufficient for analysis. Eosinophilic: eosinophils > 2.5%, neutrophils ≤ 54%; neutrophilic: eosinophils ≤ 2.5%, neutrophils > 54%; mixed: eosinophils > 2.5%, neutrophils > 54%; paucigranulocytic: eosinophils ≤ 2.5%, neutrophils ≤ 54%. IG = intervention group. CG = control group. www.nature.com/scientificreports/ (3% NaCl) for 7 min; peak flow and symptoms were evaluated before and after each inhalation. A Devilbiss Ultraneb 99 (Devilbiss Corp., Somerset, PA, USA) ultrasonic nebulizer was used. The measurement of FeNO (fractional exhaled nitric oxide) was performed by chemiluminescence (Sievers 280) according to the recommendations of the ATS 28 . The patients were instructed to blow into a Mylar balloon with an expiratory pressure supported in 12 cm H2O and a stable flow of 200 ml/s. This procedure and analysis were performed by a blinded investigator.
EBC (exhaled breath condensate) was collected for 15-20 min in the current volume to measure cytokines 29 . A condenser (Turbo DECCS System, Medivac SRL, Italy) cooled to − 20 °C for at least thirty minutes was used. The individuals were instructed to perform oral breathing using the mouthpiece of the equipment and with the aid of a nasal clip.
The levels of interleukin (IL)-4, IL-5 and IL-17A were quantified in IS supernatant and EBCs. ELISA (enzymelinked immunosorbent assay) (EL Human ISA Max Deluxe, Biolegend, San Diego, CA) 30 was utilized. The tests were performed according to the manufacturer's guidelines; the samples were analyzed in an ELISA reader (Polaris Model, Celer Biotechnology SA, Brazil).

Statistical analysis.
Descriptive analysis was performed on all patient data (means, standard deviations and medians). Differences between groups were analyzed using Student's t test, chi-square and one-way repeated measures analysis of variance (ANOVA). Intragroup analysis was compared by means of repeated measurement ANOVA. The confidence interval was 95% (p < 0.05). A statistical package was used for tests (Sigma Stat 3.11, San José, CA, USA)-descriptive analysis, differences between groups and intragroup, sample calculation) and SPSS 20.0 (odds ratio (OR) and chi-square). To calculate the odds ratio, the rates of change were compared between the groups (IG/CG) at different times of assessment: T1-T2, T2-T3, T1-T3 (95% confidence interval).
Sample size estimates were based on the estimated effect of the intervention on eosinophils in induced sputum according to a previous study 31 . The sample size calculation took into account a minimum mean difference of 67% for eosinophils, with an expected difference in means of 8 and a standard deviation of 10, test power of 80% and alpha of 0.05, thus resulting in a requirement of 13 patients in each group. Consent to participate. Informed consent was obtained from all individual participants included in the study. All participants received explanations, signed and received a copy of the written consent form.

Ethics approval. This study was approved by the HCFMUSP Research Ethics
Consent for publication. The authors affirm that human research participants provided informed consent for publication of the study data without personal identification. In the written consent form, all participants authorized the publication of the data for this study, without any individual identification related to the participants. The confidentiality and privacy of any personal data were respected in this study.  for not meeting the criteria, 115 declined to participate and 40 for other reasons. 140 subjects were distributed equally and randomly into the groups: IG (n = 70) and CG (n = 70). During the protocol, 76 patients lost followup, 29 from IG and 44 from CG. The claimed reasons for that were: lack of time to participate, other health treatments not related to this study, difficulties to get into hospital, change of address or employment, difficulty in missing work for treatment, among other reasons. Three patients were removed from IG because they presented a slight respiratory discomfort after inhalation with hypertonic saline solution. One patient was removed from CG for emotional disorder. After completing the protocol, we excluded 6 patients from IG for insufficient samples. At the end, 58 patients completed the entire study (IG n = 32) (CG n = 26) (Fig. 2).

Clinical parameters.
In evaluation of quality of life (AQLQ), we found an improvement in IG patients when comparing T1 with T2 or T3 moments (Fig. 3d, Table 2) (*p = 0.005). There were no differences between T1, T2 or T3 in CG. Regarding depression levels, IG patients presented improvement at T2 and T3 compared to T1 (*p = 0.002). In the CG, we found improvements only in T3 compared to T1 (*p = 0.006) (Fig. 3e, Table 2).
In relation to asthma control when the ACT questionnaire was applied, although IG patients presented an increase of 2.5 points, no difference was observed. Also, in the ACQ analysis, no significant differences were found in symptom-free days or daily measurement of peak flow (Table 2).
In addition, we evaluated the number of days when patients were free of the following factors: cough, wheezing, dyspnea, awakening because of asthma symptoms, rescue medication (SABA) and peak flow. In CG, patients reported more days free of cough at T3 when compared to T2 (*p = 0.028) ( Table 2). There were no differences in other parameters. www.nature.com/scientificreports/ Functional evaluation. IG presented an increase in FEV1 before (p = 0.009) and after bronchodilator (p = 0.004) in T2 and T3 when compared to T1 CG (Fig. 3b, c, Table 2). There was no difference between FEV 1 / FVC before and after bronchodilator in IG or CG (Table 2).  www.nature.com/scientificreports/ Inflammatory parameters. Both groups presented similar patient characteristics at baseline (T1). According to cellularity in endotype (induced sputum), most patients had paucigranulocytic asthma (69%) compared to neutrophilic, eosinophilic and mixed endotypes (p = 0.027*) ( Table 1). In comparison of differential cell count in sputum, a significant decrease in the eosinophil number was observed at T3 (2 months) compared to T1 in the IG (*p = 0.034) (Fig. 3a, Table 3). We did not find difference in number of neutrophils, lymphocytes and macrophages in both groups at three moments as well as in NO measurement (Table 3).
In CG, we observed decreased expression of IL-4 values at T3 compared to T2 and T1 in IS (*p = 0.001) (Fig. 4a) and at EBC analysis we found a decrease at T3 compared to T1 (*p = 0.011) (Fig. 4b). Regarding to IL-5 measurement, the CG presented decreased values at T3 compared do T2 and T1 in the IS (*p < 0.001) and also in the EBC (*p < 0.001) (Fig. 4c) (Table 3).
Regarding the EBC analysis of interleukin IL-17A, there was a decrease in the IG group in the T3 evaluation when compared with T2 (Fig. 4e, Table 3) (*p = 0.028). In IS supernatant, it was not possible to analyze the IL-17 data in either group since the values were lower than the detection curve. The data are expressed as the medians and percentiles 25-75. SABA short action beta-agonist, AQLQ Asthma Quality Life Questionnaire, BDI Beck Depression Inventory, ACT asthma control test, ACQ Asthma Control Questionnaire, FEV 1 Forced Expiratory Volume in One Second, FVC forced vital capacity, β2 beta2-adrenergic (bronchodilator). *p < 0.05 compared to T1. **Symptom Diary was delivered to patients on day 0 and day 28 and returned on day 56. One-way ANOVA.

Discussion
In the present study, we found that asthmatic patients with the disease partly controlled and uncontrolled who underwent an educational intervention showed improved clinical control of asthma, pulmonary function, markers of airway inflammation, and quality of life and decreased levels of depression.
We showed the importance of an educational activity for a short period of time. This has led to a better lung function, decreased inflammation and levels of depression, and a better quality of life. This improvement occurred even without changing the previous pharmacological treatment of the patients, thus showing the importance of the educational approach in the management of asthma.
An effective guided asthma self-management education may help patients and reduce morbidity 2,32,33 , asthmarelated hospitalizations, emergency department visits and unscheduled doctor or clinic visits, missed work/school days, and night awakening 2,34,35 . The use of inhalation devices promotes a high concentration of medication in the airways, more rapid onset of action, and fewer systemic adverse effects than systemic delivery. For this to happen, the correct use of the inhalation device must be learned and maintained. In this context, skills training to use inhaler devices effectively are essential in an individualized self-care plan for asthma patients 2 .
Some previous studies have shown that educational activities and self-management related to asthma features have been associated with better control of the disease in both adults and children 2,9,31,36 . Boulet et al. showed significant improvements after an educational program, such as a reduction in unscheduled visits, inappropriate use of medications and an increase in FEV 1 in mild to moderate asthmatic patients after 1 year of follow-up 37 . In another study, asthmatic and COPD patients showed better clinical control and lung function after submitting to a 6-8 follow-up educational program 38 . In our study, we also found an increased FEV 1 after one (before and Table 3. Inflammatory profile. The data are expressed as the medians and percentiles 25-75 (SD). CG control group, IG intervention group, T1 day 0 (baseline), T2 day 28, T3 day 56, NOex Nitric Oxide exhaled, ppb parts per billion, EBC exhaled breath condensate, IG intervention group, CG control group. *p < 0.05. One-way ANOVA. *compared to T1. **compared to T2. ***compared to T2 and T1. www.nature.com/scientificreports/ after bronchodilator) and 2 months (after bronchodilator) of educational intervention follow-up, that is, the improvement of this parameter after the educational activity. Lv et al. compared an SMS with a traditional educational program and an outpatient ordinary service (control group) 39 . They found that both groups that received an educational program presented increased scores on perceived control asthma questionnaires, although scores in the SMS group were higher than those in the traditional educational program 39 . Continuing medical program education in asthma care was shown to improve parent-reported provider communication skills, the number of days affected by asthma symptoms, and asthma health care use 40 .  44 . In our study, asthmatic patients from IG presented better quality of life after 1 and 2 months (T2 and T3) of protocol intervention. Janson et al. 36 showed that individualized asthma self-management education resulted in a decrease in nighttime awakenings, improved perceived control of asthma, better adherence to treatment, and improved clinical parameters of the disease and quality of life.
However, we did not observe differences when comparing ACT and ACQ scores in either group. In the same way, Maricoto et al. 38 also did not find differences between groups after an educational intervention regarding proper use of ICS in ACT scores. França-Pinto et al. did not show a difference in ACQ after an aerobic training program of asthmatic patients, although they presented a better quality of life and better scores of depression 27 . However, when the effect of the educational intervention was compared between the groups (IG and CG) through the odds ratio and chi-square test among the 3 study visits (T1, T2, T3) we observed a significant difference in the percentage of ACQ between T1 and T3. www.nature.com/scientificreports/ One strength of our study is that even the CG presented more cough-free days and better IL-4, IL-5, and depression levels after the protocol. We hypothesized that these positive results could be motivated by the use of peak flow to fill a diary of symptoms and by some calls received from a health professional; thus, it was enough to help them feel self-confident to better manage the disease.
Depression is an important asthma comorbidity and has been associated with worsening disease control in adults 45,46 . Plourde et al. 47 validated the BDI, one of the most widely used questionnaires to screen depression in health research areas 24,[47][48][49][50] , to be used in asthmatic adult patients.
We found better depression scores in the IG after 1 month (T2) of educational intervention and after 2 months (T3) in the CG. When invited to participate in a survey, the participant's attention is focused on the subject in question, that is, to have more health care. It is possible that the fact of having applied questionnaires about your illness, quality of life, depression, may have influenced further reflection and self-care, generating some positive results even in patients in the control group.
Stoop et al. carried out a study with people with diabetes, asthma or COPD and the subjects answered questionnaires after 3, 6, 9, 12 and 18 months. The intervention group received a 12-month stepped care treatment and monitoring of symptoms. The control group received usual care plus monitoring by filling in a postal questionnaire every 3 months. Eighteen months post screening, the difference in symptoms of depression between intervention and control group was not significant anymore 51 .
Regarding inflammatory parameters, the exact role of FeNO in asthma is still not clear 2,52 . Some studies have associated FeNO with eosinophilia in asthmatic patients 2,53,54 . However, according to GINA 2 , this measure has not been established as useful for ruling in or ruling out a diagnosis of asthma; among other factors, it is not elevated in some asthma phenotypes, such as neutrophilic asthma 3,55 . The majority of our patients had paucigranulocytic asthma, which may explain the decreased values of FeNO after educational intervention ( Table 2).
Eosinophils have an important role in the immunological response in asthma. Studies have demonstrated a relationship between eosinophils and airway hyperresponsivity and remodeling as well as its relationship with asthma severity 30,56 . A retrospective study of asthmatic adult patients followed up for 2 years showed a relationship between better disease control and a decrease in the number of eosinophils. They suggested that the control of asthma could be a consequence of intensifying treatment with ICS 57 . In our study, patients who were submitted to the educational program presented a decrease in the number of eosinophils in the sputum, which may be due to better adherence to ICS.
Another mediator related to eosinophils and the inflammatory process of asthma is Th2 cytokines, such as IL-4 and IL-5 1,30,38,58 . The ERS/ATS Task Force (2020) made recommendations on the use of novel therapies for severe asthma, specifically biologics for type 2 high asthma, such as the anti-IL-5 mepolizumab and reslizumab, the IL-5 receptor antagonist benralizumab and dupilumab, the IL-4 and IL-13 α-chain receptor antagonist. Those biologics showed efficacy for severe uncontrolled eosinophilic asthma phenotypes, particularly for those with severe corticosteroid-dependent asthma 59 . Thus, decreasing the levels of these cytokines with only a short-term educational intervention can bring benefits to patients.
In the IG group, it has already been possible to observe with one month of educational intervention an improvement of inflammation, seen through the reduction of IL-4 and IL-5, both as evaluated by IS and EBC; this reduction was also observed after 2 months of intervention. In the CG, although there has been a decrease of these parameters, this reduction only occurred after 2 months. CG presented a decrease in these cytokines but they did not present improvement in other parameters that could represent disease control, such as lung function, the number of eosinophils and quality of life. Although these cytokines are involved in asthma pathogenesis, other mediators are also important to achieve the control of asthma symptoms.
Although allergic asthmatic features are linked to Th2 cytokines, recent studies have associated Th2 with Th17 cytokines. Bullens et al. found an increase in the expression of IL-17 mRNA in the sputum of asthmatic patients compared to healthy controls 60 . Barczyk et al. also presented a correlation of increased levels of IL-17 in asthmatic patients with bronchial hyperreactivity 61 . Camargo et al. showed that therapy with anti-IL-17 could be used to control the inflammatory process in an exacerbated asthma model 62 . In addition, Fattahi et al. found that lower levels of IL-17 + cells in asthmatic patients were associated with atopy and ICS use 63 . We observed decreased expression of IL-17A at EBC only in IG, suggesting a possible pathway to explain reduced inflammation in these patients.
We believe that one of the limiting factors of this study may have been the use of dithiothreitol (DTT) for processing sputum supernatant. It is indicated for better preparation of slides 64 ; however, its use has already been described as potentially harmful for differential cell analysis 65,66 . This procedure may have influenced the analysis of cytokines, a fact that was observed only after the freezing of samples. Another factor to be considered is the sample size, which could be expanded in future studies. It should also be considered that the CG may have influenced some of the results despite not having received the educational intervention in the first two visits. The fact that CG participated in the research may have influenced the results in a beneficial way and may have inferred some of the results. Despite these limitations, our study has several strengths. We showed that an educational intervention performed for a short period of time, applied by a trained professional, can produce benefits to patients in relation to inflammation, lung function, quality of life and depression levels.
In summary, we showed that an educational program directed to asthmatic patients in addition to physician care has an important role in disease control. Identifying patients' needs, beliefs and behaviors can indicate where improvements should be focused to help people and plan future interventions. In this study, this benefit was evidenced by a reduction in airway inflammatory markers, such as IL-4, IL-5, IL-17A, FeNO and lung function, which consequently resulted in better quality of life and decreased depression levels. Finally, we suggest that health educational programs should be part of asthma management.

Data availability
All relevant data are within the paper and; the main data can also be accessed at: https:// clini caltr ials. gov/ ct2/ show/ resul ts/ NCT03 655392. Clinical Trial Registration: ClinicalTrials.gov; number: NCT03655392; URL: www. clini caltr ials. gov. It was first registered in 03/28/2018 (retrospectively recorded). Any additional data can be requested from the corresponding author or the last author.