Fraction exhaled nitric oxide and IOS as potential biomarkers for HDM immunotherapy in patients with allergic rhinitis and asthma

Background Allergen specic immunotherapy (AIT) is the only potential disease-modifying treatment of house dust mite (HDM) allergic asthma and rhinitis. The conventional treatment procedure lasted for 3 years, but biomarkers indicating early ecacy from this long-term treatment has not been well illustrated. In this single arm, observation study, we evaluated non-invasive airway inammation parameters such as Exhaled nitric oxide (FeNO), nasal NO levels (FnNO), and small airway function parameter-impulse oscillometry system (IOS) as well as serum parameters Dermatophagoides pteronyssinus (Derp) specic IgG4 and sIgG4/IgE ratio as potential biomarkers for AIT. ratio maybe good therapeutic predictors to reect the ecacy of AIT in the rst year of treatment.


Introduction
House dust mite (HDM) allergy is a major cause of respiratory allergic diseases and often implicated as a trigger for asthma and perennial allergic rhinitis, especially in East and South Asia [1]. Allergen speci c immunotherapy (AIT) provides an alternative option for treating HDM allergy that target to the underlying cause and modify the development of the disease. A treatment course of 3 years is commonly considered to achieve long-term e cacy [2][3][4]. The World Allergy Organization (WAO) recommends that the primary outcomes used in AIT include measurement of symptoms and the use of concomitant medications and/or a combination of the both of the measures. Currently there are no validated or generally accepted candidate biomarkers predictive of the clinical response to AIT. Most of the candidate biomarkers used in clinical trials of allergic rhinitis patients with/without asthma in the literature could not be used with routine clinical testing procedures [5]. Conventional AIT treatment lasts for 3-5 years. Feasible and practical biomarkers re ecting the e cacy of the treatment at a relative early phase, e.g. one year, are important for both physician and patients.
Recently some simple, rapid, non-invasive parameters have been used in evaluating the severity of airway allergic in ammation, such as exhaled nitric oxide (FeNO) and nasal NO levels (FnNO) [6][7][8][9]. As to mild allergic asthma patients, the classical lung function parameters, e.g.FEV1, FEV1/FVC, are normal and di cult to change after treatment because of celling effect. However, small airway function evaluated by impulse oscillometry system (IOS) may showed the treatment effects. The mechanisms for AIT are still not fully elucidated, especially for the lung parameters. In this study, we investigated two non-invasive airway in ammatory parameters, i.e. FeNO and FnNO, as well as IOS as the biomarkers for evaluating the e cacy with allergic asthma patients during one year of AIT.

Subjects:
All subjects were non-smoking adults with mild allergic asthma concomitant with allergic rhinitis. Patient were considered eligible for HDM AIT according to the following factors: diagnosed with asthma under control with standard pharmacotherapy (without exacerbation or systemic corticosteroid use within the last 6 months), HDM allergy and perennial asthma (medical history consistent with HDM allergy).
The study was performed with the approval by the Ruijin hospital Research Ethics Board (#2017-117). All subjects gave written informed consent.

AIT protocol
The patients were treated with subcutaneous injection of aluminium-formulated Dermatophagoides pteronyssinus Alutard® SQ (; ALK, Hørsholm, Denmark). The treatment protocol followed the recommended up-dosing schedule of 5 months with 1 injection/week and 2.5 years treatment with a maintenance dose of 100,000 SQ with injection interval of 4±2weeks.
Clinical evaluations and serum allergen speci c IgE and IgG4 measurement Allergy symptoms were recorded using the visual analog scale (VAS) of 100 mm range and the patients marked on a line the point they felt represented their perceptions of their current state. Medication score was scored on a scale of 0-5 (0, not at all; 1, occasionally seldom; 2, occasionally often; 3, almost daily; 4, continuously; 5, continuously with the maximal dose) that included oral or nasal anti-histamine, leukotriene antagonist(LTRA),nasal corticosteroid spray as well as ICS or ICS/LABA treatment for asthma.
Serum samples were collected before and after one year AIT. Der p-speci c IgE was determined by the Unicap 100 uoresence enzyme immunoassay system (Phadia, Uppsala, Sweden) according to the manufacturer's instructions. Serum Der p-speci c IgE and IgG4 levels were reported as kAU/L with a cut off value of 0.35 kAU/L and upper limit of 100 kAU/L.

Measurement of FeNO, FnNO, IOS and pulmonary function
FeNO levels were measured according to the guidelines of American Thoracic Society (ATS) by the singlebreath method (on-line measurement) using a fast response (0.02 s) chemiluminescence analyzer (NOA 280; Sievers Instruments Inc., Boulder, CO) [10]. Before FnNO measurements, the patient should blow their nose and assure free air ow in both nostrils. Aspiration is done through one nostril with the use of a tightly tting nasal olive with the other nostril open. The subject performs a deep inhalation and holds their breath to obtain velum closure, while air circulates from one naris to the other around the posterior nasal septum. Velum closure may also be obtained by oral expiration against a resistance of 10 cmH2O, by pursed-lip breathing via the mouth, or by voluntary elevation of the soft palate. Velum closure can be monitored by measuring nasal CO2. Measurement is done after a deep inhalation with NO-free gas, followed by slow exhalation through the nose (with the mouth closed) into a tightly tted mask covering the nose. [9,7] IOS measurements were obtained using a commercially available IO device (Master Screen IOS, Jaeger, Germany) according to the manufacturer's recommendations. [11] After the IOS measurements, the MasterScreen IOS-Jaeger(Germany) device was used to perform spirometry. To avoid any negative effects of forced expiration on the airway, spirometry was never performed before the IOS measurements, The percent predicted forced vital capacity(%FVC), the percent predicted forced expiratory volume in 1 s(%FEV1), the FEV1/FVC ratio, the percent predicted the maximal mid-expiratory ow(%MMEF), and the percent predicted peak expiratory ow(%PEF) were also obtained.

Statistical analysis
Statistical analysis was conducted via SPSS version 20 and R version 3.6.0. Numeric variables were expressed as means±SEM. The differences between different time point were tested by repeated measurement analysis of variance. Differences between different groups were tested via one-way analysis of variance. P <0.05 was considered statistically signi cant.

Results
Totally 22 subjects (female 59.1%) with mean age of 34.41 ± 2.13y were included in this study. The subjects suffered from well controlled or partially controlled mild allergic asthma with rhinitis (ACT score, 23.32 ± 0.22; FEV1% of predicted value, 82.10 ± 0.45%) But their airway eosinophilic in ammation were signi cant high according to FeNO evaluation (FeNO, 56.45 ± 4.47 ppb). Almost every subject has been treated with medication regularly (medication score, 3.23 ± 0.11; only one subject less than 3). Detailed baseline symptom, lung function and airway or serum in ammation indicators were listed in Table 1.

Discussion
The clinical data of the current study show that HDM AIT in patents with allergic asthma concomitant with rhinitis results in a signi cant improvement in symptom and reduction in medication. We also found that non-invasive airway in ammation parameters FeNO and FnNO (which re ect total airway and upper airway allergic in ammation) and small airway function parameter (which represented by IOS R5) decreased after both half and one-year AIT in these mild and almost well controlled asthma patients. We saw a substantial increase of IgG4, while IgE remained unchanged, but ratio of IgE/IgG4 decreased along with the rst year of immunotherapy process. The weakness of our study is lack of control group. As AIT has been a routine treatment for mild allergic asthma for more than a decade in our clinic, it is di cult to nd enough patients willing to receive placebo or delayed AIT for controlled observation. We've only found 6 patients as the control group(data not shown), but the number of cases was too small for statistical analysis.
The standard AIT e cacy is the evaluation of clinical symptoms and rescue medications during natural allergen exposure, as de ned by the EAACI task force following regulatory guidelines [5,12]. For traditional clinical e cacy evaluation, the visual analog scale (VAS) is generally recommended for subjective symptom evaluation by allergic patients. The VAS can be a good means for assessing allergic patient's feelings about the severity of their disease, especially during long-term follow-up of AR [2,3]. The traditional clinical e cacy of AIT in allergic asthma is measured using medication scores as well as lung function test. Although the medication scores were used as primary endpoints in AIT trials done both worldwide and China [12][13], the score system has not covered mild to moderate asthma routine medication, for example ICS/LABA combination or LTRA. Thus, in this study, we use the medication score system including all the medication for allergic rhinits and asthma in order to evaluate overall treatment response for both AR and asthma. ACT (asthma control test) is widely used in evaluating asthma control status according to GINA guideline and numerous publications. But in patients with different disease severity, the improvement of ACT was more predominant in moderate asthma group when receiving AIT [14,15]. In our study, most patients are well and partially controlled their asthma before treatment, so the subjects had a trend of symptom alleviation re ected by decreased ACT. With one year of HDM speci c immunotherapy, both VAS and medication score were signi cantly improved, consistent with the results of most of the previous studies [13][14][15]. In addition, for subjects with continuous improvement (there was improvement during both the rst and the second half year), most of this effect was gained during the rst half year which means early effect of AIT in these mild asthma concomitant with rhinitis patients .
Measurement of FeNO is a simple and non-invasive assessment of airway in ammation severity in asthmatic patients [16]. It has been used in epidemiological studies of allergic asthma as well as rhinitis [17], and recently it had been used as a supplemental tool for monitoring AIT treatment e cacy in asthmatic children [18]. Within the nasal region, altered nasal NO levels (FnNO) have also been described in a number of conditions, including allergic rhinitis, sinusitis, and nasal polyps [7][8][9]. Few studies have yet investigated the effect of AIT on exhaled nitric oxide concentration [18], although AIT with D. pteronyssinus and D. farinae extracts has been found to induce exhaled NO in asthmatic children with mite allergy [19]. However, the results from the previous studies are controversial and a clear demonstration of a reduction in exhaled NO in asthmatic patients taking subcutaneous immunotherapy(SCIT) is lacking [18,20]. According to our data, exhaled NO levels decreased after SCIT with HDM, no matter from the airway re ected by FeNO or from the nose re ected by FnNO. It indicates that AIT is effective in reducing airway allergic in ammation. Again, most of this effect was gained during the rst half year which means early effect of AIT.
Peripheral airway disease as indicated by increased frequency dependence of resistance and reactance measured by Impulse oscillometry system (IOS) which is a novel device for respiratory functional assessment especially in evaluation of lung mechanics [21]. Peripheral airway impairment detected by IOS or spirometry (i.e., forced expiratory ow between 25% and 75%) commonly occurs, and each measurement may be complementary in predicting loss of control even with normal forced expiratory volume in 1 second [22]. In recent years, IOS has been used in assessing airway resistance after bronchoprovocation [23,24]. In small-airway obstruction situation like mild asthma, the value of both R5 (low frequencies which represents resistance from large and small airways) and R5-R20 (resistance from more peripheral airways) would elevate. In our study, R5 decreased signi cantly after half and one-year AIT. This may indicate R5 is a sensitive biomarker for pulmonary physiologic changes during AIT process. Besides, IOS re ects changes in the caliber of the small airways occur earlier in asthma, before the abnormalities in the larger airways characterized by spirometry [25,26].
According to the indication of AIT, patients receiving HDM immunotherapy in our study were mild asthma patients with almost normal value of FEV1 and FEV1/FVC. It was di cult to improve these parameters after treatment because of lung function ceiling effect. As we know, for children, lung parameters like IOS were superior in identifying asthmatics then the conventional spirometry [27]. But until now as we know, no related study has been done in adult patients with allergic asthma and rhinitis. Our results indicate IOS evaluation may be another lung mechanics biomarker during adult AIT process.
Elevated serum allergen speci c IgE and related allergic symptom on allergen exposure to sensitized allergy like HDM are currently the sole standard for allergy diagnosis and inclusion criteria for starting AIT [28][29][30]. Serum biomarkers are important indicators re ecting AIT e cacy. Levels of speci c IgE (sIgE) transiently increase during treatment and followed by gradually decreasing. Speci c IgG subclasses especially antigen speci c IgG4 has been fully studied during AIT [31]. A correlation between allergen sIgG4 and clinical outcomes has been reported in some studies which had used immunoblotting and Pharmacia CAP system as we did in this work [32,33]. Longitudinal data on serum speci c sIgE and sIgG4 to Der p allergen during AIT are limited in Chinese populations. In the previous study, Chinese doctors found sIgE/sIgG4 ratios for Der p 1 and Der p 2 decreased continuously from 6 through 24 months of AIT in asthma and rhinits children [34]. Allergen speci c IgE/total IgE ratio is one of the biomarkers for monitoring clinical e cacy of AIT for allergic rhinoconjunctivitis and allergic asthma [5].
But it is still controversial about the usefulness of serum t-IgE levels in the clinical diagnosis of allergy to common aeroallergens [35]. As total IgE level in Chinese adult population may in uenced by more factors such as parasite exposure during younger age [36]. Thus, we combined allergen speci c immunological parameter IgE and IgG4 level and use HDM IgE/IgG4 ratio to clinical outcome. It showed that HDM speci c IgG4 increase as well as IgE/IgG4 decreased in the rst year of AIT which may be a better biomarker to evaluate the early treatment e cacy compare to the traditional allergen speci c IgE/total IgE ratio[37].
In conclusion, one-year HDM AIT improved allergic symptoms and reduced medication score in patients with allergic asthma and rhinitis. Non-invasive airway in ammation parameters such as FeNO and FnNO and small airway function parameter IOS were good parameters for evaluating early e cacy of AIT. HDM speci c IgG4 and IgE/IgG4 may be good therapeutic indicator to re ect the e cacy of AIT during the rst year of treatment. Asthma symptoms and medication use were decreased after immunotherapy. A) ACT score, B) VAS score and C) Medication score during treatment period were as shown. * P < 0.05 and ***P < 0.001.

Figure 4
Change of Serum biomarkers B) IgG4 and their ratio C) IgE/IgG4 were signi cant pre-and post-treatment while there was no signi cant difference for IgE. *P < 0.05, **P < 0.01.