Allergic rhinitis (AR) is the allergic inflammation of immune cells in the nasal mucosa, caused by an abnormal T-cell response. Interleukin (IL)-37, a unique member of the IL-1 family with broad anti-inflammatory roles in various autoimmune diseases, participates in the immune regulation of AR. However, the regulatory mechanism of IL-37 in AR has remained elusive. In the present study, a mouse model of AR was established by treating mice with ovalbumin (OVA). Following systemic administration of IL-37, the effects of the cytokine on allergic symptoms were evaluated. The nasal mucosal infiltration of eosinophils was assessed by histopathological observation. The serum and nasal lavage fluid concentrations of immunoglobulin (Ig)E, IgG1, IgG2a, interferon (IFN)-γ, IL-4, IL-13, IL-17a and C-C motif cytokine ligand (CCL)11 were determined by ELISA. Treatment with OVA resulted in allergic symptoms, including enhanced eosinophil infiltration in the nasal mucosa, increased thickness of the nasal mucosa and increased levels of IgE, IgG1, IgG2a, IL-4, IL-13, IL-17a and CCL11, but the level of IFN-γ was indicated to decrease. After IL-37 treatment, the frequency of nasal rubbing and sneezing was reduced compared with that in the OVA group. IL-37 administration also decreased the number of eosinophils in the nasal mucosa and the thickness of the nasal mucosa, as well as the serum and nasal lavage fluid levels of IgE, IgG1, IgG2a, IL-4, IL-13, IL-17a and CCL11, but the level of IFN-γ decreased. In addition, the OVA-induced increases in histamine and substance P levels were reversed by IL-37 administration. CCL11 expression levels were correlated with the expression levels of IFN-γ, IL-4, IL-13, IL-17a, histamine and substance P. In conclusion, IL-37 alleviated the OVA-induced allergic symptoms and allergic inflammatory response by reducing the serum cytokine levels via decreasing CCL11 expression levels in mice.
Allergic rhinitis (AR) is the allergic inflammation of immune cells in the nasal mucosa and is characterized by clinical symptoms and complaints including itching, allergic conjunctivitis, rhinorrhea, nasal congestion and disturbed olfaction (
The imbalance of type 2 T helper (Th2) cells, a subset of CD4+ T cells, has an essential role in the pathogenesis and immunological characteristics of AR (
IL-37 is a unique member of the IL-1 family with broad anti-inflammatory roles during autoimmune diseases, including systemic lupus erythematosus, psoriasis and asthma (
Wang
C-C motif cytokine ligand (CCL)11 is critical for attracting eosinophils during allergic asthma in mice (
On the basis of the aforementioned studies, it was hypothesized that IL-37 alleviates the allergic symptoms of AR via CCL11 signaling in Th2 cells. To further understand the role of IL-37 in CCL11 signaling, a mouse model of AR was established by treating animals with ovalbumin (OVA). The allergic symptoms were investigated and the serum and nasal lavage fluid levels of IgE, IgG1, IgG2a, IFN-γ, IL-4, IL-13, IL-17a and CCL11, as well as histamine and substance P, were determined by ELISA. In addition, the number of eosinophils was determined by histopathological observation.
A total of 40 male BALB/c mice (age, 6-8 weeks; weight, 20-23 g) were purchased from Beijing SPF Vital Laboratory Animal Technology Company. The mice were housed in a limited access rodent facility with 8 rats per polycarbonate cage in a room with a 12 h light, 12 h dark cycle (lights on from 7:00 a.m. to 7:00 p.m.), with a relative humidity maintained at 55±15%, and a constant temperature (24±2˚C). The experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Beijing Jishuitan Hospital (Beijing, China).
To induce sensitization, mice were administered with an intraperitoneal injection of 1 mg/ml OVA (Grade V; Sigma-Aldrich; Merck KGaA) and 20 mg/ml aluminum hydroxide (Sigma-Aldrich; Merck KGaA) in sterile saline (0.1 ml/mouse) on days 1, 3, 5, 7, 9, 11 and 13. To induce AR, mice were subsequently intranasally administered 60 mg/ml OVA in sterile saline (20 µl/mouse) from day 20 to day 30(
The experimental design is presented in
Nasal symptoms were evaluated by counting the frequency of nasal rubbing and sneezing (
At 24 h after the last administration of OVA, the mice were anesthetized with isoflurane. Cardiac blood samples were harvested from each mouse in EDTA-coated tubes, maintained on ice, centrifuged at 4,000 x g for 10 min at 4˚C and subsequently, serum was isolated. ELISA was performed to determine the following: Total IgE (Mouse IgE ELISA kit; cat. no. SEKM-0095; Beijing Solarbio Science & Technology Co., Ltd.), IgG1 (Mouse IgG1 ELISA kit; cat. no. SEKM-0097; Beijing Solarbio Science & Technology Co., Ltd.), IgG2a (Mouse IgG2a ELISA kit; cat. no. SEKM-0099; Beijing Solarbio Science & Technology Co., Ltd.), IFN-γ (Mouse IFN-γ ELISA kit; cat. no. PI508; Beyotime Institute of Biotechnology), IL-4 (Mouse IL-4 ELISA kit; cat. no. PI612; Beyotime Institute of Biotechnology), IL-13 (Mouse IL-13 PicoKine ELISA kit; cat. no. EK0425; Boster Biological Technology), IL-17a (Mouse IL-17a ELISA kit; cat. no. PI545; Beyotime Institute of Biotechnology) and CCL11 (Mouse CCL11 PicoKine ELISA kit; cat. no. EK0330; Boster Biological Technology). Concentrations were determined by ELISA using specific rabbit polyclonal antibodies (Abcam) according to the manufacturer's protocol.
After the collection of blood, the mice were sacrificed with an overdose of isoflurane. When the cardiac activity and respiration ceased 15 min later, nasal lavage fluid was collected using an 18-gauge catheter. In brief, the trachea was partly resected, a catheter was inserted from the trachea into the nasopharynx and the nasal passages were gently perfused with 1 ml saline. The nasal lavage fluid was centrifuged at 10,000 x g for 10 min at 4˚C. The concentrations of IFN-γ, IL-4, IL-13, IL-17a and CCL11 were determined by ELISA using the aforementioned kits. In addition, histamine (Histamine ELISA kit; cat. no. ab213975; Abcam) and substance P (Substance P ELISA kit; cat. no. ab133029; Abcam) were measured by ELISA.
After 24 h of the last challenge on day 30, a portion of the nasal mucosa was excised and fixed in 10% formalin for 3 days at room temperature. The tissues were cut into 4-µm sections and stained for 1 min at room temperature using a haematoxylin-eosin staining kit (Beyotime Institute of Biotechnology) to assess the extent of inflammatory cell infiltration. A total of 5 random fields of vision in the nasal mucosal sections were examined using an Olympus FluoView 1200 confocal microscope (Olympus Corp.) and photomicrographs (magnification, x400) of representative nasal mucosa areas were acquired. The thickness of the nasal mucosa and the number of eosinophils were calculated using Image J software (Version 1.5.1; National Institutes of Health)
Values are expressed as the mean ± standard deviation. Statistical analyses were performed using SPSS software (version 23.0; IBM Corp.) and GraphPad Prism software (version 7.0; GraphPad Software, Inc.). One-way analysis of variance followed by Bonferroni's correction was used for multiple comparisons among four groups. The correlation between two variables was assessed by Pearson correlation analysis. P<0.05 was considered to indicate a statistically significant difference.
The effects of IL-37 administration on the allergic symptoms of the mice with OVA-induced AR are presented in
Serum IgE, IgG1 and IgG2a levels in mice were measured by ELISA and the results are presented in
The thickness of the nasal mucosa and number of eosinophils were histologically determined (
As presented in
The serum cytokine levels are presented in
In the present study, the protective effect and ability of IL-37 to attenuate the allergic inflammatory response by decreasing the production of CCL11 in a mouse model of OVA-induced AR was investigated. Allergic symptoms, including nasal mucosal eosinophilia, increased thickness of the nasal mucosa, as well as altered serum IgE, IgG1 and Ig G2a levels, were reduced or reversed in AR mice treated with IL-37. Furthermore, IL-37 decreased the levels of cytokines, including IL-4, IL-13, IL-17a and CCL11, in the serum and nasal lavage fluid of mice with OVA-induced AR. However, the level of IFN-γ in the serum and nasal lavage fluid of mice could be increased by IL-37 treatment. In addition, OVA-induced increases in the levels of histamine and substance P were reversed by IL-37 administration. CCL11 levels displayed a positive correlation with the levels of IL-4, IL-13, IL-17a, histamine and substance P. CCL11 displayed a negative correlation with the expression of IFN-γ, which suggested that IL-37 may attenuate the allergic inflammatory response by decreasing CCL11 expression in an OVA-induced mouse model of AR.
Immunoglobulins have major roles in mediating allergic and inflammatory reactions. The expression of several immunoglobulin antibodies, including IgE, IgG1 and IgG2a, has been reported to be involved in B-cell immune responses controlled by cytokines produced by Th cells, such as TNF-α, IL-4, IL-5, IL-10, IL-12 and IL-13 (
The infiltration of eosinophils into the mucosa and the thickness of the nasal mucosa are important pathological features of AR. Limited chronic inflammation and a priming effect are stimulated by eosinophil infiltration into the nasal mucosa, both of which may intensify nasal hyperreactivity (
IL-4 and IL-13 are immunoregulatory cytokines secreted predominantly by activated Th2 cells and serve as key mediators of the allergic response, including the induction of isotype switching to IgE and the promotion of eosinophil migration across the endothelium of Th2 lymphocytes (
IL-17 is involved in airway hyperreactivity and mucus hypersecretion in the upper airway during AR (
Although the levels of IL-4, IL-13 and IL-17 were significantly reduced by IL-37, similar to the results reported in previous studies (
Despite the important role of IL-37 in the regulation of CCL11-mediated OVA-induced AR, IL-37 has previously been reported to inhibit STAT6 activation and STAT3 phosphorylation to alleviate pulmonary eosinophilia and airway remodeling (
The present study reported novel results on the effect of IL-37 in a mouse model of OVA-induced AR. The results suggested that IL-37 reversed allergic symptoms and the allergic inflammatory response by downregulating serum cytokine levels and inhibiting CCL11 expression.
Not applicable.
No funding was received.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HL conceived the idea and designed the study. HL, YS and SQ performed the experiments. HL and YS analyzed the data and performed the statistical analysis. HL wrote the manuscript. All authors have reviewed the manuscript and read and approved the final manuscript.
The present study was approved by the Animal Care and Use Committee of Beijing Jishuitan Hospital (Beijing, China).
Not applicable.
The authors declare that they have no competing interests.
Experimental design of the mouse model of AR. To induce sensitization, mice were administered OVA and alum i.p. on days 1, 3, 5, 7, 9, 11 and 13. To induce AR, the mice were challenged i.n. with OVA from day 20 to 30 and were subsequently administered rhIL-37 i.p. for 5 consecutive days from day 26 to 30. AR, allergic rhinitis; OVA, ovalbumin; alum, aluminum hydroxide; i.p., intraperitoneally; i.n., intranasally; rhIL-37, recombinant human interleukin 37.
Systemic administration of IL-37 attenuates OVA-induced allergic symptoms. Frequency of (A) sneezing and (B) nasal rubbing. Values are expressed as the mean ± standard deviation (n=40). *P<0.05, as indicated. IL, interleukin; OVA, ovalbumin; Sal, saline.
Systemic administration of IL-37 reduces the OVA-induced serum levels of IgE, IgG1 and IgG2a in a mouse model of allergic rhinitis. Serum levels of (A) IgE, (B) IgG1 and (C) IgG2a. Values are expressed as the mean ± standard deviation (n=40). *P<0.05, as indicated. IL, interleukin; OVA, ovalbumin; Ig, immunoglobulin; Sal, saline.
Systemic administration of IL-37 attenuates OVA-induced nasal eosinophil infiltration and alterations to the thickness of the nasal mucosa in a mouse model of allergic rhinitis. Representative photomicrographs of the nasal mucosa in the (A) control, (B) OVA, (C) OVA + Sal and (D) OVA + IL-37 groups. The nasal mucosa tissues were stained with haematoxylin & eosin and histopathologically observed (magnification, x400; scale bar, 50 µm). (E) Eosinophil count and (F) thickness of the nasal mucosa. Values are expressed as the standard deviation (n=40). The black arrows represent eosinophils. *P<0.05, as indicated. IL, interleukin; OVA, ovalbumin; Sal, saline.
Systemic administration of IL-37 reverses OVA-induced effects on the levels of cytokines or chemical mediators in the nasal lavage fluid in a mouse model of allergic rhinitis, and the correlation between CCL11 and IFN-γ, IL-4, IL-13, IL-17a, histamine and substance P. Nasal lavage fluid level of (A) IFN-γ, (B) IL-4, (C) IL-13, (D) IL-17a, (E) CCL11, (F) histamine and (G) substance P. The correlation between nasal lavage fluid levels of CCL11 and (H) IFN-γ, (I) IL-4, (J) IL-13, (K) IL-17a, (L) Histamine and (M) Substance. P Values are expressed as the mean ± standard deviation (n=40). *P<0.05, as indicated. IL, interleukin; OVA, ovalbumin; CCL11, C-C motif cytokine ligand 11; IFN-γ, interferon-γ; Sal, saline.
Systemic administration of IL-37 reverses the OVA-induced effects on serum levels of IFN-γ, IL-4, IL-13, IL-17a and CCL11 in a mouse model of allergic rhinitis, and the correlation between serum levels of CCL11 and IFN-γ, IL-4, IL-13 and IL-17a. Serum levels of (A) IFN-γ, (B) IL-4, (C) IL-13, (D) IL-17a and (E) CCL11. The correlation between serum levels of CCL11 and (F) IFN-γ, (G) IL-4, (H) IL-13 and (I) IL-17a. Values are expressed as the mean ± standard deviation (n=40). *P<0.05, as indicated. IL, interleukin; OVA, ovalbumin; IFN-γ, interferon-γ; CCL11, C-C motif cytokine ligand 11; Sal, saline.