Effects of sublingual immunotherapy for dust mite on Th 17 / Treg cells in children with asthma

Effects of sublingual immunotherapy for dust mite on Th 17 / Treg cells in children with asthma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of sublingual immunotherapy for dust mite on Th 17 / Treg cells in children with asthma Fang Li, Kun Wang, Xue Wang, Wei Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6979274/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: observing the clinical effects of dust mite sublingual immunotherapy (SLIT) on children with asthma, changes of Th17 / Treg cells and related cytokines in order to investigate the possible pathological mechanism of immune tolerance induced by SLIT. Methods: Sixty children with asthma allergic to dust mites were included, divided into SLIT group (n=30) and non-SLIT group (n=30). Clinical symptoms of asthma in each group had been scored before, 1 year and 2 years after treatment. Meanwhile we also evaluated the proportion of Th17 and CD4 + CD25 + Treg cells in peripheral blood using flow cytometry. Besides, cell culture supernatant was collected to detect the changes of IL-6, IL-10 and IL-17 levels. Results: We found that in SLIT group, asthma symptom and drug use score, Th17 cell percentages, IL-6 and IL-17 levels have significantly decreased throughout the study period (p < 0.05), while FEV1%, Treg cell percentages and IL-10 level have prominently increased throughout the study period (p < 0.05). By contrast, in non-SLIT group, asthma symptom score, lung function, Th17 cell percentages, IL-6 and IL-17 levels have all significantly improved, but on the whole lower than SLIT group (p < 0.05). However, we have observed no statistical differences in drug use score, Treg cell percentages, IL-10 level for non-SLIT group throughout the study period. Conclusions: SLIT of Dust mite drops could change T immune cell profiles whereas improve asthma symptoms. SLIT might reverse the functional imbalance of Th 17 / Treg cells and induce immune tolerance by upregulating Treg cell function and downregulating Th17 cell function. Dust mite sublingual immunotherapy Th17 cell Treg cell children Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Allergen immunotherapy (AIT) is the only method that can shorten the course of allergic disease ( 1 ) . Currently, studies of AIT mechanism have been focusing on immune tolerance ( 2 ) . However, the mechanism of how AIT induces the developing immune tolerance in T lymphocytes is not fully understood. Th17 / Treg cells are a pair of T lymphocytes with pro-inflammatory / anti-inflammatory effects who participate in maintaining the dynamic balance of the body's immune function ( 3 ) . The role of Th17 / Treg cells is also increasingly appreciated in asthma whose functional imbalance may lead to the occurrence and further development of asthma ( 4 ) . Our previous study has discovered the dysfunction of Treg cell in children who have bronchial asthma allergic to house dust mites, while the interesting point is that AIT can upregulate its function to exert therapeutic effects, yet the mechanism of this process has not been further examined ( 5 ) . We also have proved the presence of the vast majority of eosinophil accumulation in children with bronchial asthma allergic to dust mites and an imbalance of Th17 / Treg cell function in these children. Whether AIT can induce immune tolerance in children with bronchial asthma by reversing the Th17 / Treg cell function imbalance is unknown. The role of Th17/Treg cells is also increasingly appreciated in asthma, which is a hot issue in current research.There is no report about the changes of Th17 / Treg cells and their related cytokines in bronchial asthma children allergic to dust mite allergy after SLIT treatment over 2 years. Therefore, in this study, we aim to observe the effects of dust mite SLIT on children with asthma and changes of Th17 / Treg cells percentages as well as related cytokines to investigate the possible pathological mechanism of immune tolerance induced by SLIT. Patients and methods Patients Children with mild and moderate non-acute onset bronchial asthma were finally selected for the study. Each child would need to meet the following inclusion criteria: 1) diagnosed with mild to moderate allergic asthma according to diagnostic criteria for bronchial asthma(GINA 2015) ( 6 ) ; 2) allergic to dust mite diagnosed by skin prick test, wheal range ≥ ++, specific IgE ≥ 0.7Ku/L; 3) if child was also allergic to other allergens, such as spring and autumn pollen, cat, dog and animal fur or Alternaria , non-dust mite allergens must be <++ identified by skin prick test; 4) no other cardiovascular diseases, autoimmune diseases, or other underlying diseases. 5) None of the children had used systemic corticosteroids and other immunosuppressants, nor had they used antihistamines. The exclusion criteria: 1) diagnosed with chronic lung diseases, such as bronchopulmonary dysplasia, pulmonary fibrosis, congenital abnormal lung development and so on; 2) diagnosed with an immunodeficiency disease; 3) diagnosed with other serious illnesses; 4) diagnosed with severe bronchial asthma; 5) diagnosed with contraindications to pediatric pulmonary function tests. They were randomly divided into SLIT and non-SLIT groups according to the random number table method. Guardians of all children had provided the informed consent. Meanwhile, this study was approved by the Ethics Committee of the Second Hospital of HeBei Medical University. Treatment administration method Patients were treated with conventional treatment for asthma or plus SLIT over 2 years. The treatment process consisted of a dose-escalation phase and a maintenance phase for 2 years. They were evaluated before the treatment and at the end of each year. The SLIT group was given sublingual dust mite drops (Zhejiang Wolwo Biological Technology Co., Ltd, Deqing, Zhejiang Province, China) numbered 1 to 4. No. 1, 2, and 3 which would be administered in weeks 1, 2, and 3 individually, with daily doses of 1, 2, 3, 4, 6, 8, and 10 drops. From the 4th week, the No. 4 would be given 3 drops once a day until the end of 2-year-treatment. The drops are swallowed after sublingual administration for 1 ~ 3 min at night at same time under the care of parents, and oral food intake was only allowed for 15 min after each administration until the end of the course of treatment. All children treated with no systemic corticoids, other immunosuppressants and no anti-allergic drugs within 4 weeks before blood collection. Both groups had the same basic medication, and were given with small to moderate doses of inhaled corticosteroids (Budesonide inhalant), without combining long-acting antihistamines and antileukotriene drugs. Reagents and equipment The fluorescein-isothiocyanate (FITC) mouse anti-human CD4, mouse anti-human (PE Alexa Fluor 700), Alexa Fluor 647 anti-human IL-10, APC anti-human IL-17, PE anti-human Foxp3, anti-human CD25, human IL-6, human IL-10, human IL-17 ELISA kits were all purchased from eBioscience, Inc (San Diego, CA, USA). Cytofix/Cytoperm solution, lymphocyte separation medium and RPMI-1640 medium were bought from Gibco (USA). The flow cytometer (FACSCantoII) was purchased from BD Biosciences (New Jersey, USA); Automatic allergen detector (Uni- CAP100) was purchased from Pharmacia (Uppsala,Sweden); the CO 2 thermostatic incubator(MCO-175) was purchased from SANYO (Osaka, Japan); Fully automatic microplate reader (Multiskan MK3) was purchased from Thermo Fisher Labsystems (USA). Standard for clinical asthma symptom score and asthma drug use score Evaluation was performed with night and morning asthma symptom scores ( 7 ) . The night symptoms would be scored from 0 to 4 points as following: “0” for no symptoms; “1” for early or one arousal during sleep; “2” for early and more than two arousals during sleep; “3” for many arousals during sleep; “4” for no sleep at night. Daytime symptoms were scored as following: “0” for no symptoms; “1” for a little symptoms and short duration; “2” for more than twice very short symptoms; “3” for mild symptoms occurred at more times but with less affects to study and life; “4” for more time and more symptoms with affects on the study and life; “5” for the symptoms are serious and the children cannot live and study normally. The higher score indicated more severe asthma symptoms. Asthma drug use score own a total score of 7 points as below in which these following drugs could be used in combination according to the child's condition. “1” for the β2 receptor agonists or leukotriene receptor antagonists, individually; “2” for inhaled corticosteroids; “3” for inhalational glucocorticoids corticosteroids (ICS) combined with a long-acting β2 receptor agonist (LABA). Cell isolation and culture The fasting peripheral venous blood (5 mL) would be drawn from each subject before treatment, 1 year and 2 years of treatment. The blood would be placed in sterile heparin tubes separated by Ficoll density-gradient centrifugation to isolate the PBMCs. Then cells were supposed to be washed with Hank’s solution for three times and resuspend in RPMI-1640 medium with 10% fetal bovine serum (FBS). The PBMCs would be then stimulated with dust mite leaching solution at dose of 20 µg/mL for 48 hours. The cell culture supernatant would be obtained to evaluate IL-6, IL-10 and IL-17 levels by ELISA, and cells would be collected to analyze the Th17 cells (CD4 + IL-17 + T cells) and Treg cells (CD4 + CD25 + FOXP3 + IL-10 + T cells) percentages. Statistical analysis Data is analyzed using the SPSS24.0 software. All sets of measurement data are expressed as mean ± standard deviation ( ± S). The Chi-Square test (χ 2 test) is used for the comparison of categorical data. For the comparison between groups (SLIT and non-SLIT groups), t-test is performed to compare data with normally distributed variance alignment, while the unpaired Mann-Whitney U test is used for non-normal distribution or uneven variance, and paired multi-sample Friedman-test is employed for within-group (pre-treatment, 1 year of treatment, 2 years of treatment) comparisons. P < 0.05 is considered as statistically significant. Results Subject characteristics A total of 76 children with bronchial asthma were initially included in this study, but 6 cases were lost in the non-SLIT group, of whom 4 were lost to follow-up and 2 voluntarily withdrew from the study for personal reasons. The 10 cases were lost in the SLIT group, of whom 4 were lost to follow-up and 7 voluntarily withdrew from the study for personal reasons. Finally, 60 cases were included and randomly divided into SLIT and non-SLIT groups with 30 patients in each group. In the SLIT group, 19 boys and 11 girls were aged 9.22 ± 2.64 years. In the non-SLIT group, 18 boys and 12 girls were aged 8.90 ± 2.44 years. There were no differences in the two groups in age, sex, severity, duration, lung function, presence, associated disease, dust mite specific IgE level, and positive allergen distribution of the skin prick test results (See Figure.1, Tables 1 and 2 ). Table 1 General information of children SLIT group non-SLIT group t / χ 2 value P value Cases 30 30 Gender (Male/ Female) 19/11 18/12 0.071(χ 2 ) 0.791 Age (Year) 9.22 ± 2.64 8.90 ± 2.44 0.482 (t) 0.631 Disease course (Year) 4.73 ± 1.19 4.10 ± 1.99 -1.357 (t) 0.098 Dust mite specificity IgE (KU/mL)※ 134.65 ± 142.52 138.70 ± 106.64 -0.99 (t) 0.322 Family history(N/Y) 16/14 19/11 0.617 (χ 2 ) 0.432 Rhinallergosis (N/Y) 9/21 11/19 0.300 (χ 2 ) 0.584 Allergic dermatitis (N/Y) 19/11 21/9 0.300 (χ 2 ) 0.584 Data are expressed as the mean ± standard deviation, ※ P < 0.05, was considered to be statistically significant difference. Table 2 Distribution of allergen skin prick test results in the children SLIT group Non-SLIT group χ 2 value P value Cases 30 30 Family history (N/Y) 16/14 19/11 0.617 0.432 Rhinallergosis (N/Y) 9/21 11/19 0.300 0.584 Allergic dermatitis (N/Y) 19/11 21/9 0.300 0.584 House dust (N/Y) 25/5 24/6 0.111 0.739 Mould Ⅱ (N/Y) 23/7 26/4 1.002 0.317 Alternaria (N/Y) 25/5 26/4 0.131 0.718 Dog hair (N/Y) 22/8 23/7 0.089 0.766 Cat epithelium (N/Y) 20/10 23/7 0.739 0.390 Weed pollen (N/Y) 20/10 17/13 0.635 0.426 Ragweed (N/Y) 17/13 15/15 0.268 0.605 Artemisia argyi (N/Y) 16/14 18/12 0.271 0.602 Hay dust (N/Y) 26/4 25/5 0.131 0.718 ※ P < 0.05, was considered to be statistically significant difference Asthma symptom scores Mean asthma symptom scores in the SLIT group were respectively 2.72 ± 0.45, 2.12 ± 0.45 and 1.52 ± 0.46 in before treatment, 1 year and 2 years after treatment. In addition, the scores obviously decreased throughout the research period (F = 50.334, p < 0.01). Although the mean asthma symptom scores have shown the decline in the non-SLIT group, we found no statistically different changes until treatment after 2 year compared with before treatment (F = 39.800, p < 0.01). Intergroup comparison has revealed that the range ability of asthma symptoms scores in the SLIT group was remarkably higher than that of non-SLIT group (p < 0.05). See Figure.2. Asthma drug use scores Mean asthma drug use scores in the SLIT group were respectively 5.18 ± 0.47, 4.13 ± 0.62 and 3.27 ± 1.02 in before treatment, 1 year, 2 years after treatment. And the scores significantly declined throughout the research period (F = 60.000, p < 0.01). The mean drug use scores appeared to decline in the non-SLIT group, but the changes had no statistically significant differences (F = 4.467, p = 0.107). Intergroup comparison has revealed that the change amplitude in the SLIT group was significantly greater than non-SLIT group (p < 0.01). See Figure.3. Lung function Mean FEV 1% in the SLIT group were 82.83 ± 12.17%, 95.47 ± 11.68% and 102.83 ± 12.17%, before treatment, and 1 year, 2 years after treatment, respectively. And FEV 1% significantly increased over the treatment period (F = 34.889, p < 0.01). FEV 1% increased significantly in the non-SLIT group after 2 years of drug treatment compared with before treatment (F = 27.042, p < 0.01). It has shown that the change amplitude was significantly higher in the SLIT group than non-SLIT group by intergroup comparison (p < 0.01). See Figure.4. Decreasing of Th17 cells percentages after treatment To determine the effects of SLIT on the Th17 cells proportion in PBMCs, the FACs analysis was conducted to identify and quantify CD4 and cells secreting IL-17. Percentages of Th17 cells in SLIT group were 4.90 ± 0.96, 3.15 ± 1.07 and 2.68 ± 1.16 before treatment, 1 year and 2 years after treatment, individually. Th17 cell percentage gradually declined throughout the research period in SLIT group, with significantly differences (F = 47.400, p < 0.01). The percentages of Th17 cells were 4.96 ± 1.87, 4.53 ± 2.07, 4.41 ± 2.12 before treatment, 1 year and 2 year after treatment in non-SLIT group, individually. The percentage of Th17 cell also gradually declined throughout the study period in non-SLIT group (F = 44.826, p < 0.01). While the magnitude of change was obviously higher in the SLIT group than that of the non-SLIT group (p < 0.05). See Figure.5. Increase in the Treg cell percentages after treatment To determine whether SLIT alters the proportion of Treg cells in PBMCs, we further identified and quantified CD4 + CD25 + Foxp3 + IL-10 + T cells by FACs. Its percentages significantly enhanced throughout the research period in SLIT group (F = 40.267, p < 0.01), from 1.27 ± 0.37 at baseline to 1.89 ± 0.38, 2.11 ± 0.35 at 1 year and 2 years after treatment. Although percentage of Treg cell appeared to increase in the non-SLIT group, no statistically significant differences were observed (F = 3.525, p = 0.172). The percentages of Treg cells in the treated SLIT group were significantly higher than non-SLIT group, with a statistically significant difference (P < 0.01). See Figure.6. Changes of IL-6, IL-10 and IL-17 The ELISA was conducted to identify the IL-6, IL-10 and IL-17 levels in supernatants of PBMCs. IL-6 levels in SLIT group were 55.02 ± 9.16, 45.44 ± 8.18 and 36.62 ± 6.81 pg/mL before treatment, 1 year and 2 years after treatment, respectively. It gradually declined in SLIT group over the study period, with significantly differences (F = 50.400, p < 0.01, Figure.7). In contrast to IL-6, IL-10 levels were significantly increased throughout the study period in SLIT group (F = 49.043, p < 0.01), which were 44.05 ± 9.41, 48.84 ± 9.21 and 56.12 ± 8.15 pg/mL before treatment, 1 year and 2 years after treatment, respectively (Figure.8). While IL-17 levels were 6.46 ± 1.15, 5.11 ± 1.08 and 3.41 ± 1.02 pg/mL before treatment, 1 year and 2 years after treatment in SLIT group, respectively, and the differences were statistically significant (F = 59.051, p < 0.01, Figure.9). Although IL-6, IL-10 and IL-17 levels in non-SLIT group also appeared the similar tendency of changes, the magnitude of change was apparently greater in the SLIT group than that of non-SLIT group (all p < 0.01, Figure.7, Figure.8, Figure.9). Discussion The current international consensus is that allergen immunotherapy (AIT) has definite curative effects in allergic rhinitis and allergic asthma. AIT is a therapeutic vaccination used to treat IgE-mediated allergies to common allergens such as pollen, house dust mites, and aspiration insect venom. AIT has two main forms in clinical use: subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). SCIT is the classic form of administration, but the need for subcutaneous administration increases the pain and the risk of serious systemic adverse reactions. However, SLIT is easy to take, and rarely causes serious systemic adverse effects, so it attracts more and more attentions and likely to replace SCIT ( 8 ) . The mechanism of how specific immunotherapy induced immune tolerance in T lymphocytes is not fully understood. It is believed that the main mechanism of SLIT induction of immune tolerance is to promote the conversion of antigen-sensitized CD4 + Th0 cells into Treg cells and Th1 cells and thus inhibit the Th2 cell-associated immune response ( 9 ) . Dust mite drop immunotherapy is a kind of sublingual specific immunotherapy. It is made of dust mite antigen active protein, which can induce the body to generate specific antibodies, then combine with allergens, thus blocking the allergen and basophils and mast cells interaction, block the occurrence of type I allergic reaction, at the same time can inhibit T lymphocytes and eosinophils gathered in the target organ, inhibit degranulation changes in lymphocytes and exert regulatory effects on the immune system ( 10 ) . Th 17 / Treg cells play an important role in maintaining the dynamic balance of immune function in vivo. Under some specific conditions, Treg cells and Th 17 cells can interconvert, such as changes in cytokine concentration ( 11 ) . Th17 cells play an important role in the immunopathogenesis of various autoimmune diseases and tumors. IL-17 is mainly a cytokine secreted by Th 17 cells, and it cannot replicate the asthma model if IL-17 is knocked out. IL-17 is mainly involved in severe asthma and hormone-resistant asthma, which is dominated by neutrophil infiltration ( 12 ) . IL-6 is involved in the proliferation and differentiation process of Th17 cells. It has a key regulatory role in the differentiation of initial CD4 + T cells into Th17 cells and Treg cells ( 13 ) . Binding of IL-6 to cell surface receptors can trigger the lung JAK / STAT3 signaling, thus playing a key regulatory role in Th17 / Treg differentiation ( 14 ) . The activated JAK kinase phosphorylates STAT3, p-STAT3 dimerizes and transplaces into the nucleus to initiate expression of downstream related genes such as RORγt, IL-17A / F, prompting differentiation of initial CD4 + T lymphocytes to Th17. Meanwhile, STAT3 can also promote the methylation of the CpG island of the enhancer upstream of FOXP3 gene and inhibit its expression. SOCS1 and SOCS3 negatively regulate IL-6 / STAT3 signaling. Animal experiments found that downregulation of IL-6 in the respiratory tract achieved good results in treating asthmatic animals ( 15 ) . Clinical experiments found that the effect of inhaled corticosteroids in asthma was related with downregulation of IL-6 levels in serum ( 16 ) . Treg cells are a group of cells that exert a negative regulatory effect on the cellular immune function, among which the FOXP3 protein is its most important transcription factor. Treg cells mainly inhibit the T cell-mediated inflammation dominated by eosinophil infiltration in the respiratory tract, thus reducing the respiratory allergic reaction and preventing the development of asthma ( 17 ) . The Treg cells predominantly secrete IL-10 and TGF-β. IL-10 may have an immune dual effect, which promotes the development of inflammatory responses rather than anti-inflammatory under some conditions, and its dual function is not only dependent on the target cells, but also on the concentration of IL-10 ( 18 ) . IL-10 can affect the function of T cells at different stages of inflammation. Moreover, IL-10 was also able to inhibit the differentiated in Th 17 cells ( 19 ) . The imbalance of Th17 / Treg cell function plays an important role in the development of bronchial asthma, Hu Y et al reported that differentiation of Th 17 / Treg cells is implicated in the pathogenesis of allergic asthma ( 20 ) . They found a significant Th 17 / Treg imbalance in the child with bronchial asthma, in which the percentage of Th 17 cells was significantly increased while the percentage of Treg cells was significantly reduced, and the IL-17 levels were significantly increased while the IL-10 levels were significantly reduced. However, Katrien Van der Borgh et al treated the mouse model of bronchial asthma allergy with house dust mite (HDM) with prophylactic SLIT treatment, and they found the SLIT treatment could increase the levels of treg and Th 17 in the lungs of the asthma model mice ( 21 ) . Thus, the role of Th 17 / Treg in SLIT is inconclusive, and it is unclear whether SLIT can play a role in immune tolerance by reversing the imbalance of Th17 / Treg cell function. Therefore, we aimed to preliminarily explore the mechanism of SLIT by observing the changes in Th17 / Treg cells and corresponding cytokine levels in bronchial asthma children allergic to dust mite receiving SLIT. Our study found that asthma symptom score and drug use score Th17 percentages, IL-6 and IL-17 levels were significantly decreased in SLIT group throughout the study period (p < 0.05). In contrast, FEV1%, Treg cell percentages and IL-10 level significantly enhanced in SLIT group throughout the study period (p < 0.05). Asthma symptom score, lung function, Th17 cell percentages, IL-6 and IL-17 levels all significantly improved in non-SLIT group even all lower than SLIT group (p < 0.05). But no statistical difference was observed in drug use score, Treg cell percentages and IL-10 level for non-SLIT group. Our study found that Treg levels remained at a high level after 2 years of sublingual specific immunotherapy, which might be related to the child still in desensitization treatment. It was reported that the optimal treatment time of SLIT was 3 years, and it was considered that it can maintain better long-term efficacy after 3 years of drug withdrawal ( 22 ) . The 2-year SLIT course did not maintain the long-term effect. We will further explore the changes of Th 17 / Treg cells and their associated cytokines levels in children after the cessation of treatment to evaluate the long-term efficacy of specific immunotherapy. The results showed that Th17 / Treg cell function imbalance existed in children with bronchial asthma allergic to eosinophil accumulation, especially Treg cell dysfunction. Although drug treatment can improve the clinical symptoms and the level of some cytokines in children, it cannot reduce the use of asthma drugs and upregulate Treg cell function. SLIT can upregulate Treg cell function and downregulate Th17 cell function to reverse the imbalance of Th17 / Treg cell function and induce immune tolerance. However, whether it reverses Th17 / Treg cell function by inhibiting IL-6 secretion needs further investigation. Conclusion In conclusion, Our study proved that SLIT of Dust mite drops could alter T immune cell profiles and improves asthma symptoms. And SLIT might reverse the functional imbalance of Th 17 / Treg cells to induce immune tolerance by upregulating Treg cell function and downregulating Th17 cell function. Declarations Ethics approval and consent to participate This study was approved by the Institutional Review Board of Ethics Committee of the Second Hospital of Hebei Medical University. Written informed consent was obtained from all participants. Consent for publication All participants provided written consent for the publication of their data. Conflict of Interest The authors declare that there are no competing interests. Funding Not applicable Authors' contributions Fang Li: Research design and writing Wei Wang: Research design, experiment design and manuscript revision Kun Wang：Data acquisition Xue Wang: Data processing and analysis Acknowledgements This research received no external financial or non-financial support. Authors' information Fang Li: Department of pediatrics, The Second Hospital of Hebei Medical University Associate clinical professor, Master e-mail: [email protected] Telephone and fax numbers：0311-95658371 Department of Pediatrics, The Second Hospital of Hebei Medical University, Shijiazhuang, China Kun Wang： Department of pediatrics, Hebei Medical University Third Hospital Clinical professor, Doctor e-mail: [email protected] Telephone and fax numbers：0311-88603060 Department of Pediatrics, Hebei Medical University Third Hospital, Shijiazhuang, China Xue Wang： Department of pediatrics, The Second Hospital of Hebei Medical University Associate clinical professor, Master e-mail: [email protected] Telephone and fax numbers：0311-95658371 Department of Pediatrics, The Second Hospital of Hebei Medical University, Shijiazhuang, China Wei Wang (Corresponding Author): Department of pediatrics, Hebei Medical University Third Hospital Clinical professor, Doctor e-mail: [email protected] Telephone and fax numbers：0311-88603060 Department of Pediatrics, Hebei Medical University Third Hospital, Shijiazhuang, China References Shamji MH, Sharif H, Layhadi JA, Zhu R, Kishore U, Renz H. 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Front Immunol. 2022;13:862866. Pavón-Romero GF, Parra-Vargas MI, Ramírez-Jiménez F,et al.Allergen Immunotherapy: Current and Future TrendsCells. 2022 Jan 8;11(2):212. Daneshvar-Ghahfarokhi S, Rahnama A, Mohammadi-Shahrokhi V. Teucrium polium Extract Attenuates Inflammation in Asthma by Reducing RORgammat Transcription and Increasing IL-10 Secretion in an Ovalbumin-induced Murine Asthma Model. Iran J Immunol. 2023;20(2):159-66. Hu Y, Chen Z, Zeng J, Zheng S, Sun L, Zhu L, et al. Th17/Treg imbalance is associated with reduced indoleamine 2,3 dioxygenase activity in childhood allergic asthma. Allergy Asthma Clin Immunol. 2020;16:61. Van der Borght K, Brimnes J, Haspeslagh E, Brand S, Neyt K, Gupta S, et al. Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells. Mucosal Immunol. 2024; S1933-0219(24)00028-X. Creticos PS, Gunaydin FE, Nolte H,et al，Allergen Immunotherapy: The Evidence Supporting the Efficacy and Safety of Subcutaneous Immunotherapy and Sublingual Forms of Immunotherapy for Allergic Rhinitis/Conjunctivitis and Asthma.J Allergy Clin Immunol Pract. 2024 Jun;12(6):1415-1427. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6979274","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489098643,"identity":"a6469c3f-25db-487a-998e-2c3ce1c849dd","order_by":0,"name":"Fang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBAC+/nPDz6Q4KmRY2NvIFKLAUNOsoGFzDFjfp4DRGtJMJOosGFOnDkjgUgt5gwH0iRu5LAxbrj5eOMNhhqbaIJaLBsbD1vOOCPDbHA7rdiC4VhabgNBPYcZEm9L9rCxGdzOMZNgbDhMhJZjDAbSf/8x8xjcPEOkFoMzDEYSEjzMEpIzeIjUAlSZbCDBc8yAnwfolwRi/MIvwQ6Oyvo29sMbb3yosSHCL8iOlEggRTlEC6k6RsEoGAWjYGQAAMxWPXghimKCAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Fang","middleName":"","lastName":"Li","suffix":""},{"id":489098644,"identity":"30d3098e-ca45-47db-b083-340a5182694f","order_by":1,"name":"Kun Wang","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Wang","suffix":""},{"id":489098645,"identity":"bff851e2-298d-4cf8-9600-5fd8a70af48f","order_by":2,"name":"Xue Wang","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Wang","suffix":""},{"id":489098646,"identity":"46a65290-9002-4b92-9c0d-dbc98c80f5d4","order_by":3,"name":"Wei Wang","email":"","orcid":"","institution":"The Third Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-06-26 03:59:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6979274/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6979274/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87553074,"identity":"1c8ca3e4-58ad-40f6-9d5d-acdb236757a3","added_by":"auto","created_at":"2025-07-25 06:35:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":212430,"visible":true,"origin":"","legend":"\u003cp\u003eCONSORT flowchart. CONSORT, Consolidated Standards of Reporting Trials.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/b02b41b1bb6663ac9482a537.png"},{"id":87552353,"identity":"42dc12fe-c3d5-46f9-94d5-a74ce2f27c72","added_by":"auto","created_at":"2025-07-25 06:27:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":392653,"visible":true,"origin":"","legend":"\u003cp\u003eAsthma symptom scores in SLIT group and non-SLIT group. Mean asthma symptom scores obviously decreased in SLIT group over the study period (F = 50.334, p \u0026lt; 0.01); symptom scores did not differ within the non-SLIT group until treatment after 2 year compared with before treatment (F = 39.800, p \u0026lt; 0.01). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/ea34f7258d94a8277fd0b031.png"},{"id":87552356,"identity":"bc3888ce-03cf-46e3-a28a-9b72777c98df","added_by":"auto","created_at":"2025-07-25 06:27:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":339225,"visible":true,"origin":"","legend":"\u003cp\u003eDrug scores in SLIT group and non-SLIT group. Mean drug scores obviously decreased in SLIT group over the study period (F = 60.000, p \u0026lt; 0.01); drug scores did not differ within the non-SLIT group (F = 4.467, p = 0.107). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/08a061bd313f877d926ee7e5.png"},{"id":87553072,"identity":"0deffdea-22a4-4f64-b48a-7ab64e630917","added_by":"auto","created_at":"2025-07-25 06:35:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":418936,"visible":true,"origin":"","legend":"\u003cp\u003eFEV 1% in SLIT group and non-SLIT group. Mean FEV 1% significantly increased over the treatment period in SLIT group (F=34.889, p \u0026lt; 0.01). And it also increased significantly in the non-SLIT group after 2 years of drug treatment compared with before treatment (F = 27.042, p \u0026lt; 0.01). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/a637c278b9d744e98975c172.png"},{"id":87553071,"identity":"aa367171-b08d-4bc7-8b98-2bd5c700a63d","added_by":"auto","created_at":"2025-07-25 06:35:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":458844,"visible":true,"origin":"","legend":"\u003cp\u003eDecreases in Th17 cells following treatment. FACS was used to detect IL-17-posititive PBMCs in patients of SLIT group and non-SLIT group. Th17 cell percentage significantly declined throughout the research period in SLIT group (F = 47.400, p \u0026lt; 0.01); Th17 cell also gradually declined throughout the study period in non-SLIT group (F = 44.826, p \u0026lt; 0.01).While the magnitude of change was obviously higher in the SLIT group than that of the non-SLIT group (p \u0026lt; 0.05). *p \u0026lt; 0.05, **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/2bc679074436c9f43be00abd.png"},{"id":87554491,"identity":"e5bdea3a-3b11-497f-ac4c-f630781c2188","added_by":"auto","created_at":"2025-07-25 06:43:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":408970,"visible":true,"origin":"","legend":"\u003cp\u003eIncreases in Treg cells following treatment. FACS was used to detect CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e T cells in patients of SLIT group and non-SLIT group. Treg cell percentage significantly increased throughout the research period in SLIT group (F = 40.267, p \u0026lt; 0.01); Treg cell were unchanged throughout the study period in non-SLIT group (F = 3.525, p = 0.172).\u0026nbsp; **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/a8ca8abf9ae39c32ccf60a27.png"},{"id":87552362,"identity":"e492d0a4-eb17-4baa-aac3-9566441d9efc","added_by":"auto","created_at":"2025-07-25 06:27:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":443441,"visible":true,"origin":"","legend":"\u003cp\u003eDecreases in IL-6 following treatment. ELISA was used to identify the IL-6 levels in supernatants of PBMCs in patients of SLIT group and non-SLIT group. IL-6 level significantly declined throughout the research period in SLIT group (F=50.400, p \u0026lt; 0.01); IL-6 also gradually declined throughout the study period in non-SLIT group (F = 24.267, p \u0026lt; 0.01). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/037f7e2b5b6832a5ce72e484.png"},{"id":87553076,"identity":"db2b5eb8-ee05-426f-8d5c-30f0eaa54018","added_by":"auto","created_at":"2025-07-25 06:35:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":426790,"visible":true,"origin":"","legend":"\u003cp\u003eIncreases in IL-10 following treatment. ELISA was used to identify the IL-10 levels in supernatants of PBMCs in patients of SLIT group and non-SLIT group. IL-10 level significantly increased throughout the research period in SLIT group (F = 49.043, p \u0026lt; 0.01); while IL-10 level was unchanged throughout the study period in non-SLIT group (F = 5.765, p = 0.056). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/1fb3f9491ccbad650a746937.png"},{"id":87552365,"identity":"63bfa542-34bb-4061-833e-25d69bcd5c50","added_by":"auto","created_at":"2025-07-25 06:27:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":415368,"visible":true,"origin":"","legend":"\u003cp\u003eDecreases in IL-17 following treatment. ELISA was used to identify the IL-17 levels in supernatants of PBMCs in patients of SLIT group and non-SLIT group. IL-17 level significantly decreased throughout the research period in SLIT group (F = 59.051, p \u0026lt; 0.01); IL-17 level also gradually declined throughout the study period in non-SLIT group (F = 38.000, p \u0026lt; 0.01). **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/89854f243ddea5ebfafe858b.png"},{"id":92740049,"identity":"093bdecb-7f8f-4c79-a2fd-682b3e05f746","added_by":"auto","created_at":"2025-10-03 17:17:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4173602,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6979274/v1/da3e0c11-cafc-4a11-ba39-51d4bbfab38b.pdf"}],"financialInterests":"","formattedTitle":"Effects of sublingual immunotherapy for dust mite on Th 17 / Treg cells in children with asthma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAllergen immunotherapy (AIT) is the only method that can shorten the course of allergic disease\u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/sup\u003e. Currently, studies of AIT mechanism have been focusing on immune tolerance\u003csup\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. However, the mechanism of how AIT induces the developing immune tolerance in T lymphocytes is not fully understood.\u003c/p\u003e\u003cp\u003eTh17 / Treg cells are a pair of T lymphocytes with pro-inflammatory / anti-inflammatory effects who participate in maintaining the dynamic balance of the body's immune function\u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e. The role of Th17 / Treg cells is also increasingly appreciated in asthma whose functional imbalance may lead to the occurrence and further development of asthma\u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e. Our previous study has discovered the dysfunction of Treg cell in children who have bronchial asthma allergic to house dust mites, while the interesting point is that AIT can upregulate its function to exert therapeutic effects, yet the mechanism of this process has not been further examined\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe also have proved the presence of the vast majority of eosinophil accumulation in children with bronchial asthma allergic to dust mites and an imbalance of Th17 / Treg cell function in these children. Whether AIT can induce immune tolerance in children with bronchial asthma by reversing the Th17 / Treg cell function imbalance is unknown. The role of Th17/Treg cells is also increasingly appreciated in asthma, which is a hot issue in current research.There is no report about the changes of Th17 / Treg cells and their related cytokines in bronchial asthma children allergic to dust mite allergy after SLIT treatment over 2 years. Therefore, in this study, we aim to observe the effects of dust mite SLIT on children with asthma and changes of Th17 / Treg cells percentages as well as related cytokines to investigate the possible pathological mechanism of immune tolerance induced by SLIT.\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003cp\u003e\u003cb\u003ePatients\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChildren with mild and moderate non-acute onset bronchial asthma were finally selected for the study. Each child would need to meet the following inclusion criteria: 1) diagnosed with mild to moderate allergic asthma according to diagnostic criteria for bronchial asthma(GINA 2015)\u003csup\u003e(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/sup\u003e; 2) allergic to dust mite diagnosed by skin prick test, wheal range ≥ ++, specific IgE ≥ 0.7Ku/L; 3) if child was also allergic to other allergens, such as spring and autumn pollen, cat, dog and animal fur or \u003cem\u003eAlternaria\u003c/em\u003e, non-dust mite allergens must be \u0026lt;++ identified by skin prick test; 4) no other cardiovascular diseases, autoimmune diseases, or other underlying diseases. 5) None of the children had used systemic corticosteroids and other immunosuppressants, nor had they used antihistamines. The exclusion criteria: 1) diagnosed with chronic lung diseases, such as bronchopulmonary dysplasia, pulmonary fibrosis, congenital abnormal lung development and so on; 2) diagnosed with an immunodeficiency disease; 3) diagnosed with other serious illnesses; 4) diagnosed with severe bronchial asthma; 5) diagnosed with contraindications to pediatric pulmonary function tests. They were randomly divided into SLIT and non-SLIT groups according to the random number table method. Guardians of all children had provided the informed consent. Meanwhile, this study was approved by the Ethics Committee of the Second Hospital of HeBei Medical University.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTreatment administration method\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePatients were treated with conventional treatment for asthma or plus SLIT over 2 years. The treatment process consisted of a dose-escalation phase and a maintenance phase for 2 years. They were evaluated before the treatment and at the end of each year. The SLIT group was given sublingual dust mite drops (Zhejiang Wolwo Biological Technology Co., Ltd, Deqing, Zhejiang Province, China) numbered 1 to 4. No. 1, 2, and 3 which would be administered in weeks 1, 2, and 3 individually, with daily doses of 1, 2, 3, 4, 6, 8, and 10 drops. From the 4th week, the No. 4 would be given 3 drops once a day until the end of 2-year-treatment. The drops are swallowed after sublingual administration for 1 ~ 3 min at night at same time under the care of parents, and oral food intake was only allowed for 15 min after each administration until the end of the course of treatment.\u003c/p\u003e\u003cp\u003eAll children treated with no systemic corticoids, other immunosuppressants and no anti-allergic drugs within 4 weeks before blood collection. Both groups had the same basic medication, and were given with small to moderate doses of inhaled corticosteroids (Budesonide inhalant), without combining long-acting antihistamines and antileukotriene drugs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReagents and equipment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe fluorescein-isothiocyanate (FITC) mouse anti-human CD4, mouse anti-human (PE Alexa Fluor 700), Alexa Fluor 647 anti-human IL-10, APC anti-human IL-17, PE anti-human Foxp3, anti-human CD25, human IL-6, human IL-10, human IL-17 ELISA kits were all purchased from eBioscience, Inc (San Diego, CA, USA). Cytofix/Cytoperm solution, lymphocyte separation medium and RPMI-1640 medium were bought from Gibco (USA). The flow cytometer (FACSCantoII) was purchased from BD Biosciences (New Jersey, USA); Automatic allergen detector (Uni- CAP100) was purchased from Pharmacia (Uppsala,Sweden); the CO\u003csub\u003e2\u003c/sub\u003e thermostatic incubator(MCO-175) was purchased from SANYO (Osaka, Japan); Fully automatic microplate reader (Multiskan MK3) was purchased from Thermo Fisher Labsystems (USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStandard for clinical asthma symptom score and asthma drug use score\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEvaluation was performed with night and morning asthma symptom scores\u003csup\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/sup\u003e. The night symptoms would be scored from 0 to 4 points as following: “0” for no symptoms; “1” for early or one arousal during sleep; “2” for early and more than two arousals during sleep; “3” for many arousals during sleep; “4” for no sleep at night. Daytime symptoms were scored as following: “0” for no symptoms; “1” for a little symptoms and short duration; “2” for more than twice very short symptoms; “3” for mild symptoms occurred at more times but with less affects to study and life; “4” for more time and more symptoms with affects on the study and life; “5” for the symptoms are serious and the children cannot live and study normally. The higher score indicated more severe asthma symptoms.\u003c/p\u003e\u003cp\u003eAsthma drug use score own a total score of 7 points as below in which these following drugs could be used in combination according to the child's condition. “1” for the β2 receptor agonists or leukotriene receptor antagonists, individually; “2” for inhaled corticosteroids; “3” for inhalational glucocorticoids corticosteroids (ICS) combined with a long-acting β2 receptor agonist (LABA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell isolation and culture\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe fasting peripheral venous blood (5 mL) would be drawn from each subject before treatment, 1 year and 2 years of treatment. The blood would be placed in sterile heparin tubes separated by Ficoll density-gradient centrifugation to isolate the PBMCs. Then cells were supposed to be washed with Hank’s solution for three times and resuspend in RPMI-1640 medium with 10% fetal bovine serum (FBS). The PBMCs would be then stimulated with dust mite leaching solution at dose of 20 µg/mL for 48 hours. The cell culture supernatant would be obtained to evaluate IL-6, IL-10 and IL-17 levels by ELISA, and cells would be collected to analyze the Th17 cells (CD4\u003csup\u003e+\u003c/sup\u003e IL-17\u003csup\u003e+\u003c/sup\u003e T cells) and Treg cells (CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e IL-10\u003csup\u003e+\u003c/sup\u003e T cells) percentages.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData is analyzed using the SPSS24.0 software. All sets of measurement data are expressed as mean ± standard deviation (\u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003e± S). The Chi-Square test (χ\u003csup\u003e2\u003c/sup\u003e test) is used for the comparison of categorical data. For the comparison between groups (SLIT and non-SLIT groups), t-test is performed to compare data with normally distributed variance alignment, while the unpaired Mann-Whitney U test is used for non-normal distribution or uneven variance, and paired multi-sample Friedman-test is employed for within-group (pre-treatment, 1 year of treatment, 2 years of treatment) comparisons. P \u0026lt; 0.05 is considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSubject characteristics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 76 children with bronchial asthma were initially included in this study, but 6 cases were lost in the non-SLIT group, of whom 4 were lost to follow-up and 2 voluntarily withdrew from the study for personal reasons. The 10 cases were lost in the SLIT group, of whom 4 were lost to follow-up and 7 voluntarily withdrew from the study for personal reasons. Finally, 60 cases were included and randomly divided into SLIT and non-SLIT groups with 30 patients in each group. In the SLIT group, 19 boys and 11 girls were aged 9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64 years. In the non-SLIT group, 18 boys and 12 girls were aged 8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44 years. There were no differences in the two groups in age, sex, severity, duration, lung function, presence, associated disease, dust mite specific IgE level, and positive allergen distribution of the skin prick test results (See Figure.1, Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGeneral information of children\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSLIT group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003enon-SLIT group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003et / χ\u003csup\u003e2\u003c/sup\u003e value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCases\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender (Male/ Female)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19/11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18/12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.071(χ\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.791\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (Year)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.482 (t)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.631\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDisease course (Year)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-1.357 (t)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.098\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDust mite specificity\u003c/p\u003e\u003cp\u003eIgE (KU/mL)※\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e134.65\u0026thinsp;\u0026plusmn;\u0026thinsp;142.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e138.70\u0026thinsp;\u0026plusmn;\u0026thinsp;106.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-0.99 (t)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.322\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily history(N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16/14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19/11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.617 (χ\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.432\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRhinallergosis (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9/21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11/19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.300 (χ\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.584\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAllergic dermatitis\u003c/p\u003e\u003cp\u003e(N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19/11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21/9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.300 (χ\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.584\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, ※ \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, was considered to be statistically significant difference.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of allergen skin prick test results in the children\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSLIT group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNon-SLIT group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCases\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFamily history (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16/14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19/11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.617\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.432\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRhinallergosis (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9/21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11/19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.584\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAllergic dermatitis (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19/11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21/9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.300\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.584\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHouse dust (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24/6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.111\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.739\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMould Ⅱ (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23/7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.317\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlternaria (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.718\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDog hair (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e22/8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23/7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.089\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.766\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCat epithelium (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20/10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23/7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.739\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.390\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWeed pollen (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20/10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17/13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.635\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.426\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRagweed (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17/13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15/15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.268\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.605\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eArtemisia argyi (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16/14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18/12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.271\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.602\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHay dust (N/Y)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26/4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25/5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.718\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e※ P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, was considered to be statistically significant difference\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAsthma symptom scores\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMean asthma symptom scores in the SLIT group were respectively 2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45, 2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 and 1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 in before treatment, 1 year and 2 years after treatment. In addition, the scores obviously decreased throughout the research period (F\u0026thinsp;=\u0026thinsp;50.334, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Although the mean asthma symptom scores have shown the decline in the non-SLIT group, we found no statistically different changes until treatment after 2 year compared with before treatment (F\u0026thinsp;=\u0026thinsp;39.800, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Intergroup comparison has revealed that the range ability of asthma symptoms scores in the SLIT group was remarkably higher than that of non-SLIT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Figure.2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAsthma drug use scores\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMean asthma drug use scores in the SLIT group were respectively 5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47, 4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 and 3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 in before treatment, 1 year, 2 years after treatment. And the scores significantly declined throughout the research period (F\u0026thinsp;=\u0026thinsp;60.000, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The mean drug use scores appeared to decline in the non-SLIT group, but the changes had no statistically significant differences (F\u0026thinsp;=\u0026thinsp;4.467, p\u0026thinsp;=\u0026thinsp;0.107). Intergroup comparison has revealed that the change amplitude in the SLIT group was significantly greater than non-SLIT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). See Figure.3.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLung function\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMean FEV 1% in the SLIT group were 82.83\u0026thinsp;\u0026plusmn;\u0026thinsp;12.17%, 95.47\u0026thinsp;\u0026plusmn;\u0026thinsp;11.68% and 102.83\u0026thinsp;\u0026plusmn;\u0026thinsp;12.17%, before treatment, and 1 year, 2 years after treatment, respectively. And FEV 1% significantly increased over the treatment period (F\u0026thinsp;=\u0026thinsp;34.889, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). FEV 1% increased significantly in the non-SLIT group after 2 years of drug treatment compared with before treatment (F\u0026thinsp;=\u0026thinsp;27.042, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). It has shown that the change amplitude was significantly higher in the SLIT group than non-SLIT group by intergroup comparison (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). See Figure.4.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDecreasing of Th17 cells percentages after treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the effects of SLIT on the Th17 cells proportion in PBMCs, the FACs analysis was conducted to identify and quantify CD4 and cells secreting IL-17. Percentages of Th17 cells in SLIT group were 4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96, 3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 and 2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 before treatment, 1 year and 2 years after treatment, individually. Th17 cell percentage gradually declined throughout the research period in SLIT group, with significantly differences (F\u0026thinsp;=\u0026thinsp;47.400, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The percentages of Th17 cells were 4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87, 4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07, 4.41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12 before treatment, 1 year and 2 year after treatment in non-SLIT group, individually. The percentage of Th17 cell also gradually declined throughout the study period in non-SLIT group (F\u0026thinsp;=\u0026thinsp;44.826, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). While the magnitude of change was obviously higher in the SLIT group than that of the non-SLIT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Figure.5.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIncrease in the Treg cell percentages after treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine whether SLIT alters the proportion of Treg cells in PBMCs, we further identified and quantified CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003eIL-10\u003csup\u003e+\u003c/sup\u003e T cells by FACs. Its percentages significantly enhanced throughout the research period in SLIT group (F\u0026thinsp;=\u0026thinsp;40.267, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), from 1.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 at baseline to 1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38, 2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 at 1 year and 2 years after treatment. Although percentage of Treg cell appeared to increase in the non-SLIT group, no statistically significant differences were observed (F\u0026thinsp;=\u0026thinsp;3.525, p\u0026thinsp;=\u0026thinsp;0.172). The percentages of Treg cells in the treated SLIT group were significantly higher than non-SLIT group, with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). See Figure.6.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges of IL-6, IL-10 and IL-17\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ELISA was conducted to identify the IL-6, IL-10 and IL-17 levels in supernatants of PBMCs. IL-6 levels in SLIT group were 55.02\u0026thinsp;\u0026plusmn;\u0026thinsp;9.16, 45.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.18 and 36.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.81 pg/mL before treatment, 1 year and 2 years after treatment, respectively. It gradually declined in SLIT group over the study period, with significantly differences (F\u0026thinsp;=\u0026thinsp;50.400, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Figure.7). In contrast to IL-6, IL-10 levels were significantly increased throughout the study period in SLIT group (F\u0026thinsp;=\u0026thinsp;49.043, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which were 44.05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.41, 48.84\u0026thinsp;\u0026plusmn;\u0026thinsp;9.21 and 56.12\u0026thinsp;\u0026plusmn;\u0026thinsp;8.15 pg/mL before treatment, 1 year and 2 years after treatment, respectively (Figure.8). While IL-17 levels were 6.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15, 5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 and 3.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 pg/mL before treatment, 1 year and 2 years after treatment in SLIT group, respectively, and the differences were statistically significant (F\u0026thinsp;=\u0026thinsp;59.051, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Figure.9). Although IL-6, IL-10 and IL-17 levels in non-SLIT group also appeared the similar tendency of changes, the magnitude of change was apparently greater in the SLIT group than that of non-SLIT group (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Figure.7, Figure.8, Figure.9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current international consensus is that allergen immunotherapy (AIT) has definite curative effects in allergic rhinitis and allergic asthma. AIT is a therapeutic vaccination used to treat IgE-mediated allergies to common allergens such as pollen, house dust mites, and aspiration insect venom. AIT has two main forms in clinical use: subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). SCIT is the classic form of administration, but the need for subcutaneous administration increases the pain and the risk of serious systemic adverse reactions. However, SLIT is easy to take, and rarely causes serious systemic adverse effects, so it attracts more and more attentions and likely to replace SCIT\u003csup\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe mechanism of how specific immunotherapy induced immune tolerance in T lymphocytes is not fully understood. It is believed that the main mechanism of SLIT induction of immune tolerance is to promote the conversion of antigen-sensitized CD4\u003csup\u003e+\u003c/sup\u003e Th0 cells into Treg cells and Th1 cells and thus inhibit the Th2 cell-associated immune response\u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e. Dust mite drop immunotherapy is a kind of sublingual specific immunotherapy. It is made of dust mite antigen active protein, which can induce the body to generate specific antibodies, then combine with allergens, thus blocking the allergen and basophils and mast cells interaction, block the occurrence of type I allergic reaction, at the same time can inhibit T lymphocytes and eosinophils gathered in the target organ, inhibit degranulation changes in lymphocytes and exert regulatory effects on the immune system\u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTh 17 / Treg cells play an important role in maintaining the dynamic balance of immune function in vivo. Under some specific conditions, Treg cells and Th 17 cells can interconvert, such as changes in cytokine concentration\u003csup\u003e(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e. Th17 cells play an important role in the immunopathogenesis of various autoimmune diseases and tumors. IL-17 is mainly a cytokine secreted by Th 17 cells, and it cannot replicate the asthma model if IL-17 is knocked out. IL-17 is mainly involved in severe asthma and hormone-resistant asthma, which is dominated by neutrophil infiltration\u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIL-6 is involved in the proliferation and differentiation process of Th17 cells. It has a key regulatory role in the differentiation of initial CD4\u003csup\u003e+\u003c/sup\u003e T cells into Th17 cells and Treg cells\u003csup\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e. Binding of IL-6 to cell surface receptors can trigger the lung JAK / STAT3 signaling, thus playing a key regulatory role in Th17 / Treg differentiation\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e. The activated JAK kinase phosphorylates STAT3, p-STAT3 dimerizes and transplaces into the nucleus to initiate expression of downstream related genes such as RORγt, IL-17A / F, prompting differentiation of initial CD4\u003csup\u003e+\u003c/sup\u003e T lymphocytes to Th17. Meanwhile, STAT3 can also promote the methylation of the CpG island of the enhancer upstream of FOXP3 gene and inhibit its expression. SOCS1 and SOCS3 negatively regulate IL-6 / STAT3 signaling. Animal experiments found that downregulation of IL-6 in the respiratory tract achieved good results in treating asthmatic animals\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e. Clinical experiments found that the effect of inhaled corticosteroids in asthma was related with downregulation of IL-6 levels in serum\u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTreg cells are a group of cells that exert a negative regulatory effect on the cellular immune function, among which the FOXP3 protein is its most important transcription factor. Treg cells mainly inhibit the T cell-mediated inflammation dominated by eosinophil infiltration in the respiratory tract, thus reducing the respiratory allergic reaction and preventing the development of asthma\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e. The Treg cells predominantly secrete IL-10 and TGF-β. IL-10 may have an immune dual effect, which promotes the development of inflammatory responses rather than anti-inflammatory under some conditions, and its dual function is not only dependent on the target cells, but also on the concentration of IL-10\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e. IL-10 can affect the function of T cells at different stages of inflammation. Moreover, IL-10 was also able to inhibit the differentiated in Th 17 cells\u003csup\u003e(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe imbalance of Th17 / Treg cell function plays an important role in the development of bronchial asthma, Hu Y et al reported that differentiation of Th 17 / Treg cells is implicated in the pathogenesis of allergic asthma\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e. They found a significant Th 17 / Treg imbalance in the child with bronchial asthma, in which the percentage of Th 17 cells was significantly increased while the percentage of Treg cells was significantly reduced, and the IL-17 levels were significantly increased while the IL-10 levels were significantly reduced. However, Katrien Van der Borgh et al treated the mouse model of bronchial asthma allergy with house dust mite (HDM) with prophylactic SLIT treatment, and they found the SLIT treatment could increase the levels of treg and Th 17 in the lungs of the asthma model mice\u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e. Thus, the role of Th 17 / Treg in SLIT is inconclusive, and it is unclear whether SLIT can play a role in immune tolerance by reversing the imbalance of Th17 / Treg cell function. Therefore, we aimed to preliminarily explore the mechanism of SLIT by observing the changes in Th17 / Treg cells and corresponding cytokine levels in bronchial asthma children allergic to dust mite receiving SLIT. Our study found that asthma symptom score and drug use score Th17 percentages, IL-6 and IL-17 levels were significantly decreased in SLIT group throughout the study period (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, FEV1%, Treg cell percentages and IL-10 level significantly enhanced in SLIT group throughout the study period (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Asthma symptom score, lung function, Th17 cell percentages, IL-6 and IL-17 levels all significantly improved in non-SLIT group even all lower than SLIT group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). But no statistical difference was observed in drug use score, Treg cell percentages and IL-10 level for non-SLIT group. Our study found that Treg levels remained at a high level after 2 years of sublingual specific immunotherapy, which might be related to the child still in desensitization treatment. It was reported that the optimal treatment time of SLIT was 3 years, and it was considered that it can maintain better long-term efficacy after 3 years of drug withdrawal\u003csup\u003e(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/sup\u003e. The 2-year SLIT course did not maintain the long-term effect. We will further explore the changes of Th 17 / Treg cells and their associated cytokines levels in children after the cessation of treatment to evaluate the long-term efficacy of specific immunotherapy.\u003c/p\u003e\u003cp\u003eThe results showed that Th17 / Treg cell function imbalance existed in children with bronchial asthma allergic to eosinophil accumulation, especially Treg cell dysfunction. Although drug treatment can improve the clinical symptoms and the level of some cytokines in children, it cannot reduce the use of asthma drugs and upregulate Treg cell function. SLIT can upregulate Treg cell function and downregulate Th17 cell function to reverse the imbalance of Th17 / Treg cell function and induce immune tolerance. However, whether it reverses Th17 / Treg cell function by inhibiting IL-6 secretion needs further investigation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, Our study proved that SLIT of Dust mite drops could alter T immune cell profiles and improves asthma symptoms. And SLIT might reverse the functional imbalance of Th 17 / Treg cells to induce immune tolerance by upregulating Treg cell function and downregulating Th17 cell function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Review Board of Ethics Committee of the Second Hospital of Hebei Medical University. Written informed consent was obtained from all participants.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written consent for the publication of their data.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFang Li: Research design and writing\u003c/p\u003e\n\u003cp\u003eWei Wang: Research design, experiment design and manuscript revision\u003c/p\u003e\n\u003cp\u003eKun Wang：Data acquisition\u003c/p\u003e\n\u003cp\u003eXue Wang: Data processing and analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external financial or non-financial support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFang Li:\u003c/p\u003e\n\u003cp\u003eDepartment of pediatrics, The Second Hospital of Hebei Medical University Associate clinical professor, Master\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eTelephone and fax numbers：0311-95658371\u003c/p\u003e\n\u003cp\u003eDepartment of Pediatrics, The Second Hospital of Hebei Medical University, Shijiazhuang, China\u003c/p\u003e\n\u003cp\u003eKun Wang：\u003c/p\u003e\n\u003cp\u003eDepartment of pediatrics, Hebei Medical University Third Hospital\u003c/p\u003e\n\u003cp\u003eClinical professor, Doctor\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eTelephone and fax numbers：0311-88603060\u003c/p\u003e\n\u003cp\u003eDepartment of Pediatrics, Hebei Medical University Third Hospital, Shijiazhuang, China\u003c/p\u003e\n\u003cp\u003eXue Wang：\u003c/p\u003e\n\u003cp\u003eDepartment of pediatrics, The Second Hospital of Hebei Medical University Associate clinical professor, Master\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eTelephone and fax numbers：0311-95658371\u003c/p\u003e\n\u003cp\u003eDepartment of Pediatrics, The Second Hospital of Hebei Medical University, Shijiazhuang, China\u003c/p\u003e\n\u003cp\u003eWei Wang (Corresponding Author):\u003c/p\u003e\n\u003cp\u003eDepartment of pediatrics, Hebei Medical University Third Hospital\u003c/p\u003e\n\u003cp\u003eClinical professor, Doctor\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e\n\u003cp\u003eTelephone and fax numbers：0311-88603060\u003c/p\u003e\n\u003cp\u003eDepartment of Pediatrics, Hebei Medical University Third Hospital, Shijiazhuang, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShamji MH, Sharif H, Layhadi JA, Zhu R, Kishore U, Renz H. 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Teucrium polium Extract Attenuates Inflammation in Asthma by Reducing RORgammat Transcription and Increasing IL-10 Secretion in an Ovalbumin-induced Murine Asthma Model. Iran J Immunol. 2023;20(2):159-66.\u003c/li\u003e\n\u003cli\u003eHu Y, Chen Z, Zeng J, Zheng S, Sun L, Zhu L, et al. Th17/Treg imbalance is associated with reduced indoleamine 2,3 dioxygenase activity in childhood allergic asthma. Allergy Asthma Clin Immunol. 2020;16:61.\u003c/li\u003e\n\u003cli\u003eVan der Borght K, Brimnes J, Haspeslagh E, Brand S, Neyt K, Gupta S, et al. Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells. Mucosal Immunol. 2024; S1933-0219(24)00028-X.\u003c/li\u003e\n\u003cli\u003eCreticos PS, Gunaydin FE, Nolte H,et al，Allergen Immunotherapy: The Evidence Supporting the Efficacy and Safety of Subcutaneous Immunotherapy and Sublingual Forms of Immunotherapy for Allergic Rhinitis/Conjunctivitis and Asthma.J Allergy Clin Immunol Pract. 2024 Jun;12(6):1415-1427. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dust mite, sublingual immunotherapy, Th17 cell, Treg cell, children","lastPublishedDoi":"10.21203/rs.3.rs-6979274/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6979274/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e observing the clinical effects of dust mite sublingual immunotherapy (SLIT) on children with asthma, changes of Th17 / Treg cells and related cytokines in order to investigate the possible pathological mechanism of immune tolerance induced by SLIT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Sixty children with asthma allergic to dust mites were included, divided into SLIT group (n=30) and non-SLIT group (n=30). Clinical symptoms of asthma in each group had been scored before, 1 year and 2 years after treatment. Meanwhile we also evaluated the proportion of Th17 and CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003e+\u003c/sup\u003e Treg cells in peripheral blood using flow cytometry. Besides, cell culture supernatant was collected to detect the changes of IL-6, IL-10 and IL-17 levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We found that in SLIT group, asthma symptom and drug use score, Th17 cell percentages, IL-6 and IL-17 levels have significantly decreased throughout the study period (p \u0026lt; 0.05), while FEV1%, Treg cell percentages and IL-10 level have prominently increased throughout the study period (p \u0026lt; 0.05). By contrast, in non-SLIT group, asthma symptom score, lung function, Th17 cell percentages, IL-6 and IL-17 levels have all significantly improved, but on the whole lower than SLIT group (p \u0026lt; 0.05). However, we have observed no statistical differences in drug use score, Treg cell percentages, IL-10 level for non-SLIT group throughout the study period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e SLIT of Dust mite drops could change T immune cell profiles whereas improve asthma symptoms. SLIT might reverse the functional imbalance of Th 17 / Treg cells and induce immune tolerance by upregulating Treg cell function and downregulating Th17 cell function.\u003c/p\u003e","manuscriptTitle":"Effects of sublingual immunotherapy for dust mite on Th 17 / Treg cells in children with asthma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 06:27:03","doi":"10.21203/rs.3.rs-6979274/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e91b9ae-5da1-481e-99fb-55b30ec95fc5","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-19T16:38:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 06:27:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6979274","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6979274","identity":"rs-6979274","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}