A pilot study to evaluate the role of circulation CD4+ CCR6+ CRTh2+ cell in predicting risk of asthma in wheezing children

Background Wheezing is common in younger children and often related to viral infection. It is lack of reliable indicators for asthma prediction. Objective To evaluate the relationship between circulation CD4+CCR6+CRTh2+ memory Th2 cells and asthma diagnosis in wheezing children. Methods A prospective study was performed in children under 5 years old presented with wheezing or at last one episode of documented wheezing history. After inclusion, the level of serum allergen-specific serum IgE (sIgE) and circulating CD4+CCR6+CRTh2+cells were detected. The patients’ personal and family histories of allergic disease were acquired by questionnaire. The children were followed up over 2 years. Diagnosis of asthma was assessed at the end follow-up. The risk factors in predicting asthma diagnosis were evaluated. Results A total of 43 children completed follow-up. Higher wheezing frequency were found in children with asthma diagnosis. The mean of circulating CD4+CCR6+CRTh2+cells in children diagnosed with or without asthma was 1.6 %±0.8 and 0.8 %±0.6 %, respectively, and was significantly higher in children diagnosed with asthma (p < 0.01). There was no significant difference between children with and without allergic diseases history or family allergic diseases in level of circulating CD4+CCR6+CRTh2+ cells. Logistic regression analysis indicated that circulating CD4+CCR6+CRTh2+ cells (EXP, 8.986; 95 % CI,1.886–42.816) and wheezing frequency(EXP, 0.127; 95 % CI, 0.023–0.703)were high risk factors for asthma. Conclusions Our exploratory study shown that circulating CD4+CCR6+CRTh2+ memory Th2 cells increased in asthma diagnosed children and it was a high-risk factor for asthma. Detection of this type of cells could be helpful in predicting the risk of asthma in wheezing children.


Background
Asthma is a common chronic inflammatory disease of the airway in children. The most typical symptom of asthma is recurrent wheezing. However, wheezing is common in younger children which is commonly related to acute respiratory tract infection. The Modified Asthma Predictive Index (mAPI) is a practical method commonly used in the clinic; however, its sensitivity and specificity are limited. Therefore, it is of great clinical value to explore biological indicators based on the immunology of bronchial asthma to guide the diagnosis and treatment of asthma more effectively.
Pathogenic memory type Th2 cells play an important role in allergic airway inflammation. CD4 + memory T cells can not only cause or aggravate allergic airway inflammation by inducing eosinophilic aggregation, mucus hypersecretion [1] and IL-17 secretion [2], but also by inducing airway hyperresponsiveness without eosinophil accumulation [3]. Conversely, inhibition of memory Th2 cells can alleviate allergic airway inflammation and improve the symptoms of asthma [4]. Those studies suggest that memory Th2 cells reflect previous allergic inflammation and can distinguish wheezing caused by non-allergic inflammation versus an immunological mechanism. CD4 + CCR6 + CRTh2 + cell is an important pathogenic memory type Th2 cells which is mainly resident in airway [5].
In this study, a cohort of wheezing children was established. The patient and family histories of allergic diseases were recorded in detail. In addition, the proportion of circulating CD4 + CCR6 + CRTh2 + memory Th2 cells were measured, and the wheezing children were followed up for 2 years to confirm whether they were diagnosed with asthma. Statistical methods were used to determine the correlation between the level of circulating CD4 + CCR6 + CRTh2 + memory Th2 cells and a diagnosis of asthma, thereby providing a theoretical basis for the early diagnosis of asthma in children.

Subject characteristics and demographics
A total of 45 children were enrolled in this study. Among them, 43 cases (95.6 %) underwent blood tests and completed follow-up. 14 (32.6 %) of the included patients were girls. The median age of children at followup was 3.0 years and the age distribution was as follows: 21 cases were aged under 3-year-old (48.8 %) and 22 cases were 3-5 years old (51.2 %). 22 cases (51.2 %) presented lower respiratory tract infection (LRTI). 10 cases (23.3 %) of the children used systemic glucocorticoid because of wheezing. A total of 76.7 % of the subjects had other allergic diseases. 32 cases (74.4 %) positive for at least one food or inhalants sIgE. 23 cases (53.5 %) had a family history of allergic diseases. 36 cases (83.7 %) had more than two wheezing episodes at the end of followup (recurrent wheezing). 24 cases (55.8 %) accepted inhaled corticosteroids or montelukast therapy for more than 3 months. Characteristics of the patients are listed in Table 1. There was no significant difference between children with or without asthma diagnosis in age and sex (p > 0.05) ( Table 2).
Similar personal atopic features and family allergic diseases history between children with or without asthma diagnosis, but higher positive rate of loose API in children with asthma diagnosis The percentage of AD and AR in asthma diagnosis group and without asthma diagnosis group were 65.4 %, 52.9 and 57.7 %, 41.2 % respectively (p > 0.05). There was no significant difference between those two groups in percentage of AR or AD. There was also no significant difference in family history of allergic diseases between the asthma (65.4 %) and the non-asthma (35.3 %) groups (p > 0.05) The positive rates of inhaled-allergen-specific IgE and food allergen-specific IgE in asthma group were 69.6 and 65.2 %, respectively, while the positive rates in non-asthma group were 47.1 and 47.1 %, respectively. The serum level of total IgE in wheezing children with or without asthma diagnosis were 46 (8.6, 204.0) IU/ml and 192.5 (60.9, 805.0) IU/ml, respectively. There was no significant difference in the presence of inhaled allergen-specific IgE (p > 0.05), food allergen-specific IgE (p > 0.05) and total IgE (p > 0.05) between the two groups. The positive rate of loose API in asthma diagnosis group and without asthma diagnosis group were 88.5 and 52.9 %, respectively. The positive rate of loose API was higher in asthma diagnosis group than without asthma diagnosis group (p < 0.05) ( Table 2).
The duration of wheezing onset to enroll in asthma diagnosis group and without asthma diagnosis group were 0.9(0.0,1.5) years and years 2.1(0.4, 4.8) years, Controller Therapy a n (%) 24 (55.8) a Controller Therapy: treated with ICS or montelukast for more than 3 months respectively. There was no significant difference between those two groups (p > 0.05) ( Table 2).
Similar positive rate of AD or AR history in children with or without recurrent wheezing Thirty-six cases (83.7 %) had more than two wheezing episodes (recurrent wheezing) at the end of follow-up. 28 of 36 (77.8 %) cases with recurrent wheezing had either AD or AR history, whereas 5 of 7 (71.4 %) cases without recurrent wheezing had either AD or AR history. There was no significant difference between those two groups (p > 0.05, Pearson Chi-Square test).
Higher circulation CD4 + CCR6 + CRTH2 + memory Th2 cells were associated with asthma diagnosis but not with AD or AR history CD4 + CCR6 + CRTH2 + cell is regarded as one type of pathologic memory Th2 cell which mainly resident in lung tissue. To investigate the difference of this type of memory Th2 cells between wheezing children diagnosed with or without asthma, peripheral blood CD4 + CCR6 + CRTH2 + memory Th2 cells were measured by flow cytometer at time of inclusion. Data were analyzed in CD4 + subset using CytExpert software. The results were used to correlate with diagnosis after the 2-year follow-up. In wheezing children diagnosed with asthma, the level of peripheral blood CD4 + CCR6 + CRTH2 + was 1.6 %±0.8 %, while the level in children without asthma was 0.8 %±0.6 %.There was a significant difference in the level of CD4 + CCR6 + CRTH2 + memory Th2 cells children with asthma compared with the non-asthma group (p < 0.01) ( Table 2; Fig. 1).
We further explored the relationship between the level of CD4 + CCR6 + CRTh2 + memory Th2 cells and history of allergic diseases such as AR and AD. There was no significant difference in the level of CD4 + CCR6 + CRTh2 + cells between the AR group and the non-AR group(1.3 %±0.6 and 1.2 %±0.8 %) (p > 0.05), as well as between the AD group and the non-AD group(1.2 %±0.8 and 1.3 %±0.8 %) (P > 0.05, Fig. 1). The level of CD4 + CCR6 + CRTh2 + memory Th2 cells and wheezing frequency were effective indexes for the diagnosis of asthma in wheezing children Binary logistic regression analysis and the Wald X 2 test were performed to identify the effective indexes for the diagnosis of asthma. We first analyzed several factors could affect the outcome of asthma diagnosis during follow-up using Pearson Chi-Square test and t-test (listed in Table 2). We found that there were significant differences in wheezing frequency,level of CD4 + CCR6 + CRTh2 + memory Th2 cells and API between children diagnosed with asthma and children with non-asthma outcomes. Binary logistic regression analysis and the Wald X 2 test of each regression  (Fig. 2). The cut-off value for CD4 + CCR6 + CRTh2 + memory Th2 cells is 0.575 %.
The level of CD4 + CCR6 + CRTh2 + memory Th2 cells displayed higher specificity and PPV in predicting asthma diagnosis We calculated NPV and PPV for API and CD4 + CCR6 + CRTH2 + memory Th2 cells level in predicting asthma diagnosis. We regard the cases whose circulation CD4 + CCR6 + CRTh2 + cells level was higher than cut-off value as positive cases. The sensitivity, specificity, PPV and NPV of API in predicting asthma outcome were 88.5 %, 47.1 %, 71.9 and 72.7 % respectively. The same parameters of CD4 + CCR6 + CRTh2 + cells were 84.6 %, 76.5 %, 84.6 and 76.5 % respectively. The results indicated that circulation CD4 + CCR6 + CRTh2 + memory Th2 cells displayed higher specificity and PPV in predicting asthma diagnosis (Table 4).

Discussion
At present, the diagnosis and treatment of asthma under 6 years of age is mainly based on the risk of asthma. mAPI is a practical tool to evaluate the risk of asthma in clinic [6][7][8]. However, the positive predictive rate of mAPI usually around 60-70 % [9][10][11] which means 30-40 % of wheezing children with atopic feature will not develop asthma although atopy is a high-risk factor for asthma. In current study, we evaluated potential risk factor for asthma diagnosis. First, we evaluated atopic features such as serum sIgE, total IgE, patient allergic disease history and family allergic disease between asthma diagnosed and without asthma diagnosed wheezing children since allergic diseases and family allergic diseases were used as an important indicator in evaluating the risk of asthma. In this study, 76.7 % of patients had history of AR or AD, 74.4 % of patients were positive for inhalants sIgE or food sIgE, which meant most of those patients had atopic feature. We found that none of those factors were significant difference between asthma diagnosed and without asthma diagnosed groups. Those results indicated that the value of above single factors in prediction of asthma in highly atopic children may limited. On the contrary, we compared wheezing profiles between asthma diagnosed and without asthma diagnosed wheezing children. We found that those diagnosed with asthma shown higher wheezing frequency during follow-up. Thus, wheezing frequency was more valuable in predicting of asthma diagnosis in atopic children. API is a practical tool for management younger wheezers in clinics. In this study, we evaluated loose API in predicting future asthma risk since part of participants had less than four wheezing episodes. The sensitivity and specificity of loose API were 88.5 and 47.1 %, respectively, indicated that API showed insufficient specificity in predicting asthma risk.
Next, we compared the difference of circulation CD4 + CCR6 + CRTh2 + memory Th2 cells between those two groups. We found that the level of CD4 + CCR6 + CRTh2 + memory Th2 cells in the asthma diagnosed group was significantly higher than those in the non-asthma diagnosed group. Logistic regression analysis further indicated that CD4 + CCR6 + CRTh2 + memory Th2 cells and wheezing frequency were the independent risk factor for the outcome of asthma diagnosis. As compared to loose API, circulation CD4 + CCR6 + CRTh2 + memory Th2 cells had better efficiency in predicting risk of asthma diagnosis based on higher specificity, higher PPV and larger area under the ROC curve.
Interestingly, there was no significant difference in the proportion of circulation CD4 + CCR6 + CRTh2 + memory Th2 cells in children with AR or AD history. These results suggest that the increase in circulation CD4 + CCR6 + CRTh2 + memory Th2 cells seemed specifically related to asthma but not AR or AD. Basic research indicated that CD4 + CCR6 + CRTh2 + memory Th2 cell is a type of pathogenic memory-type Th2 cell. Pathogenic memory-type Th2 cells are mainly distributed in local tissues for long periods of time and play key roles in the maintenance of chronic allergic inflammation [5,12,13]. CD4 + CRTH2 + CCR6 + cells were mainly found in the airway. CD4 + CRTH2 + CCR6 + cells secrete IL-17, IL-4 and IL-13, leading to the infiltration neutrophils and eosinophils, which is responsible for chronic inflammation associated with asthma. A small number of pathogenic memory-type Th2 cells could enter the circulation [5,12]. Therefore, increasement of circulating CD4 + CRTH2 + CCR6 + memory Th2 cells may reflect the chronic airway allergic inflammation, an important immunological feature of asthma.
In summary, despite of small sample size, our exploratory study shown that higher wheezing frequency, but not history of allergic diseases or family allergic diseases were related to asthma diagnosis after 2 years follow-up in wheezing children who had atopic features. Moreover, circulation CD4 + CCR6 + CRTh2 + memory Th2 cells, which have certain lung tissue specificity, was increased in asthma diagnosed children and was risk factor for the outcome of asthma diagnosis. Detecting of CD4 + CCR6 + CRTh2 + memory Th2 cells in BALF would be more valuable in predict future asthma since those cells are mainly resident in airway. However current study had its limitation in sample size and short follow-up duration, so expansion of the sample size and prolonging the follow-up time is required to further validate the current results.

Conclusions
Our exploratory study shown that circulating CD4 + CCR6 + CRTh2 + memory Th2 cells increased in asthma diagnosed children and it was a high-risk factor for asthma. Detection of this type of cells could be helpful in predicting the risk of asthma in wheezing children.

Study design and subjects
We recruited forty-three children under five years old who presented with recurrent wheezing or lower respiratory tract infection in respiratory ward or in clinic of

Follow up
The included children were followed up for 2 years in clinic. Patients required controller therapy visited clinic monthly. Otherwise, patients visited clinic when they had respiratory symptoms. All patients visited clinic at least once a year and received an annual telephone interview with their parents through questionnaires covered the frequency of wheezing episodes, pediatric respiratory specialists diagnosed asthma and medication for recurrent wheezing. Asthma diagnosis was made by pediatric respiratory specialists according to guidelines for the diagnosis and management of asthma in children (2016) which developed by the Chinese Pediatric Society, Chinese Medical Association [14] and Global Strategy for Asthma Management and Prevention(2020)(Chap. 6, diagnosis and management of asthma in children 5 years and younger). For children older than five years old, asthma diagnosis was based on typical respiratory symptom, lung function test and FeNO. For children less than five years old, asthma diagnosis was evaluated at the end of 2-year-follow-up by recurrent wheezing, response to bronchodilator, history of allergic disease, allergen sensitization, asthma in first-degree relatives and clinical improvement during 3 months of controller treatment (ICS or montelukast). Since part of participants had less than four wheezing episodes, loose API was used to predict future risk of asthma. Loose API (asthma predictive index) were assessed according to detailed questionnaires, medical record information, serum sIgE, blood eosinophilic cell count. Participants with one major or two minor risk factors were defined as API-positive, otherwise were defined as API-negative. Briefly, physician-diagnosed atopic dermatitis, parental asthma, and sensitization to inhalant allergen were defined as major risk factors. Minor risk factors included sensitization to food allergens, eosinophil levels in the peripheral blood eosinophilic cell ≥ 4 %, and wheezing unrelated to a cold.

Statistical analysis
All statistical analysis was carried out using SPSS v. 22.0. Continuous variables were represented by mean ± SD or median (interquartile range, IQR). Classification variables were represented by frequency. T test was used to compare the normally distributed data. Mann-Whitney rank sum test was used to compare the non-normally distributed data. Chi-Square test was used to compare the difference of qualitative variables between children diagnosed with or without asthma. Binary logistic regression analysis and the Wald X 2 test was used for correlation analysis. The area under the curve was calculated by the ROC of the subjects. Bilateral significance level was set at P < 0.05.