Decreased Expression of Interleukin‑37 and Its Anti‑Inflammatory Effect in Allergic Rhinitis
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MOLECULAR MEDICINE REPORTS 17: 1333-1339, 2018 Decreased expression of interleukin‑37 and its anti‑inflammatory effect in allergic rhinitis YANG SHEN1*, XIA KE1*, LIU YUN2, GUO‑HUA HU1, HOU‑YONG KANG1 and SU‑LING HONG1 1Department of Otorhinolaryngology, The First Affiliated Hospital of Chongqing Medical University; 2Chongqing Key Laboratory of Ophthalmology, Chongqing 400016, P.R. China Received November 22, 2016; Accepted July 12, 2017 DOI: 10.3892/mmr.2017.7988 Abstract. Interleukin-37 (IL-37), a novel member of the IL-1 Introduction cytokine family has been identified as a natural suppressor of innate immunity and inflammatory responses. The present Allergic rhinitis (AR) is an immunoglobulin E-mediated study aimed to determine the expression of IL‑37 in periph- type of inflammation of the upper airway, which is induced eral blood mononuclear cells (PBMCs) from patients with by allergens and regulated by T cells. AR has an estimated allergic rhinitis (AR), and examine the possible immuno- worldwide incidence rate of 10‑20% (1). The authors' previous suppressive effect of IL‑37 on inflammatory mediators and epidemiological investigations demonstrated that, in western CD4+ T cells in the pathogenesis of AR. The expression levels China, the prevalence of self‑reported AR was 32.30% in of IL‑37 were determined in PBMCs from 39 patients with Chongqing, 34.3% in Chengdu, 37.9% in Urumqi, and 30.3% in AR and 43 controls using reverse transcription‑quantitative Nanning (2). AR has a major effect on quality of life by causing polymerase chain reaction (RT‑qPCR) analysis and flow symptoms of sneezing, nasal congestion, nasal pruritus, rhinor- cytometry. Cytokines in the supernatants of the PBMCs rhea and obstruction of the nasal passages. Furthermore, AR is and CD4+ T cells, which were stimulated with lipopolysac- a known risk factor for comorbid conditions, including asthma, charide in the presence or absence of IL‑37, were assayed rhinosinusitis, nasal polyposis and sleep disorders, resulting using enzyme‑linked immunosorbent assays and RT‑qPCR in important medical and social problems (3,4). Over the last analysis. The results showed that the patients with AR exhib- 20 years, the pathogenesis of AR has been widely investigated ited significantly decreased expression of IL‑37, and increased and the majority of studies have focused on proinflammatory expression levels of interleukin (IL)‑1β and IL‑6 in PBMCs. cytokines. However, anti‑inflammatory cytokines in AR have Recombinant IL‑37 (rIL‑37) inhibited the production of IL‑1p received less attention. and IL‑6, and enhanced the production of IL‑27 in PBMCs Interleukin (IL)-37, a novel member of the IL-1 family from the patients with AR and the control individuals. rIL‑37 and originally termed IL‑1F7, has been shown to be a also markedly decreased the expression of IL‑17 by CD4+ T natural suppressor of innate immunity and inflammatory cells in the patients with AR and controls. These results responses (5,6). IL‑1F7b is the largest variant, and is referred suggested that IL‑37 may be an important cytokine in the to as IL‑37 in the present study. IL‑37 interacts with the cell pathogenesis of AR. It may have a protective role in AR by surface receptors, IL‑18Ro and IL‑18‑binding protein (BP), inhibiting the production of proinflammatory cytokines and and has five splice variants (IL‑1F7a‑e) (7). It is expressed through suppressive regulation of the Th17 response. in human peripheral blood mononuclear cells (PBMCs) and various tissues at low levels, and can be induced by inflam- matory stimulation, including Toll‑like receptor agonists. A previous study demonstrated that the expression of IL‑37 in dendritic cells (DCs) promoted the generation of semimature tolerogenic DCs and suppressed antigen‑specific immune responses of the skin, which revealed IL‑37 as an inhibitor of Correspondence to: Professor Su‑Ling Hong or Professor the adaptive immune response (8). The abnormal expression Hou‑Yong Kang, Department of Otorhinolaryngology, The First of IL‑37 has been reported in several inflammation‑related Affiliated Hospital of Chongqing Medical University, 1 Yixueyuan diseases, including Vogt‑Koyanagi‑Harada disease (9‑11), Road, Chongqing 400016, P.R. China E‑mail: [email protected] inflammatory bowel disease (12), systemic lupus erythema- E‑mail: [email protected] tosus (13), Graves' disease (14), ankylosing spondylitis (15) and rheumatoid arthritis (16,17). In addition, Lunding et al (18) *Contributed equally demonstrated that IL-37 ablated a Th2 cell-directed allergic inflammatory response and the hallmarks of experimental Key words: allergic rhinitis, interleukin-37, interleukin-27, CD4+ T cells asthma in mice, suggesting that IL-37 may be critical for the pathogenesis of asthma. It has also been reported that IL‑37 is an important cytokine in the control of asthma by suppressing 1334 SHEN et al: IL‑37 IN ALLERGIC RHINITIS the production of inflammatory cytokines, tumor necrosis Darmstadt, Germany) in the presence of recombinant IL‑37 factors, IL-β, IL‑6 and thymic stromal lymphopoietin (19,20). (rIL‑37) at different concentrations (0, 50, 100 and 200 ng/ml; These findings indicate that IL‑37 may be involved in inflam- R&D Systems, Inc., Minneapolis, MN, USA) for 72 h at room matory responses, and may have an immunosuppressive role temperature. Following incubation, the cells and culture super- in allergic disease. However, whether there are any correla- natants were collected to analyze the RNA and protein levels tions between IL‑37 and AR remain to be elucidated, and the of cytokines. The duration of 72 h was selected according to function of IL‑37 in AR warrants further investigation. a previous study; therefore, the cytokines were not measured The present study aimed to determine the mRNA and at different time‑points (10). Subsequently, the PBMCs from protein levels of IL‑37 in PBMCs from patients with AR, in seven patients with AR and six controls were re‑suspended at a order to evaluate the expression of IL‑37 in AR. This was concentration of 1x106 cells/ml and stimulated with or without followed by examination of the possible immunosuppressive 100 ng/ml rIL‑37 in the presence of 100 ng/ml LPS for 72 h effect of IL‑37 on inflammatory mediators and CD4+ T cells at room temperature, following which the culture supernatants in the pathogenesis of AR. were harvested and frozen at ‑80˚C for cytokine analysis using an enzyme‑linked immunosorbent assay (ELISA). The cells Materials and methods in the control groups were stimulated with the same volume of PBS alone. Subjects. Overall, 39 patients (23 women and 16 men) between To investigate the effect of IL-37 on effector cytokine 12 and 52 years of age were recruited, between April 2015 and production by CD4+ T cells, the CD4+ T cells were separated September 2015. All patients were identified by and treated from the PBMCs obtained the AR patients and controls using at the outpatient clinic of the Department of Otolaryngology, magnetic microbeads (CD4+ cell purity ≥98%; Miltenyi Head and Neck Surgery at the First Affiliated Hospital of Biotec, Inc., Cambridge, MA, USA). The CD4+ T cells were Chongqing Medical University (Chongqing, China). The re ‑susp ende d at a concent rat ion of 1x10 6 cel ls/m l a nd st i mu lat e d diagnosis of AR was based on the patients' medical history, with anti‑CD3/CD28 (eBioscience; Thermo Fisher Scientific, symptoms and the presence of a positive skin prick test (SPT; Inc.) in the presence or absence of 100 ng/ml rIL‑37 for 72 h to Allergopharma, Hamburg, Germany) in response to a panel detect the levels of IL‑10 and IL‑17 in the supernatants. of common allergens defined by the Allergic Rhinitis and its Impact on Asthma 2008 guidelines (21). The SPT results Reverse transcription‑quantitative polymerase chain reaction were diagnosed in accordance with these commendations (RT‑qPCR) analysis. The mRNA levels of IL‑37 and proin- of the Subcommittee on Allergen Standardization and Skin flammatory cytokines from the PBMCs were determined using Tests of the European Academy of Allergy and Clinical RT‑qPCR analysis. Total RNA was extracted from the isolated Immunology (22). A positive SPT result was defined as the PBMCs using TRIzol extraction (Invitrogen; Thermo Fisher formation of a wheal measuring ≥50% of the diameter of the Scientific, Inc.) according to the manufacturer's instructions, histamine control wheal, and ≥3 mm larger than the diameter and was reverse transcribed to cDNA using random hexamer of the negative control wheal. A total of 18 inhaled allergens primers and RNase H‑reverse transcriptase (Invitrogen; were assessed, including house dust, grass, tree, mold, food, Thermo Fisher Scientific, Inc.). The expression levels of and cat and dog dander. Patients with accompanying systemic mRNA were determined using the ABI Prism 7500 Sequence disease were excluded from the study. An additional 43 healthy Detection system (Applied Biosystems; Thermo Fisher volunteers of the same ethnicity as the patients were recruited Scientific, Inc.) and SYBR Premix Taq (Takara Biotechnology as a control group. Co., Ltd., Dalian, China). The PCR primer sequences are summarized in Table I. PRISM samples contained 1X SYBR Ethics statement. The local Ethics Committee of the First Green Master Mix, 1.5 µl, 5 µM primers, and 25 ng synthesized Affiliated Hospital of Chongqing Medical University provided cDNA in a 25 µl volume. Reaction mixtures were heated to permission and assisted in obtaining informed consent from 95˚C for 10 min, followed by 40 cycles of denaturation at 95˚C all participants. Written informed consent was obtained from for 10 sec, and annealing extension at 60˚C for 60 sec. All PCR all participants.