RESEARCH ARTICLE Total and specific immunoglobulin E in induced sputum in allergic and non-allergic asthma Astrid Crespo-Lessmann ID*, Elena Curto, Eder Mateus, Lorena Soto, Alba Garcı́a-Moral, Montserrat Torrejón, Alicia Belda, Jordi Giner, David Ramos-Barbón, Vicente Plaza Department of Respiratory Medicine and Allergology, Hospital de la Santa Creu i Sant Pau, Institute of Sant Pau Biomedical Research (IBB Sant Pau), Department of Medicine of Universitat Autònoma de Barcelona (UAB), CIBER of Respiratory Diseases (CIBERES), Barcelona, Spain a1111111111 a1111111111 * acrespo@santpau.cat a1111111111 a1111111111 a1111111111 Abstract Background OPEN ACCESS Most patients with nonallergic asthma have normal serum immunoglobulin E (IgE) levels. Citation: Crespo-Lessmann A, Curto E, Mateus E, Recent reports suggest that total and aeroallergen-specific IgE levels in induced sputum Soto L, Garcı́a-Moral A, Torrejón M, et al. (2020) may be higher in nonallergic asthmatics than in healthy controls. Our objective is to compare Total and specific immunoglobulin E in induced total and dust-mite specific (Der p 1) IgE levels in induced sputum in allergic and nonallergic sputum in allergic and non-allergic asthma. PLoS ONE 15(1): e0228045. https://doi.org/10.1371/ asthmatics and healthy controls. journal.pone.0228045 Editor: Aleksandra Barac, Clinic for Infectious and Methods tropical diseases, Clinical centre of Serbia, SERBIA Total and Der p 1-specific IgE were measured in induced sputum (ImmunoCAP immunoas- Received: September 27, 2019 say) from 56 age- and sex-matched asthmatics (21 allergic, 35 nonallergic) and 9 healthy controls. Allergic asthma was defined as asthma with a positive prick test and/or clinically- Accepted: January 6, 2020 significant Der p 1-specific serum IgE levels. Published: January 29, 2020 Peer Review History: PLOS recognizes the Results benefits of transparency in the peer review process; therefore, we enable the publication of Patients with allergic asthma presented significantly higher total and Der p 1-specific serum all of the content of peer review and author IgE levels. There were no significant between-group differences in total sputum IgE. How- responses alongside final, published articles. The ever, Der p 1-specific sputum IgE levels were significantly higher (p = 0.000) in the allergic editorial history of this article is available here: asthmatics, but without differences between the controls and nonallergic asthmatics. Serum https://doi.org/10.1371/journal.pone.0228045 and sputum IgE levels were significantly correlated, both for total IgE (rho = 0.498; p = Copyright: © 2020 Crespo-Lessmann et al. This is 0.000) and Der p 1-specific IgE (rho, 0.621; p = 0001). an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and Conclusions reproduction in any medium, provided the original Total IgE levels measured in serum and induced sputum are significantly correlated. No sig- author and source are credited. nificant differences were found between the differents groups in total sputum IgE. Neverthe- Data Availability Statement: The dataset less, the levels of Der p 1-specific sputum IgE levels were significantly higher in the allergic supporting the conclusions of this article is available for consultation at www.figshare.com asthmatics, but without differences between the controls and nonallergic asthmatics. Proba- (DOI 10.6084/m9.figshare.11499162.v1). bly due to the lack of sensitivity of the test used, but with the growing evidence for local PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 1 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Funding: Funding sources: Leti Grant. Fundació allergic reactions better methods are need to explore its presence. The Clinical Trials Identi- Catalana de Pneumologia, 2016. The funders had fier for this project is NCT03640936. no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing interests: AC. in the last three years received honoraria for speaking at sponsored meetings from AstraZeneca, Chiesi, Esteve Introduction Laboratories, Faes Farma, Ferrer, GlaxoSmithKline, Novartis, Teva, Zambon. Received help assistance Although the causes of allergic asthma are well-understood, much less is known about the to meeting travel from Bial, Novartis. Act as a pathophysiology of nonallergic asthma. Non-allergic asthma is generally defined as nonatopic consultant for AstraZeneca, Boehringer, asthma with or without normal serum levels of immunoglobulin E (IgE) antibodies. Unlike GlaxoSmithKline, Novartis. And received funding/ allergic asthma, which is triggered by a specific allergen, nonallergic asthma may be triggered grant support for research projects from a variety of Government agencies and not-for-profit by a wide range of factors, including smoke, viruses, and other nonspecific stimuli[1]. foundations, as well as AstraZeneca. EC has Despite their differences, these two types of asthma may share many similarities, including received funding to travel to and attend training airway inflammation with eosinophilia, increased Th2 cytokine production (interleukin [IL]- activities from ALK, Menarini, Teva, AstraZeneca, 4, IL-5, and IL-13), hyperreactivity, and airway-induced exacerbations. Although most patients Chiesi, Boehringer, and Novartis. LS, EM and with nonallergic asthma present normal total serum IgE levels, in some cases IgE may be ele- declare no conflict of interests. JG has received vated when compared to healthy controls [2,3], with some reports suggesting that approxi- funding to travel and attend to training activities from Menarini, Teva, AstraZeneca, Chiesi, GSK, mately 30% of asthmatic patients with a negative skin prick test have high total circulating IgE Mundipharma, Boehringer. In the last three years, (>150 U/mL) [4,5]. These shared features suggest that unidentified environmental allergens VP has received honoraria for speaking at could be involved in “nonallergic” asthma, causing a local allergic reaction in these patients[6]. sponsored meetings from AstraZeneca, Mouthuy et al. previously demonstrated that patients with nonallergic asthma present elevated Boehringer-Ingelheim, Chiesi, GSK, and Novartis. levels of total IgE and Dermatophagoides pteronyssinus (Der p 1) specific IgE in induced spu- VP has also received financial support to travel to meetings organized by Chiesi and Novartis. VP is a tum versus healthy controls[7]. Those findings suggest that nonallergic asthma patients may consultant for ALK, AstraZeneca, Boehringer, present localized allergic inflammation. This hypothesis is further supported by several studies MundiPharma, and Sanofi. VP has also received that have demonstrated symptom relief after the administration of omalizumab—an anti-IgE funding/grant support for research projects from a treatment—in nonatopic patients[8–10]. The positive effect of omalizumab in these patients variety of governmental agencies and not-for-profit could be explained by the presence of unidentified allergens[11]. Additional support for this foundations, as well as from AstraZeneca, Chiesi hypothesis comes from the relatively recent identification of a new phenotype of rhinitis— and Menarini. denominated local allergic rhinitis (LAR)—in patients with chronic rhinitis [12–16], charac- terized by local production of specific IgE with a nasal cellular Th2 immune response to nasal allergen provocation test but with negative skin prick test and undetectable serum IgE. Never- theless, more research is needed to confirm the existence of this potential new asthma pheno- type characterized by a local allergic reaction. In this context, we hypothesized that patients with nonallergic asthma would present higher levels of IgE in induced sputum than healthy controls. To test this hypothesis, we measured total IgE and Der p 1-specific IgE in serum and induced sputum in three different groups: 1) patients with a confirmed diagnosis of allergic asthma, 2) patients diagnosed with nonallergic asthma, and 3) healthy controls. Secondary aims were to assess the correlation between total and Der p 1-specific IgE levels in serum and induced sputum and to establish a preliminary estimate of total IgE and Der p 1-specific IgE in the induced sputum of healthy individuals. Materials and methods Study design and participants This was a comparative cross-sectional study to measure and compare total IgE and Der p 1-specific IgE levels in the induced sputum of asthmatics and in a group of healthy volunteers. Patients and controls were matched for age, sex, and disease severity; and asthma control for allergic and non-allergic groups. Patients were consecutively enrolled from the outpatient PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 2 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma asthma unit of our institution, a tertiary referral university hospital in Spain, between January and December 2013. Total and Der p 1-specific IgE were measured in both serum and induced sputum. The IgE levels in serum and sputum were compared to determine the correlation between IgE levels in these two fluids. Definition of allergic and nonallergic asthma We defined asthma as a history of variable respiratory symptoms and evidence of variable expiratory airflow limitation. All patients had a positive bronchodilator test or a document positive methacholine challenge test. Asthma severity was defined according to the Global Ini- tiative for Asthma Management (GINA)[17]. Allergic asthma was defined as asthma with 1) positive skin prick test to aeroallergens and/ or 2) clinically-significant Der p 1-specific IgE according to the recommendations of the Com- mittee of Skin Tests of the European Academy of Allergy and Clinical Immunology (EAACI) international task force[18]. Patients sensitized to various allergens were included only if dust mite allergy was the only clinically relevant one; if they showed symptoms in relation to other exposures they were excluded. Nonallergic asthma was defined as asthma with: 1) negative prick test, 2) negative Der p 1-specific IgE in serum; and 3) negative Phadiatop test (Immuno- CAP immunofluoroassay; Phadia ThermoFisher Scientific)[19]. D. pteronyssinus was the selected allergen because is the perennial allergen more prevalent in our geographic area. Inclusion and exclusion criteria Inclusion criteria were: 1) age 18–70 years; 2) continuous residence (> 4 years) in the geo- graphic region of the study; 3) diagnosis of stable bronchial asthma according to GINA criteria [17]; 4) non-smoker; 5) no respiratory infections in the month prior to enrolment; 6) no oral corticosteroids in the last month; 7) no biological treatment with anti-IgE monoclonal anti- bodies; 8) no allergenic immunotherapy. Exclusion criteria were: 1) pregnancy; 2) moderate to severe active alcohol use; 3) severe atopic dermatitis; 4) presence of any lung disease, autoimmune disease or systemic inflamma- tory disease, or cancer. Control group The control group consisted of healthy, non-smoking volunteers age 18 to 79 years, without rhinitis, allergic asthma, or other allergic symptoms (GINA criteria) or other lung disease. Controls were recruited from among staff members at our hospital. Participation was completely voluntarily. All controls were required to present a negative prick test for aeroaller- gens and Der p 1-specific IgE, and negative Phadiatop test. Assessments and study procedures Upon enrolment, demographic and clinical variables were assessed and recorded for all partic- ipants. On the same day, the following assessment were performed: FeNO (exhaled nitric oxide test); forced spirometry; inflammatory cell count in induced sputum; eosinophil count in peripheral blood; total serum IgE levels; and skin prick test for common aeroallergens. Patients also completed the validated Spanish-language version of the Asthma Control Test (ACT)[20]. FeNO was measured before spirometry using an electrochemical equipment (NO Vario Analyzer; FILT Lungen and Thorax Diagnostic GmbH, Berlin, Germany) and an expiratory maneuver providing a sustained 50 mL/s flow from total lung capacity, following the 2005 PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 3 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma recommendations of the American Thoracic Society/European Respiratory Society[21]. Spi- rometry was performed using a Daptospir-600 spirometer (Sibelmed, S.A., Barcelona, Spain) in accordance with the 2003 recommendations of the Spanish Society of Pneumology and Thoracic Surgery (SEPAR), with FEV1 (forced expiratory volume in 1 second) in the reference range when 80% of the predicted value [22,23]. Skin prick testing was performed according to standard procedures, with wheal diameters � 4 mm considered positive. Allergic asthma was defined as the presence of asthma symptoms for one or more allergens, with positive skin prick tests for these allergens. Well- controlled asthma was defined as ACT � 20. Induction and analysis of sputum Induced sputum was evaluated following the procedures previously described by our group [24] and by Pizzichini et al.[25]. Briefly, mucus plugs were manually selected and weighed, and incubated at room temperature for 15 min in 4 times the weight (in ml) of the selected plug (in mg) in 0.1% dithiothreitol (Calbiochem, San Diego, Calif., USA), washed with 4 times the plug weight (in ml) in Dulbecco’s PBS, and gravity filtered through a 41-μm-pore nylon net filter (Millipore Inc; Billerica, Mass., USA). Each specimen was homogenised and aliquoted into two equal volumes. A Neubauer hemocytometer was used to determine total cell count; visu- ally identifiable squamous epithelial cells were not included in the total cell count. Samples with insufficient sputum cell numbers (<1000 × 106 cells/g) were excluded. Cell viability was determined by light microscopic assessment using trypan blue exclusion staining. The cells underwent centrifugation to obtain a cell pellet and a supernatant. The cell pellet was used for differential cell counts (macrophages, eosinophils, neutrophils, lymphocytes, and bronchial epithelial cells) performed on May-Grünwald-Giemsa-stained preparations. A differential leu- kocyte analysis of nonsquamous cells (Diff-Quik stained) was performed on a minimum of 400 cells. Differential cell counts are expressed as the percentage of total nonsquamous nucle- ated cells. Reference values for the cell counts were performed as described in other publica- tions[26]. Measurement of total and specific IgE antibodies Total and specific IgE in induced sputum supernatants were measured by the ImmunoCAP fluoroenzyme immunoassay (Phadia ThermoFisher Scientific) following the manufacturer’s instructions. The test was considered positive at > 2kU/L for total IgE and > 0.35kU/L for spe- cific IgE, according to manufacturer’s recommendation. Statistical analysis Categorical variables were expressed as absolute and relative frequencies and quantitative vari- ables as mean and standard deviation (SD). Groups were compared using ANOVA or chi- square test as appropriate. Given that the distribution of total and specific IgE values in serum and sputum were not normal, non-parametric tests (Mann-Whitney or Kruskal-Wallis for independent samples) were applied; the values were expressed as medians with interquartile ranges and ranges. The significance values in the case of non-parametric tests were adjusted by the Bonferroni correction. For the correlation analyses, Spearman’s Rho test was used. Statisti- cal significance was set at p<0.05. Statistical analysis was performed with Statistical Package for the Social Sciences version 18.0 (SPSS, Chicago, IL). PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 4 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Ethics approval and consent to participate The study design complied with the principles of the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee at the Hospital de la Santa Creu i Sant Pau in Barcelona (COD 26/2012). All participants provided written informed consent. All patient-related data were anonymised, with the identity of the participating known only by the treating physicians. The clinicaltrials.gov identifier is NCT03640936. Results A total of 56 asthmatic patients—21 (37.5%) with allergic asthma and 35 (62.5%) with nonal- lergic asthma—met all inclusion criteria and completed the study. The control group consisted of nine healthy volunteers. Table 1 shows the clinical, functional, and inflammatory character- istics of the patients and controls. There were no significant differences between the three groups with regard to age, sex, or body mass index (BMI). There were significant differences between the groups in: FEV1 and need for more than one round of oral corticosteroids last 12 months. FEV1 values in the aller- gic (83.4%) and nonallergic (80%) patient groups were similar, but substantially higher (100.2%) in the healthy controls (p = 0.013) and the need for more than one round of oral cor- ticosteroids was significantly higher (p = 0.022) in the nonallergic group. As for other not clini- cally relevant sensitizations detected by prick test in the group of patients with allergic asthma, the most frequent were polysensitized patients (17), sensitized to D. farinae (8) and pet epithe- lia (4). A single patient had a positive prick test for molds. Table 2 shows the total and Der p 1-specific levels of IgE in serum and sputum for all three groups. Significant between-group differences in serum IgE levels were observed for both total and Der p 1-specific IgE, with the allergic asthma group presenting significantly higher levels of total IgE (mean, 1702.3 KU/L) and Der p1-specific IgE levels (15.5 KU/L) than the nonaller- gic group and healthy controls. In the sputum samples, no significant between-group differ- ences in total IgE were observed. However, Der p 1-specific IgE levels were significantly higher Table 1. Clinical, functional, and inflammatory characteristics of patients and controls. Variables Nonallergic asthma (n = 21) Allergic asthma (n = 35) Healthy controls (n = 9) P Age (years) 54.8 (14.8) 51.7(13.6) 41.88 0.076 Sex (% females) 57.1% 51.4% 88.8% 0.125 BMI (kg/m2) 28.7 (4.1) 27.3 (5.9) 25.5 (6.48) 0.353 FEV1 (%) 83.4 (13.9) 80 (20.8) 100.2 (10.74) 0.013 9 Serum eosinophils (x10 /L) 0.302 (0.310) 0.271 (0.239) 0.095 (0.068) 0.113 Sputum eosinophils (%) 10.33 (19.54) 11.2 (13.01) 0.77 (1.09) 0.166 Rhinitis 61.9% 82.8% n/a 0.080 Nasal polyposis (%) 28.5% 20% n/a 0.462 Severe persistent asthma (%) 42.8% 42.8% n/a 0.870 GINA 4.0 scale, grade 5–6 (%) 52.4% 48.5% n/a 0.590 Good asthma control (ACT >20%) 19% 20% n/a 0.357 Emergency room visits last 12 months (%) 28.5% 11.4% n/a 0.182 >1 round of oral corticosteroids last 12 months 38% 8.5% n/a 0.022 Inflammatory phenotype in induced sputum (%) Paucigranulocytic: 28.6% Paucigranulocytic: 22.4% n/a 0.425 Mixed: 9.5% Mixed: 8.6% Eosinophilic: 33.3% Eosinophilic: 54.3% Neutrophilic: 28.6% Neutrophilic: 14.3% https://doi.org/10.1371/journal.pone.0228045.t001 PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 5 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Table 2. Total and dust-mite specific IgE n serum and sputum in patients with allergic asthma, non-allergic asthma, and healthy controls. VARIABLE NONALLERGIC ASTHMA ALLERGIC ASTHMA HEALTHY CONTROLS p (N = 21) (N = 35) (N = 9) High quality induced sputum (%)� 61.9% 69.7% 44% 0.294 Total IgE (KU/L) in serum, median (IQR) 55.5 (177.93) 233.5 (368.25) 14.25 (38.30) <0.0001 Total IgE (KU/L) in serum, average range 423.85 1702.3 87.78 Der p-specific IgE (KU/L) in serum, median 0.01 (0.03) 15.45 (32.68) 0.02 (0.04) <0.0001 (IQR) Der p-specific IgE (KU/L) in serum, average 0.08 99.97 0.10 range Total IgE (kU/L) in sputum, median (IQR) 2.69 (4.55) 4.5 (2.18) 3.16 (1.41) 0.188 Total IgE (kU/L) in sputum, average range 8.11 7.45 4.12 Der p-specific IgE in sputum (kU/L), median 0.055 (0.03) 0.095 (0.09) 0.06 (0.02) <0.0001 (IQR) Der p-specific IgE in sputum (kU/L), average 0.04 0.54 0.05 range � High quality defined as > 40% viability, <20% epithelial cells, > 1x106 cells https://doi.org/10.1371/journal.pone.0228045.t002 (p<0.0001) in the allergic asthma group compared to the other two groups. Fig 1 shows the total sputum IgE levels in the three groups, with no significant differences between the groups. Fig 2 shows the specific Der p 1-specific IgE levels in sputum for each group. The allergic asthma group presented significantly higher Der p 1-specific IgE levels than both the nonaller- gic patients (p<0.0001) and the healthy controls (p = 0.006). There were no significant differ- ences between the nonallergic patients and the healthy controls. In the group of patients with non-allergic asthma, a subgroup analysis was performed among those with total sputum IgE >2 or <2, with no statistically significant differences either at clinical characteristics or regard- ing the other IgE measurements. Correlations Using data from the whole sample (patients and controls), we calculated the correlations between total and Der p 1-specific IgE levels in sputum and serum. Table 3 shows a matrix with all significant correlations (p<0.0001) in descending order from strongest to weakest. As that table makes clear, the strongest correlation was between total IgE and Der p 1-specific IgE in serum. Discussion In the present study, we sought to test the hypothesis that the airways of patients with nonaller- gic asthma exhibit a local inflammatory response by determining total and dust mite-specific IgE antibody levels in induced sputum. Our results showed that patients with allergic asthma presented significantly higher total and Der p 1-specific IgE levels in serum compared to both nonallergic asthmatics and healthy controls. However, contrary to our expectations, there were no significant differences among the three groups in total sputum IgE levels. Moreover, there were no differences in Der p 1-specific sputum IgE levels between healthy controls and nonallergic asthma patients. Overall, the lack of significant differences in total sputum IgE lev- els between the three groups was surprising. However, diverse factors could explain this unex- pected finding, as we discuss in detail below. The hypothesis that patients with nonallergic asthma may present a local allergic response in the airways derives from the mounting evidence for a new phenotype of local allergic PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 6 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Fig 1. Total IgE in sputum in patients with allergic asthma, non-allergic asthma, and healthy controls. https://doi.org/10.1371/journal.pone.0228045.g001 rhinitis in nonatopic rhinitis[12–15]. Studies have demonstrated local production of specific IgE in the nostrils of patients with negative skin prick test and undetectable serum IgE[12]. Given the similarities between asthma and rhinitis, it seems highly plausible that patients with nonatopic asthma could also present a local allergic response, particularly considering the lack of a clear physiopathologic explanation for nonallergic asthma. Although there are differences between allergic and nonallergic asthma, these two clinical entities share many similarities, including airway inflammation with eosinophilia, increased Th2 cytokine production, airway- induced exacerbations[5,27]. In addition, up to 30% of patients with nonallergic asthma may present elevated total serum IgE levels[4,5,16]. These shared features suggest that unidentified environmental allergens—which stimulate a local allergic reaction—may be responsible for the symptoms experienced by patients with nonallergic asthma, a hypothesis supported by a small but growing body of evidence showing local airway synthesis of IgE[27], even in patients with- out any allergen-specific serum IgE7. Moreover, the findings from multiple studies that the anti-IgE treatment omalizumab provides symptom relief in nonatopic patients implies that an inflammatory reaction does, in fact, play a role in these patients[8,9,10]. Mouthuy et al.[7] found that nonallergic asthmatics present elevated levels of total and Der p 1-specific IgE in induced sputum compared to healthy controls, a finding that supports the concept of local air- way inflammation in those patients. However, we were unable to confirm those findings, as we found no significant differences between the nonallergic asthmatics and healthy controls in IgE levels (both total and Der p 1-specific) in induced sputum or in serum. Our findings were PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 7 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Fig 2. Der p 1-specific IgE levels in sputum in patients with allergic asthma, non-allergic asthma and healthy controls. https://doi.org/10.1371/journal.pone.0228045.g002 closer to those reported by Manise et al.[28], who—in contrast to Mouthuy et al.—found that total sputum IgE levels were higher in atopic than in nonatopic asthmatics and that there were no differences between nonatopic asthmatics and nonatopic healthy subjects. In view of the heterogeneous findings of these three studies, it is clear that more work will be needed to clar- ify whether or not there are truly differences among nonatopic and atopic asthmatics and healthy controls with regard to IgE levels in sputum. There are several reasons that could explain the discrepancy between our results and those reported by Mouthuy et al. First, the lack of any significant differences in IgE levels between Table 3. Significant correlations between IgE values in serum and induced sputum. Serum Sputum Correlation† Total IgE Der-p IgE Total IgE Der-p IgE X X 0.658 X X 0.621 X X 0.538 X X 0.498 X X 0.454 †Spearman’s rho; P = 0.000 for all correlations https://doi.org/10.1371/journal.pone.0228045.t003 PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 8 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma the nonallergic asthmatics and healthy controls in our study could potentially be attributed to the size of the control group in our study (n = 9) versus the large control group (n = 25) in the study by Mouthy and colleagues. Another explanation could be related to the ImmunoCAP method, which may not be sufficiently sensitive to detect very small differences in IgE levels. In our sample, although the test did detect higher Der p 1-specific IgE levels in the sputum of allergic asthmatics verus both nonallergic asthmatics and healthy controls, this is probably because there were large differences in Der p 1-specific IgE levels in the allergic asthmatics ver- sus the nonallergic asthmatics and healthy controls given that the study inclusion criteria for those latter two groups specifically required that they have a negative skin prick and negative serum Der p 1. By contrast, the differences in total IgE may have been less marked, making it more difficult to detect using the ImmunoCap technique. Local IgE production has been documented previously in nonallergic asthma patients using bronchial biopsy[16,29,30]. In this regard, the lack of a significant difference between the three groups in our study with regard to total sputum IgE was surprising, especially considering that —at the very least—allergic asthmatics would be expected to present substantially higher IgE levels than healthy controls. Although the reason for this unexpected finding is not clear, it could be due to the relatively small sample size or to the limited capacity of the ImmunoCap technique to detect small differences in total IgE. In this regard, larger studies will be needed, perhaps using alternative methods to measure sputum IgE. Correlation between IgE in sputum and serum In the present study, we used the sputum induction and analysis methods described by Araujo et al.[31], who validated laboratory measurements of total and Der p 1-specific IgE in induced sputum supernatant versus serum levels, but in a heterogeneous sample involving patients with diverse pathologies, not only asthma. In recent years, several studies have correlated total IgE levels in induced sputum and serum, with sometimes contradictory findings[32]. In the present study, we found a highly significant correlation (rho, 0.498; p = 0.000) between total IgE levels in serum and sputum in our overall sample, a finding that is consistent with the results described by Manise et al. in asthmatic patients[28]. By contrast, other authors, includ- ing Mouthuy et al.[7] and Ahn et al[33] have not found any correlation between total IgE levels in sputum and serum. These contrasting findings raise further doubts about the sensitivity of the ImmunoCap technique used to measure IgE in sputum, potentially providing an additional explanation for the differences between our results and those of Mouthuy et al. Data from a recent study conducted by Pillai et al.[34] could help to explain why we were unable to detect significant between-group differences in total IgE in induced sputum. Those authors suggest that IgE produced in the bronchial mucosa of nonatopic asthmatic patients may remain confined to the mucosa, bound to cells that carry those receptors. If this hypothe- sis is correct, it would explain why IgE is not readily detectable in induced sputum. Pillai and colleagues posited that both atopic and nonatopic asthmatics would have greater total IgE con- centrations in the airways than in serum. To test this, they determined IgE levels in the blood and bronchial mucosa of 10 atopic and 10 nonatopic asthmatic patients and 10 nonatopic con- trols, finding that median total IgE levels were significantly elevated in both the atopic and nonatopic asthmatic patients versus controls. These data are consistent with the hypothesis that IgE synthesis, sequestration, or both are ongoing in the bronchial mucosa of both nonato- pic and atopic asthmatic patients. Interestingly, Pillai et al. also suggest, as other several authors have previously proposed[10,35,36], that increased bronchial mucosal IgE production in nonatopic asthmatic patients may be directed against targets other than allergens, including PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 9 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma possible “autoallergens”, or that there are allergen-independent roles for IgE in the pathophysi- ology of asthma. Overall, the findings reported in studies which use more invasive but more sensitive meth- ods (e.g., bronchial biopsy) strongly suggest the presence of elevated total IgE production in the airways of both allergic and nonallergic asthma patients[10,34–36]. The fact that we were unable to confirm the findings reported by Mouthuy et al. with regard to detecting significant differences in sputum IgE levels between nonatopic asthmatics and healthy controls, together with the contradictory data reported to date regarding the correlation between IgE levels in serum and induced sputum, suggests that more sensitive methods of measuring IgE in sputum may be required. Total and Der p-specific IgE in induced sputum in healthy individuals A secondary aim of this study was to determine, on a pilot basis, the standard levels of total and Der p 1-specific IgE in the induced sputum of healthy controls. We found the following values: median (IQR) total IgE in sputum in the controls was 3.16 (1.41) KUA/L. The Der p 1-specific values were 0.06 (0.02). Evidently, the small sample (n = 9) of healthy controls are insufficient to define standard levels, but these data provide an initial estimate. Nevertheless, more data from larger studies will be needed to confirm these initial levels, especially consider- ing the small sample and the potential limitations in the assay technique used to measure IgE in the sputum supernatant. Study strengths and limitations The main limitation of the present study is the limited number of healthy controls. Another limitation may be related to the lack of significant differences between the groups in total IgE in induced sputum, which points to limitations in the measurement technique that may have influenced our findings. An important strength of the study is that this is, to our knowledge, only the second study conducted to date to specifically determine total and Der p-specific IgE in induced sputum of allergic, nonallergic, and healthy controls. Given the conflicting results of our study and those reported by Mouthuy et al., additional studies are needed. Finally, another important strength is the well-selected and well-defined sample of patients and con- trols; we used very strict diagnostic criteria (based on the most recent clinical guidelines) to define both allergic and nonallergic asthma, as well as for the healthy controls. Conclusions The findings of this study show that total IgE levels measured in serum and induced sputum are significantly correlated. The significantly higher levels of Der p 1-specific IgE detected in the induced sputum of the allergic asthmatics underscores the potential value of measuring aeroallergen-specific IgE in induced sputum. Nevertheless, the lack of significant between- group differences in total sputum IgE levels suggests that the ImmunoCAP immunoassay tech- nique used in this study may not be sufficiently sensitive to detect small differences in total sputum IgE. To support the results of this work, a larger sample size would be necessary for future studies. A growing body of evidence indicates that both allergic and nonallergic asthmatics present elevated airway inflammation. Measuring IgE levels in induced sputum is a non-invasive, cost- effective approach that could provide valuable clinical data to help individualize the treatment of nonallergic asthma. However, more sensitive methods are needed to measure IgE levels in induced sputum. Nonetheless, there exists a clear potential to use total and/or aeroallergen- specific IgE levels measured in induced sputum as a marker of treatment efficacy. PLOS ONE | https://doi.org/10.1371/journal.pone.0228045 January 29, 2020 10 / 13 Total and dust-mite specific IgE in induced sputum and serum in asthma Acknowledgments The authors wish to thank the patients and volunteers who generous contributed to this study. We also thank Bradley Londres for editing the manuscript. Author Contributions Conceptualization: Lorena Soto, Alba Garcı́a-Moral, David Ramos-Barbón, Vicente Plaza. Data curation: Elena Curto, Lorena Soto, Alba Garcı́a-Moral, Vicente Plaza. Formal analysis: Astrid Crespo-Lessmann, Vicente Plaza. Investigation: Astrid Crespo-Lessmann, Elena Curto, Eder Mateus, Lorena Soto, Alba Garcı́a- Moral, Montserrat Torrejón, Alicia Belda, Jordi Giner, David Ramos-Barbón. Methodology: Astrid Crespo-Lessmann, Elena Curto, Eder Mateus, Montserrat Torrejón, Ali- cia Belda, Jordi Giner, David Ramos-Barbón. Project administration: Astrid Crespo-Lessmann, Montserrat Torrejón, Alicia Belda, Jordi Giner, Vicente Plaza. Software: Elena Curto. Supervision: Astrid Crespo-Lessmann, David Ramos-Barbón. Validation: Astrid Crespo-Lessmann, Vicente Plaza. Visualization: Vicente Plaza. Writing – original draft: Astrid Crespo-Lessmann, Elena Curto. Writing – review & editing: Astrid Crespo-Lessmann, Elena Curto. References 1. Peters SP. Asthma Phenotypes: Nonallergic (Intrinsic) Asthma. 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