Inflammation ( # 2015) DOI: 10.1007/s10753-015-0204-3
Cytokine Responses to Specific Immunotherapy in House Dust Mite-Induced Allergic Rhinitis Patients Hong Li,1 Enxiu Xu,2 and Mingqiang He1,3
Abstract—Allergen-specific immunotherapy is the only immunomodulatory treatment that may alter the natural course of allergic disease. However, cytokine responses accompanying sublingual immunotherapy (SLIT) responder phenotypes have not been fully understood. Herein, we examined the level of crucial plasma cytokines during SLIT and evaluated whether their changes correlated to symptom scores. We observed that the levels of interleukin (IL)-17 and complement components C3a and C5a as well as IL-4 at year 3 of SLIT were significantly decreased than those at baseline. In contrast, there was no significant difference in the levels of IL-5, IL-13, and interferon (IFN)-γ. Notably, a significant positive correlation was found between the levels of IL-17 and the symptom scores at year 3. These results suggest that IL-17 could be considered a potential biomarker for the therapeutic effect of SLIT in allergic rhinitis caused by house dust mite. KEY WORDS: sublingual immunotherapy; IL-17; house dust mite; allergic rhinitis.
INTRODUCTION Allergic rhinitis (AR) is a common inflammatory disease of the nasal mucosa that results from seasonal or perennial responses to allergens. Meta-analyses have found sublingual immunotherapy (SLIT) to be effective in adults [1–4] and children [5, 3] with allergic rhinitis, but immunologic markers accompanying a responder phenotype after SLIT have not yet been reported. There are more studies of clinical efficacy in patients receiving SLIT for pollen allergies than for house dust mite (HDM) sensitivity [2]; thus, recent reviews [1–3] have called for more clinical trials of HDM SLIT vaccines. SLIT for mites has been reported to be effective for rhinoconjunctivitis [6]. In a study of SLIT in rhinitis, Passalacqua et al. [7] observed a significant reduction in Hong Li and Enxiu Xu contributed equally to this work. 1
Department of Otolaryngology-Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China 2 Department of Paediatrics, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China 3 To whom correspondence should be addressed at Department of Otolaryngology-Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China. E-mail: [email protected]
the total symptom scores in the first year but not in the second year of treatment, although relief of nasal obstruction was significantly improved in both years. The mechanisms of action of SLIT also include early desensitization effects, modulation of T cell and B cell responses, and induction of blocking antibody, as well as decreased migration of eosinophils, basophils, and mast cells to tissues and the release of their mediators [8]. Regulatory T cells have also been identified as key regulators of immunologic processes in peripheral tolerance to allergens [9]. Little is known about the natural history of disease in patients with HDM-induced persistent allergic rhinitis. It is clear from previous placebo-controlled HDM SLIT studies that some patients improve, even with placebo treatment [7, 2], but other studies have found a lack of improvement [10]. Because patients sensitive to HDM vary with respect to severity, organ involvement, and immune responses to different HDM allergens, the World Allergy Organization [11] has outlined published guidelines for conducting clinical trials using immunotherapy and emphasized the need for the determination of other outcomes of efficacy, such as quality-of-life (QOL) analysis, and the need for more intensive studies of immunologic parameters accompanying immunotherapy, which could be identified as markers of response.
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Li, Xu, and He In this study, we performed SLIT for patients with allergic rhinitis caused by house dust mite. We investigated plasma cytokines to screen molecules as candidate biomarkers, and evaluate whether their changes correlated to symptom scores during SLIT.
MATERIALS AND METHODS Patient Selection Twenty-eight Chinese patients aged 22 to 60 years, 13 males and 15 females, with HDM-sensitive persistent or perennial rhinitis for at least 2 years, who manifested blocked nose, runny nose, itchy throat, and/or itchy eyes or palate were enrolled. Patients with mild asthma symptoms were not excluded. At the screening visit, patients underwent skin prick tests for six allergens (Der p, Bermuda grass, cat, dog, Alternaria, and a five-grass mix [Stallergenes, Antony, France]). Only patients who tested positive for Der p sensitivity to HDMs as confirmed by a positive skin prick test result, manifesting as a wheal of 4 mm or greater to a D pteronyssinus extract (Der p 1 mix), were randomized. Patients were excluded if they had a history of seasonal allergic rhinitis due to spring or summer allergens (tree or grass pollens), were known to be polysensitive, were pregnant, required daily corticosteroid treatment for asthma, or had cardiovascular disease, hypertension, or treatment with β-blockers. The serum-specific IgE (sIgE) to Dermatophagoides pteronyssinus and Dermatophagoides farinae was measured by the ImmunoCap test (Phadia) and a value of more than 0.35 kUA/l was considered a positive allergic response. In addition, 10 healthy volunteers (6 males, 4 females, median age 32 years) without any allergy history were selected as a control group. The non-atopic status of healthy controls was double confirmed by a negative SPT to all common inhalant allergens and a negative result of Phadiatop test (Phadia). All recruited subjects (both AR patients and controls) did not have an infection, asthma, autoimmune diseases, or other upper airway diseases such as septal deviation, nasal polyps, or sinusitis before enrolment. Furthermore, the patients and control subjects had not received any form of steroids or SLIT at least 6 months before the study. The demographic information of the AR patients and controls at baseline is summarized in Table 1. More than 50 % of the AR patients demonstrated highest sensitivity to HDM (i.e., grade 4 for skin prick test or grade 6 for sIgE test). Both patients and healthy controls were given written consent to undergo the clinical study. All
Table 1. Characteristics of AR Patients and Controls at Baseline
Number of subjects Age (year) (median) Gender (male/female) SPT (wheal diameter)a Negative (15 mm) HDM sIgEb Grade 4 (17.5–50 KU/l) Grade 5 (50–100 KU/l) Grade 6 (>100 KU/l) Phadiatop Negative (0.35 KU/l)
AR patients
Controls
28 33.5 13/15
10 32 6/4
0 11 17
10 0 0
4 10 15
NA NA NA
0 28
10 0
NA not applicable a A wheal diameter greater than 3 mm of the negative saline control was considered SPT positive b The serum sIgE to HDM (Der p and Der f) was measured by the ImmunoCap test and a value of more than 0.35 kUA/l (Grade 1) was considered a positive response
patients agreed with our data utilization, and they also signed informed, written consent forms before joining the experiment. The individual in this manuscript has given written informed consent to publish these case details. The study was approved by the ethics committee of Provincial Hospital affiliated to Shandong University. Study Design Twenty-eight patients (age range, 22–60 years) received Der p HDM SLIT using Staloral 300 index of reactivity units (IR)/milliliter (Stallergenes) and stepping up treatment with daily doses during the first 4 weeks (titrating 1 to 100 IR/ml during this period) until the maintenance dose of 10 sprays (0.1 ml per spray) of 300 IR/ml of SLIT was achieved and continued to receive treatment 3 days a week for the rest of the 3-year study period. Study participants were recruited during a 6-month period. They were allowed to continue taking their usual baseline medications of intranasal corticosteroids, antihistamines, or bronchodilators as needed. Symptoms were scored daily by the patients using a diary card for 2 weeks before the commencement of active and placebo treatment (randomization visit) and taken as baseline. Patients were followed up, and symptom scores assessed at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, and 36 months. The diary cards were completed daily during the first 6 weeks after randomization and on 3 days
Cytokine Responses to Specific Immunotherapy a week during the 2 weeks before the subsequent follow-up visits (8-week to 36-month visits). Adherence for the study population was assessed during the first 6 weeks by summing the daily consumption of IRs. At each visit, a physical examination was performed including careful examination of the nose and throat. The following were recorded: current rhinitis treatment, review of the diary card recordings of the Total 5 Symptom Score (T5SS; mean daily total of five individual graded scores for sneezing; runny nose; itchy ear, nose, or throat; ocular symptoms; and nasal congestion; each scored on a range of 0–5, with 0 indicating absent and 5 indicating intolerable; maximum score, 25), the Total Nonnasal Symptom Score (TNNSS; including headache, fatigue, concentration, and irritability scores; maximum score, 20), adverse events, and concomitant medications. Global evaluation of rhinitis symptoms by the investigator and the patient used a visual analog scoring system on a scale of 0 to 10, with 0 indicating absent and 10 indicating very severe. Sample Collection Blood samples from AR patients were collected before the initiation of SLIT (baseline, SLIT-untreated) and after the end of the SLIT course (the third year, SLITtreated). Control patients’ blood was also collected at the beginning of the study. PBMCs (interface) and plasma (upper layer) were isolated from blood samples by using a density centrifugation method with Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Enzyme-Linked Immunosorbent Assay Blood samples were obtained and centrifuged immediately to prevent degradation of complement components, and the plasma was stored at −80 °C within 1 h of blood collection. We conducted enzyme-linked immunosorbent assay (ELISA) or a multiple assay system using kits (IL17: R & D Systems, Minneapolis, MN; C3a, C5a: BD Biosciences; IL-4, IL-5, IL-13, IFN-γ: Multiplex Assays) according to the manufacturers’ protocols. Briefly, 96-well plates were coated by kit capture antibody at 4 °C overnight, washed, and blocked in assay diluent. After washing, samples with controls and standards were incubated on the plates for 2 h at room temperature. Plates were then washed, followed by incubation with kit detection antibody, streptavidin horseradish peroxidase (HRP), and substrate solution. Absorbance was measured within 30 min at 450 nm after the addition of stop solution using the absorbance at 570 nm as a base line.
In Vitro Cell Stimulation PBMCs were cultured with condition medium containing RPMI-1640 (Sigma, St. Louis, MO), 10 % heatinactivated fetal bovine serum (Sigma), and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA). PBMCs were stimulated with HDM allergen for 72 h at the optimized concentration (50 μg/ml). Allergen mixture is a standardized mite depot-allergoid (50 % D. pteronyssinus and 50 % D. farinae) (Allergopharma, Rheinbek, Germany). At the end of the stimulation, monensin (eBioscience, San Diego, CA) (2 μmol/l), PMA (eBioscience) (20 μg/l), and ionomycin (eBioscience) (1 mg/l) were added to both HDM-treated and HDM-untreated cultures for 5 h to enhance the expression of the cytokines. Quantitative PCR Total RNA was extracted from PBMCs by using Trizol (Invitrogen) according to the manufacture’s protocol. RNA was reversely transcribed and real-time RT PCR measurement was performed by using ABI 7300 PCR system (Applied Biosystems, Foster City, CA). Relative gene expression was analyzed using the comparative 2−ΔΔCt method. Primer sequences of IL17 and GAPDH were designed by using Primer Exress 2.0 (Applied Biosystems) and the sequences were as follows: IL17 (NCBI Reference Sequence: NM_002190), forward primer 5′-GGGCCTGGCTTCTGTCTGA-3′ and reverse primer: 5′-AAGTTCGTTCTGCCCCATCA-3′ and GAPDH (NCBI Reference Sequence: NM_002046.3), forward primer 5′-CCACTCCTCCACCTTTGACG-3′, and reverse primer 5′-CCAGCAAGAGCACAAGAGGA A-3′. Statistical Analysis The statistical significance of the difference between the values of control and treatment groups was determined by two-tailed Student t test using Prism version 5 (GraphPad Software, Inc.). Correlation analysis was performed using Spearman’s correlation coefficient by rank. p
Cytokine Responses to Specific Immunotherapy in House Dust Mite-Induced Allergic Rhinitis Patients Hong Li,1 Enxiu Xu,2 and Mingqiang He1,3
Abstract—Allergen-specific immunotherapy is the only immunomodulatory treatment that may alter the natural course of allergic disease. However, cytokine responses accompanying sublingual immunotherapy (SLIT) responder phenotypes have not been fully understood. Herein, we examined the level of crucial plasma cytokines during SLIT and evaluated whether their changes correlated to symptom scores. We observed that the levels of interleukin (IL)-17 and complement components C3a and C5a as well as IL-4 at year 3 of SLIT were significantly decreased than those at baseline. In contrast, there was no significant difference in the levels of IL-5, IL-13, and interferon (IFN)-γ. Notably, a significant positive correlation was found between the levels of IL-17 and the symptom scores at year 3. These results suggest that IL-17 could be considered a potential biomarker for the therapeutic effect of SLIT in allergic rhinitis caused by house dust mite. KEY WORDS: sublingual immunotherapy; IL-17; house dust mite; allergic rhinitis.
INTRODUCTION Allergic rhinitis (AR) is a common inflammatory disease of the nasal mucosa that results from seasonal or perennial responses to allergens. Meta-analyses have found sublingual immunotherapy (SLIT) to be effective in adults [1–4] and children [5, 3] with allergic rhinitis, but immunologic markers accompanying a responder phenotype after SLIT have not yet been reported. There are more studies of clinical efficacy in patients receiving SLIT for pollen allergies than for house dust mite (HDM) sensitivity [2]; thus, recent reviews [1–3] have called for more clinical trials of HDM SLIT vaccines. SLIT for mites has been reported to be effective for rhinoconjunctivitis [6]. In a study of SLIT in rhinitis, Passalacqua et al. [7] observed a significant reduction in Hong Li and Enxiu Xu contributed equally to this work. 1
Department of Otolaryngology-Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China 2 Department of Paediatrics, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China 3 To whom correspondence should be addressed at Department of Otolaryngology-Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, 250021, People’s Republic of China. E-mail: [email protected]
the total symptom scores in the first year but not in the second year of treatment, although relief of nasal obstruction was significantly improved in both years. The mechanisms of action of SLIT also include early desensitization effects, modulation of T cell and B cell responses, and induction of blocking antibody, as well as decreased migration of eosinophils, basophils, and mast cells to tissues and the release of their mediators [8]. Regulatory T cells have also been identified as key regulators of immunologic processes in peripheral tolerance to allergens [9]. Little is known about the natural history of disease in patients with HDM-induced persistent allergic rhinitis. It is clear from previous placebo-controlled HDM SLIT studies that some patients improve, even with placebo treatment [7, 2], but other studies have found a lack of improvement [10]. Because patients sensitive to HDM vary with respect to severity, organ involvement, and immune responses to different HDM allergens, the World Allergy Organization [11] has outlined published guidelines for conducting clinical trials using immunotherapy and emphasized the need for the determination of other outcomes of efficacy, such as quality-of-life (QOL) analysis, and the need for more intensive studies of immunologic parameters accompanying immunotherapy, which could be identified as markers of response.
0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York
Li, Xu, and He In this study, we performed SLIT for patients with allergic rhinitis caused by house dust mite. We investigated plasma cytokines to screen molecules as candidate biomarkers, and evaluate whether their changes correlated to symptom scores during SLIT.
MATERIALS AND METHODS Patient Selection Twenty-eight Chinese patients aged 22 to 60 years, 13 males and 15 females, with HDM-sensitive persistent or perennial rhinitis for at least 2 years, who manifested blocked nose, runny nose, itchy throat, and/or itchy eyes or palate were enrolled. Patients with mild asthma symptoms were not excluded. At the screening visit, patients underwent skin prick tests for six allergens (Der p, Bermuda grass, cat, dog, Alternaria, and a five-grass mix [Stallergenes, Antony, France]). Only patients who tested positive for Der p sensitivity to HDMs as confirmed by a positive skin prick test result, manifesting as a wheal of 4 mm or greater to a D pteronyssinus extract (Der p 1 mix), were randomized. Patients were excluded if they had a history of seasonal allergic rhinitis due to spring or summer allergens (tree or grass pollens), were known to be polysensitive, were pregnant, required daily corticosteroid treatment for asthma, or had cardiovascular disease, hypertension, or treatment with β-blockers. The serum-specific IgE (sIgE) to Dermatophagoides pteronyssinus and Dermatophagoides farinae was measured by the ImmunoCap test (Phadia) and a value of more than 0.35 kUA/l was considered a positive allergic response. In addition, 10 healthy volunteers (6 males, 4 females, median age 32 years) without any allergy history were selected as a control group. The non-atopic status of healthy controls was double confirmed by a negative SPT to all common inhalant allergens and a negative result of Phadiatop test (Phadia). All recruited subjects (both AR patients and controls) did not have an infection, asthma, autoimmune diseases, or other upper airway diseases such as septal deviation, nasal polyps, or sinusitis before enrolment. Furthermore, the patients and control subjects had not received any form of steroids or SLIT at least 6 months before the study. The demographic information of the AR patients and controls at baseline is summarized in Table 1. More than 50 % of the AR patients demonstrated highest sensitivity to HDM (i.e., grade 4 for skin prick test or grade 6 for sIgE test). Both patients and healthy controls were given written consent to undergo the clinical study. All
Table 1. Characteristics of AR Patients and Controls at Baseline
Number of subjects Age (year) (median) Gender (male/female) SPT (wheal diameter)a Negative (15 mm) HDM sIgEb Grade 4 (17.5–50 KU/l) Grade 5 (50–100 KU/l) Grade 6 (>100 KU/l) Phadiatop Negative (0.35 KU/l)
AR patients
Controls
28 33.5 13/15
10 32 6/4
0 11 17
10 0 0
4 10 15
NA NA NA
0 28
10 0
NA not applicable a A wheal diameter greater than 3 mm of the negative saline control was considered SPT positive b The serum sIgE to HDM (Der p and Der f) was measured by the ImmunoCap test and a value of more than 0.35 kUA/l (Grade 1) was considered a positive response
patients agreed with our data utilization, and they also signed informed, written consent forms before joining the experiment. The individual in this manuscript has given written informed consent to publish these case details. The study was approved by the ethics committee of Provincial Hospital affiliated to Shandong University. Study Design Twenty-eight patients (age range, 22–60 years) received Der p HDM SLIT using Staloral 300 index of reactivity units (IR)/milliliter (Stallergenes) and stepping up treatment with daily doses during the first 4 weeks (titrating 1 to 100 IR/ml during this period) until the maintenance dose of 10 sprays (0.1 ml per spray) of 300 IR/ml of SLIT was achieved and continued to receive treatment 3 days a week for the rest of the 3-year study period. Study participants were recruited during a 6-month period. They were allowed to continue taking their usual baseline medications of intranasal corticosteroids, antihistamines, or bronchodilators as needed. Symptoms were scored daily by the patients using a diary card for 2 weeks before the commencement of active and placebo treatment (randomization visit) and taken as baseline. Patients were followed up, and symptom scores assessed at 2 weeks, 4 weeks, 8 weeks, 12 weeks, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, and 36 months. The diary cards were completed daily during the first 6 weeks after randomization and on 3 days
Cytokine Responses to Specific Immunotherapy a week during the 2 weeks before the subsequent follow-up visits (8-week to 36-month visits). Adherence for the study population was assessed during the first 6 weeks by summing the daily consumption of IRs. At each visit, a physical examination was performed including careful examination of the nose and throat. The following were recorded: current rhinitis treatment, review of the diary card recordings of the Total 5 Symptom Score (T5SS; mean daily total of five individual graded scores for sneezing; runny nose; itchy ear, nose, or throat; ocular symptoms; and nasal congestion; each scored on a range of 0–5, with 0 indicating absent and 5 indicating intolerable; maximum score, 25), the Total Nonnasal Symptom Score (TNNSS; including headache, fatigue, concentration, and irritability scores; maximum score, 20), adverse events, and concomitant medications. Global evaluation of rhinitis symptoms by the investigator and the patient used a visual analog scoring system on a scale of 0 to 10, with 0 indicating absent and 10 indicating very severe. Sample Collection Blood samples from AR patients were collected before the initiation of SLIT (baseline, SLIT-untreated) and after the end of the SLIT course (the third year, SLITtreated). Control patients’ blood was also collected at the beginning of the study. PBMCs (interface) and plasma (upper layer) were isolated from blood samples by using a density centrifugation method with Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Enzyme-Linked Immunosorbent Assay Blood samples were obtained and centrifuged immediately to prevent degradation of complement components, and the plasma was stored at −80 °C within 1 h of blood collection. We conducted enzyme-linked immunosorbent assay (ELISA) or a multiple assay system using kits (IL17: R & D Systems, Minneapolis, MN; C3a, C5a: BD Biosciences; IL-4, IL-5, IL-13, IFN-γ: Multiplex Assays) according to the manufacturers’ protocols. Briefly, 96-well plates were coated by kit capture antibody at 4 °C overnight, washed, and blocked in assay diluent. After washing, samples with controls and standards were incubated on the plates for 2 h at room temperature. Plates were then washed, followed by incubation with kit detection antibody, streptavidin horseradish peroxidase (HRP), and substrate solution. Absorbance was measured within 30 min at 450 nm after the addition of stop solution using the absorbance at 570 nm as a base line.
In Vitro Cell Stimulation PBMCs were cultured with condition medium containing RPMI-1640 (Sigma, St. Louis, MO), 10 % heatinactivated fetal bovine serum (Sigma), and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA). PBMCs were stimulated with HDM allergen for 72 h at the optimized concentration (50 μg/ml). Allergen mixture is a standardized mite depot-allergoid (50 % D. pteronyssinus and 50 % D. farinae) (Allergopharma, Rheinbek, Germany). At the end of the stimulation, monensin (eBioscience, San Diego, CA) (2 μmol/l), PMA (eBioscience) (20 μg/l), and ionomycin (eBioscience) (1 mg/l) were added to both HDM-treated and HDM-untreated cultures for 5 h to enhance the expression of the cytokines. Quantitative PCR Total RNA was extracted from PBMCs by using Trizol (Invitrogen) according to the manufacture’s protocol. RNA was reversely transcribed and real-time RT PCR measurement was performed by using ABI 7300 PCR system (Applied Biosystems, Foster City, CA). Relative gene expression was analyzed using the comparative 2−ΔΔCt method. Primer sequences of IL17 and GAPDH were designed by using Primer Exress 2.0 (Applied Biosystems) and the sequences were as follows: IL17 (NCBI Reference Sequence: NM_002190), forward primer 5′-GGGCCTGGCTTCTGTCTGA-3′ and reverse primer: 5′-AAGTTCGTTCTGCCCCATCA-3′ and GAPDH (NCBI Reference Sequence: NM_002046.3), forward primer 5′-CCACTCCTCCACCTTTGACG-3′, and reverse primer 5′-CCAGCAAGAGCACAAGAGGA A-3′. Statistical Analysis The statistical significance of the difference between the values of control and treatment groups was determined by two-tailed Student t test using Prism version 5 (GraphPad Software, Inc.). Correlation analysis was performed using Spearman’s correlation coefficient by rank. p
