Proles of sensitization and comorbidity in asthma patients with markedly increased serum total immunoglobulin E level (>1000 kUA/L)

Background Immunoglobulin E (IgE) plays an important role in asthma, but a few cases exhibited extremely high levels of serum total IgE. This study aimed to investigate the proles of complications, severity, and sensitizations in asthma patients with serum total IgE level >1000 kU A /L. Methods We retrospectively analyzed 170 asthma patients with serum total IgE levels >1000 kU A /L from the in-patient database of First Aliated Hospital of Guangzhou Medical University from January 2014 to June 2019. Available information including age, gender, body mass index (BMI), diagnosis, results of blood routine, pulmonary function, fractional exhaled nitric oxide (FeNO), induced sputum (if any), IgE (both total and specic) and medication records were analyzed.


Background
Asthma is a chronic in ammatory disease of the airways with the typical characteristics of reversible air ow limitation and airway hyperresponsiveness (AHR) [1]. Its clinical symptoms vary from individuals, including wheeze, shortness of breath, dyspnea, chest tightness and/or cough. Asthma has been a public health issue over all ages worldwide, affecting 1-18% of the population in different countries, and the economic burden of the disease is still on the rise [2,3]. Allergic asthma is the most common phenotype [4], in which T helper type 2 in ammation is considered to be the predominant immune response against the allergens encountered [5]. Antigen presenting cells (APCs) with captured allergen leads to the activation of T helper 2 (Th2) cells, resulting in the secretion of Th2 cytokines such as interleukin-4 (IL- 4) and IL-13. Afterwards, B cell is stimulated for immunoglobulin E (IgE) synthesis and secretion. The secreted IgE can not only binds to mast cells, which triggers degranulation, consequently causing allergic symptoms, but binds to APCs and activates more Th2 cells.
In fact, most cases of asthma contributes to IgE-mediated reaction, and the increased levels of total and speci c IgE in the peripheral is involved in the onset as well as the chronic phase of the disease [6].
Studies have indicated that elevated serum total IgE levels could occur in atopic and non-atopic asthma patients, and it was associated with the risk of asthma as well as with AHR [7][8][9]. However, the correlation between IgE level and asthma severity is controversy. Borish L et al.'s study found that children with severe asthma had high serum IgE level, while another did not nd a signi cant association between total IgE levels and disease severity [10,11]. This can be affected by several factors, such as polysensitization, non-allergenic factors (e.g. infection) and persistent in ammation caused by allergens [12]. Despite of that, anti-IgE treatment has been applied in clinical practice for some cases of poorly controlled moderate-to-severe asthma, which improves asthma related symptoms and reduces severe exacerbations [13,14].
There are a few patients exhibited extremely high levels of serum total IgE (> 1000 kU A /L). Conditions like atopic eczema, helminthic infection and rare primary immunode ciencies can lead to markedly upregulated total IgE levels (> 1000 kU A /L) [15]. In some of these cases with sensitization of Aspergillus fumigatus, they might develop allergic bronchopulmonary aspergillosis (ABPA) as Th2 immunity against the fungi increased [16,17]. The prevalence was estimated to be about 6% in chronic cases of asthma, and total IgE > 1000 kU A /L is considered as one of the diagnostic criteria of ABPA [18,19]. However, certain amount of asthma patients with total IgE > 1000 kU A /L do not meet the diagnostic criteria of ABPA. To date, few study has been performed to investigate these asthma cases. In order to better understand the characteristic of these cases, we aimed to analyze the pro les of complications, severity, and sensitizations in asthma patients with serum total IgE > 1000 kU A /L.

Study subjects
This is a retrospective analysis of the in-patient database generated by First A liated Hospital of Guangzhou Medical University, from January 2014 to June 2019. We screened the database for patients who were clinically diagnosed as asthma and had serum total IgE levels > 1000 kU A /L (n=202). Patients met the diagnostic criteria of ABPA based on The International Society of Human and Animal Mycology (ISHAM) working group were excluded [18]. A total of 170 asthma patients with total IgE > 1000 kU A /L were screened out from the database ( Figure S1). Among the included subjects, 24 patients had hospitalization records for more than one time during the study period. The data used for analysis was the data of the patients when total IgE >1000 kU A /L was rst recorded among their hospitalizations. None of the included patients had diagnosis of primary immunode ciency diseases. Available information of each patient were collected for analysis, including age, gender, body mass index (BMI), diagnosis, results of blood routine, pulmonary function, fractional exhaled nitric oxide (FeNO), induced sputum (if any), IgE (both total and speci c) and medication records.

Diagnosis criteria and disease classi cation
The diagnosis of asthma was based on the Global Initiative for Asthma (GINA) guidelines, including asthma clinical features of the patient as well as presence of air ow limitation. The diagnosis of chronic obstructive pulmonary disease (COPD) was based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, including presence of clinical features like dyspnea, chronic cough or sputum production and meeting the spirometry standard. The diagnosis of eosinophilic granulomatosis with polyangiitis (EGPA) was based on the criteria developed by Lanham et al., including asthma, peak eripheral blood eosinophil count > 1.5 ×10 9 cells/L and systemic vasculitis involving two or more extrapulmonary organs [20]. The de nition of rhinosinusitis was according to the European position paper on Rhinosinusitis and nasal polyps, which contains characterized symptoms, endoscopic signs and CT changes [21]. Cardiovascular diseases (CDVs) included coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease and deep vein thrombosis and pulmonary embolism. Hypertension was diagnosed following WHO's instruction, which is on two different days, the systolic blood pressure readings was ≥ 140 mmHg and/or the diastolic blood pressure readings was ≥ 90 mmHg. The diagnosis of cancer was con rmed via pathological diagnosis of the lesions. Airway infection includes pneumonia, bronchi infections and lung infections caused by microorganisms.

Grouping
Since some of the complications (e.g. COPD) as well as sensitization pro les (food sensitization may decrease as children grew up [22]) varied from ages, patients were classi ed into minors (age <18, n=32) and adults (age ≥18, n=138) for our analysis. The assessment of asthma severity was based on the level of treatment required to control symptoms and exacerbations of the past 4 weeks following the instruction of GINA guideline [3]. Brie y, mild asthma is well controlled with Step 1 or Step 2 treatment; moderate asthma is well controlled with Step 3 treatment; severe asthma requires Step 4 or 5 treatment to prevent it from becoming 'uncontrolled', or remains 'uncontrolled' despite this treatment. The severity assessment was recorded in the medical records of each patient by specialists, and those who lacked such information were not available for assessing severity. Among the enrolled patients, 27 (15.88%) were with mild asthma, 68 (40%) were with moderate asthma, 55 (32.35%) were with severe asthma and 20 patients (11.76%) lacked information for asthma severity assessment.

IgE detection
Serum samples were obtained from venous blood collecting with separation gel containing vacutainer tubes by centrifuged at 3000rpm for 10min. Serum total IgE and speci c IgE (including Phadiatop test [23], allergen mixtures such as house dusts mix (hx2) and speci c allergens) were analyzed by ImmunoCAP 1000 system (Thermo Fisher Scienti c Inc., California, USA) according to the procedures offered by the manufacture. The tests of patients undergoing were depended on the allergy history of each patient and were prescribed by specialists. A positive result of speci c IgE was considered as ≥ 0.35 kU A /L. For those patients who were positive for Phadiatop or allergen mixtures but did not take further detection of speci c IgE of typical allergens, they were considered as sensitized to at least one inhaled allergen or at least one of the mixture allergens, respectively. IgE detection was done during the hospitalization.

Statistical analysis
Parametric quantitative data were expressed as mean ± standard deviation and non-parametric quantitative data were expressed as median (interquartile range). For Parametric quantitative data, ordinary one-way ANOVA was applied for comparison of three groups and student t-test was used for two-group comparison. For non-parametric data, differences of the three groups were analyzed using the non-parametric Kruskall-Wallis test and Mann-Whitney test was used for comparison between two groups. Spearman rho test was applied for correlation analysis. Proportions were compared between groups with chi-square tests (c 2 ). Optimal scaling was carried out with categorical principle component analysis. Statistical analysis was performed with SPSS 22.0 (SPSS, Chicago, IL) and GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). A value of P < 0.05 was considered statistically signi cant.

Results
Characteristics of the study subjects were shown in Table 1. Compared with minors, adults had lower proportion of mild asthma and higher proportion of severe asthma. This resulted in worse lung function results (%FEV 1 , FEV 1 /FVC and %PEF) in adults. Besides, the number of sIgE positives among minors was higher than that of adults (4 [3, 5] vs. 2 [1,5], P < 0.05). Age and BMI were given as mean ± standard deviation, other non-parametric quantitative data were given as medians with interquartile range (IQR). BMI: body mass index; OCS: oral corticoid steroid; ICS: inhaled corticoid steroid; WBC: white blood cell; *P < 0.05 compared with Minors; **P < 0.01 compared with Minors We investigated the complication status in these patients ( Table 2). There were 4 patients (2.35%) complicated with conditions that may associate with serum total IgE levels > 1000 kU A /L, which were helminthic infection (3 cases, 1.76%) and atopic dermatitis (1 cases, 0.59%). In patients complicated with other conditions, airway infections (76 cases, 44.71%) and rhinosinusitis (70 cases, 41.18%) were the most common two complications, followed by hypertension and/or CDVs (35 cases, 20.59%) and COPD (22 cases, 12.94%). We further analyzed the complications in patients who were not sensitized to any detected allergens, and the pattern of complication was similar to the overall study subjects (Table S1). The overlaps of complications in minors and adults were shown in Fig. 1. Only two complications were observed in minors and airway infections accounted for the most common complication in the minors group (Fig. 1A), while rhinosinusitis was the most common one in adults (Fig. 1B). In the analysis of serum total IgE levels, we performed the comparisons from various aspects, including age, asthma severity, complications, number of complications, sensitization status and number of con rmed sensitization. No signi cant differences were found in any of the comparison. (Fig. 2).
Results from optimal scaling illustrated a close association among lung function results, among peripheral neutrophil counts, percentage of neutrophil in sputum and white blood cell counts, between number of con rmed speci c IgE positivity and house dust mite speci c IgE, between peripheral eosinophil counts and percentage of eosinophil in sputum, as well as between asthma severity and A. fumigatus speci c IgE levels. (Cronbach's α = 93.7%) (Fig. 4).
In the comparison of asthma patients with different severities (Table 3), except for the lung function results, which were worsen as asthma severity increased, younger age was found in mild asthma group compared with moderate and severe group (P < 0.01). Similarly, mild asthma group had lower BMI compared with severe group (P < 0.05). Meanwhile, the peripheral blood neutrophil counts were also increased in moderate and severe asthma group compared with mild group (P < 0.05). Regarding complications, no signi cant difference was found in number of complications among the three groups (P = 0.09). In the mild group, lower proportion of COPD complication compared with the other two groups (P < 0.05). No statistical signi cance was found in serum total IgE levels (P = 0.84) and number of con rmed speci c IgE positivity (P = 0.73) among the three groups. Age and BMI were given as mean ± standard deviation, other non-parametric quantitative data were given as medians with interquartile range (IQR). BMI: body mass index; COPD: chronic obstructive pulmonary disease; EGPA: eosinophilic granulomatosis with polyangiitis; CDVs: Cardiovascular diseases. *P < 0.05 compared with mild asthma; **P < 0.01 compared with mild asthma; †P < 0.05 compared with severe asthma; † †P < 0.01 compared with severe asthma

Discussion
In this study, we described the pro les of comorbidity and sensitization of a typical group of asthma patients (total IgE > 1000 kU A /L). This is the rst study investigating the clinical information in asthma patients with such high level of serum total IgE. Our results demonstrated that in these patients, less than 5% patients were complicated with conditions associated with serum total IgE level > 1000 kU A /L and nearly 80% patients were positive for at least one speci c IgE test. Airway infections and rhinosinusitis were accounted for the most popular complications in minors and adults, respectively. Moreover, sensitization to A. fumigatus was associated with asthma severity.
In general, elevation of serum total IgE is usually found in allergic diseases. Extremely high level of serum total IgE (> 1000 kU A /L) could attribute to conditions like ABPA, helminthic infection and atopic dermatitis. Patients with ABPA were not included in our study, because it is a different entity of disease that exhibits asthma symptoms and may affects the analysis of IgE in asthma patients. Helminth infection induces strongly Th2-skewed responses in host defense, resulting in mastocytosis, eosinophilia and IgE production [24]. And in atopic dermatitis, the immune deviation towards Th2 and Th22 expansion, which also leads to generation of IgE against both exogenous and self allergens, and the persisting in ammation contributes to markedly elevated IgE level [25,26].
It has been reported that asthma accounted for 26.1% in patients with serum total IgE level > 1000 kU A /L in a 15-year retrospective analysis [15]. We further investigated the comorbidity of asthma patients with such high IgE level. There were only about 2% patients complicated with helminth infection or atopic dermatitis, and the highest two proportions of complications were airway infection (44.71%) and rhinosinusitis (41.18%). The frequency of patient complicated atopic dermatitis was low in our cohort. As patients complicated atopic dermatitis may not require hospitalization and the included patients were screened from in-patient system, the proportion might be underestimated. In patients without sensitization, only one patient complicated conditions associated with the raised total IgE (helminthic infection). Rihosinusitis and airway infections still accounted for the most common two complications.
Asthma patients complicated with rhinitis, whether allergic or non-allergic, or airway infections could lead to elevation of total IgE levels because of chronic in ammation and the reactivity of IgE against microorganisms [27][28][29][30]. In addition to infection, air pollutants such as diesel exhaust particles can also impair the integrity of airway epithelium, which promotes in ltration of pathogens via the damaged epithelium, causing in ammation responses [31].Regarding other conditions such as COPD, EGPA, type II diabetes, CVDs and hypertension, studies have also observed up-regulated levels of serum total IgE in these diseases [32][33][34][35][36]. However, whether these diseases could affect total IgE levels remains unclear and the underlining mechanism of IgE overproduction in nonatopic asthma requires further investigation.
In the analysis of sensitization pro les, despite the difference between age groups, mites had the highest positivity overall allergens, followed by food. This may due to our study population, as the top ve most sensitized allergens in southern China were house dust mite, cockroach and three types of food allergens [37]. There was no signi cant difference in total IgE levels between patients with and without sensitization, neither between patients with 1-2 speci c IgE sensitization and more than 3 speci c IgE sensitization. This suggested that the increase in sensitizations may have less impact on these total IgE level of these patients, because their IgE levels were already high.
Asthma severity is associated with various factors. Although IgE is the primarily responsible for both acute and chronic phase of in ammation characteristic of bronchial asthma, it is not an effective indicator of asthma severity [38]. In our study cohort, the proportion of mild asthma (15.88%) was the lowest, and the average age was younger compared with the other two groups. There are several risk factors of severe asthma, including allergens, environmental and occupational sensitizers, smoking, aspirin sensitivity, behavior and infection [39]. As people grow, they might have higher chance as well as longer duration of exposure to the risk factors above, which could lead to chronic and/or persisted in ammation. Indeed, compared with the mild group, a higher proportion of COPD complications were observed in the moderate and severe groups, and the peripheral blood neutrophil counts were also higher. Evidences showed that non-type 2 in ammation (with/without type 2 in ammation) is likely to contribute to severe asthma, particularly the neutrophilic airway in ammation driven by type 1 responses and IFN-γ [40]. Since there was no signi cant difference in serum total IgE levels, number of sIgE positivity or eosinophil counts among the three groups, indicating that the type 2 in ammation was at a similar level.
Hence, neutrophilic in ammation could be a major factor that increases asthma severity.
Correlation analysis showed associations between serum total IgE levels and house dust mite sIgE levels.
Since the majority of our study subjects were positive for mites, the markedly increased total IgE levels may partly attribute to the increase of house dust mite sIgE. Additionally, we found positive correlation between asthma severity and A. fumigatus speci c IgE levels. There is an entity of worse controlled asthma namely severe asthma with fungal sensitization (SAFS). Patients with SAFS could have similar symptoms as ABPA, even though they did not meet the diagnostic criteria of ABPA. In asthma patients sensitized to fungi, the long lasted in ammation caused by fungi leads to increase of Th2 immune responses, and as a result, the severity of asthma increases, which could even develop ABPA [16,17]. In fact, our result of optimal scaling also showed that A. fumigatus speci c IgE levels were related to asthma severity, and both of them were at the opposite site of lung function data in the scale, suggesting a negative correlation between these two sets of parameters. Therefore, management of these asthma patients should be focus on those sensitized to fungal allergens to prevent from developing severe asthma or ABPA.
There were several limitations in our study. First, the inclusion criteria was set as total serum IgE > 1000 kU A /L, we cannot compare with patients with lower IgE levels. Second, this study was conducted in a single center in Southern China. Thus, our results may not be representable to other populations. Third, the allergen screening may not be comprehensive. However, we have screened for suspicious allergens of each patient based on their medical history and living environment. Last but not least, the cause of the rise of IgE level was not fully explained. Further studies are required to determine the underlining mechanism of markedly increased serum IgE level in asthma patients.

Conclusion
In conclusion, in asthma patients with markedly increased serum total IgE levels (> 1000 kU A /L), the most common two complications were airway infections and rhinosinusitis despite of sensitization status. The majority were sensitized to mite allergens and house dust mite speci c IgE levels partly contributed to the remarkably raised total IgE levels. In addition, A. fumigatus speci c IgE levels were closely associated with total IgE levels and asthma severity.   Optimal scale analysis of clinical data in asthma patients with total IgE >1000 kUA/L. A closer distance between the dots indicated a stronger correlation of the corresponding data (Cronbach's α = 93.7%