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Introduction

Allergic rhinitis (AR) is defined as an inflammation of the lining of the nose and affects up to 40% of the population. It is characterized by nasal symptoms, including congestion, sneezing, itching and rhinorrhea. AR is the most common cause of inflammation of the nasal mucosa (1,2). Immunoglobulin E (IgE) is a classical antibody that mediates allergic reactions by sensitizing mast cells and basophils to allergen activation through binding tightly to a specific receptor (High-affinity receptor I, FcεRI) (3) on these cells (4). As the most common form of non-infectious rhinitis, it is also associated with an IgE-mediated immune response against allergens (5).

Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation is an integral part of the innate immune response in inflammatory disease (6). The NLRP3 inflammasome is present in variety of cells, including macrophages, neutrophils, T cells and B cells (7). The NLRP3 inflammasome is a protein scaffolding complex consisting of NLRP3, caspase-1 and the adaptor molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain that induces secretion of interleukin-1β (IL-1β) and IL-18 (8,9). Numerous mechanisms underlying the activation of the NLRP3 have been identified, including ion channel gating and excessive reactive oxygen species (ROS) generation (10). The idea that the activation of the NLRP3 inflammasome was caused by an increase in ROS production was accepted and it was hypothesized that the NLRP3 inflammasome may be a general sensor for changes in cellular oxidative stress (11,12).

AR involves systemic inflammation in addition to nasal inflammation (13). Levels of acute phase reactants, including high sensitivity C-reactive protein (14–16), fibrinogen, alpha 1-glycoprotein, alpha 1-antichymotrypsin (16), are not statistically different between patients with AR and healthy controls (5,7–9). However, serum amyloid A (15) and ceruloplasmin oxidase activity (17) were higher in AR.

IL-1β is a member of the IL-1 family of ligands. Although IL-1-targeted drugs are effective against autoinflammatory disease, they are less effective against autoimmune diseases. Both AR and asthma are allergen-mediated disorders, so AR shares several pathogenic similarities with asthma (18). Previous studies have reported on the pathological roles of IL-1β and the NLRP3 inflammasome in the development of allergic asthma, though the results were conflicting (19–23). Nevertheless, the involvement of IL-1β in AR has not been clearly examined. The present study was designed to examine the differences in serum IL-1β and PBMCs between patients with AR and the healthy controls, in order to assess whether IL-1β participates in the pathological process of AR.

Patients and methods

Patients

A total of 45 patients (Table I), with persistent moderate-severe AR, and 23 healthy controls were included in the present study. Between March 2016 and October 2016, these participants were screened at the Ear, Nose and Throat Department, First Affiliated Hospital of Jinan University (Guangdong, China). Written, informed consent was provided according to the procedure approved by the Research Ethics Committee of the First Affiliated Hospital of Jinan University. Inclusion criteria for the present study were as follows: Patients had a detailed clinical history of nasal obstruction, sneezing, itching, and/or rhinorrhea, sensitization to a minimum of one perennial allergen and had experienced these symptoms for ≥2 years. Exclusion criteria were: Nasal polyposis, chronic rhinosinusitis, excessive septal deviation, bronchial asthma, current smoking of cigarettes, cardiovascular disease, obesity, diabetes, and other systemic diseases. Blood samples were gathered from each participant. Whole blood was centrifuged at 2,451 × g for 15 min at 4°C twice, and sera was collected from the supernatant. Sera samples were stored at −80°C for subsequent analysis.

Table I. Patient clinical characteristics.

Table I.

Patient clinical characteristics.

Characteristic	Healthy control (n)	Allergic rhinitis (n)
Sex
Male	13	13
Female	10	32
Age (years)	28.5±8.2	32.3±12.4
AR duration (years)	Not applicable	6.31±2.12

[i] AR, allergic rhinitis.

PBMCs collection and stimulation

PBMCs were isolated by Ficoll gradient centrifugation and diluted with an equal amount of PBS, overlaid on Ficoll medium (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and centrifuged at 600 × g for 30 min at 20°C. The PBMCs bands were aspirated, washed twice with PBS, and re-suspended in cell freezing medium (90% FBS, Lanzhou Minhai Bio-Engineering, Gansu, China; cat. no. SA201.02, 10% DMSO; cat. no. D2660, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) prior to gradient cooling and being stored in liquid nitrogen for later use. Following the collection of PBMCs samples, PBMCs were stimulated with 100 ng/ml LPS (cat. no. L4516; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) alone, or with LPS (100 ng/ml) plus Brefeldin A (dilution, 1:1,000; cat no. 420601; BioLegend, Inc., San Diego, CA, USA) for 5 h at 37°C. PBMCs then were collected and stained (30 min, 4°C) with PerCP-A-conjugated anti-human CD14 (dilution, 1:200; cat no. 325631; BioLegend, Inc.) and Pacific Blue-conjugated anti-human IL-1β (dilution, 1:200; cat no. 511710; BioLegend, Inc.) prior to flow cytometric analysis, which stimulated with LPS added Brefeldin A. For other samples, which were stimulated with LPS alone, the cell culture supernatants were gathered and stored at −80°C for later IL-1β concentration detection.

Flow cytometric analysis

IL-1β levels in lipopolysaccharides (LPS) stimulated PBMCs were measured through flow cytometry using a BD Cytofix/Cytoperm™ Fixation/Permeablization kit (cat no. 554714; BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Once LPS was stimulated, PBMCs were harvested for immunofluorescent staining. A total of ~106 cells were stained using 50 µl staining buffer (PBS+1% FBS) with PerCP-A-conjugated anti-human CD14 dilution (1:200; cat no. 325631; BioLegend, Inc.) for 30 min at 4°C. Cells were protected from light throughout staining and storage. The cells were washed twice with PBS and pelleted using centrifugation for 10 min at 4°C (250 × g). The cells were resuspended and 250 µl Fixation/Permeabilization solution was added and incubated for 20 min at 4°C. The cells were washed twice with 1× BD Perm/Wash™ buffer staining in tubes. Then the cells were resuspended and fixed/permeabilized in 50 µl 1× BD Perm/Wash buffer with a pre-determined optimal concentration of IL-1β antibody (dilution, 1:200; cat no. 511710; BioLegend, Inc.) and incubated at 4°C for 30 min in the dark. The cells were washed again twice with 1× BD Perm/Wash™ buffer and resuspended in staining buffer prior to flow cytometric analyzed using the BD FACSVerse™ and BD FACSuite™ Software v1.0.5 (BD Biosciences) and FlowJo software v 10.0.6 (FlowJo LLC, Ashland, OR, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

RNA was extracted from PBMCs using TRIzol® reagent (cat no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described in our previous study (24), RNA was analyzed by RT-qPCR using Power SYBR Green PCR Master Mix on a Real-Time PCR system (cat no. RR820A; Takara Bio, Inc., Otsu, Japan). The reaction mixture (20 µl) contained 0.5 µl sense primer (10 µmol/l), 0.5 µl antisense primer (10 µmol/l), 10 µl SYBR-Green, 8 µl H2O and 1 µl cDNA. The thermocycling conditions were as follows: 95°C for 5 sec and 60°C for 30 sec for 40 circulations. Analysis of the relative gene expression data using RT-qPCR and the 2−ΔΔCq method (25). Relative expression levels were normalized to those of GAPDH. The primer sequences are listed in Table II. Expression was calculated as relative to GAPDH. All primers were purchased from Sangon Biotech Co., Ltd., (Shanghai, China).

Table II. Primer sequences used for RT-qPCR analysis.

Table II.

Primer sequences used for RT-qPCR analysis.

Primer	Sense (5′-3′)	Antisense (5′-3′)
NLRP3	5′-TAGGTTGAGGTGCTTTGCCA-3′	5′-AGACACACTTCCCCAGCATT-3′
IL-1β	5′-TGAACTGAAAGCTCTCCACC-3′	5′-TCTTTCAACACGCAGGACAG-3′
GAPDH	5′-TCACCAGGGCTGCTTTTAAC-3′	5′-TGACGGTGCCATGGAATTTG-3′

[i] RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Mitochondrial ROS levels detected in PBMCs

PBMCs isolated from AR (n=11) and healthy controls (n=11) were primed with LPS (100 ng/ml) for 5 h, and then stimulated with ATP (1 mmol/l; cat no. A6419; Sigma-Aldrich, Merck KGaA) for 1 h in the absence or presence of Mito-TEMPO (200 µmol/l; cat no. SML0737; Sigma-Aldrich; Merck KGaA) at 37°C. Following this, the cells were stained with 5 µmol/l MitoSOX (cat no. M36008, Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 37°C, protected from light, gated for the CD14+ population, and analyzed by flow cytometry.

Cytokine analysis

The plasma samples and cell culture supernatants were analyzed using ELISA kits (IL-1β, cat no. 437004; IL-17A cat no. 433914; BioLegend, Inc.). The sera were brought to room temperature, according to the manufacturer's protocol, for the simultaneous detection of IL-1β and IL-17 in a single sample.

Statistical analysis

The results are expressed as the means ± standard error, except the age of participants, which were expressed as the means ± standard deviation. Differences between groups were evaluated by one-way analysis of variance. Spearman's correlation analysis was used to assess associations between variables. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using IBM SPSS version 20.0 software (IBM Corp., Armonk, NY, USA).

Results

The expression of NLRP3 and proinflammatory cytokines increased in the PBMCs of patients with AR

The mRNA expression of IL-1β and NLRP3 in PBMCs from the 45 patients with AR and 23 healthy controls was detected prior to and following stimulation with LPS (a TLR4 ligand). Basal levels of IL-1β and NLRP3 mRNA expression in PBMCs were significantly upregulated in patients with AR (Fig. 1A). Following LPS stimulation, IL-1β and NLRP3 mRNA expression levels were significantly increased in PBMCs from patients with AR, compared with in cells from the healthy controls (Fig. 1A). Basal and LPS-induced production of IL-1β was significantly elevated in PBMCs (Fig. 1B) from patients with AR. These data suggest that patients with AR exhibit upregulated inflammatory cytokine production and NLRP3 expression in their PBMCs, compared with in those from healthy controls.

Figure 1. Expression profiles of NLRP3 and IL-1β in PBMCs and sera from patients with AR and HCs. PBMCs were obtained from 45 patients with AR and 23 HCs. The PBMCs were incubated with media containing FBS and stimulated with ultrapure LPS (100 ng/ml) for 5 h. (A) The mRNA expression of IL-1β and NLRP3 were analyzed by reverse transcription quantitative polymerase chain reaction. (B) IL-1β production in sera was measured by an ELISA. Data are expressed as the means ± SEM. *P<0.05 vs. HCs. IL-1β, interleukin-1β; PBMCs, peripheral blood mononuclear cells; AR, allergic rhinitis; HC, healthy controls; U, untreated; LPS, lipopolysaccharides.

Upregulated IL-1β activation in monocytes/macrophages and PBMCs in patients with AR

Human monocytes exhibit constitutive inflammasome activation (26), thus the activation of IL-1β in monocytes/macrophages was assessed. As shown in Fig. 2A and B, patients with AR exhibited a significantly elevated level of IL-1β in LPS-primed monocytes/macrophages compared with the non-AR healthy controls. Production of IL-1β was significantly upregulated in PBMCs from patients with AR, as determined using ELISAs (Fig. 1B). The results indicate that IL-1β activation is upregulated in the monocytes/macrophages and PBMCs of patients with AR.

Figure 2. Upregulated maturation of IL-1β activation in monocytes/macrophages. (A) PBMCs were obtained from 45 patients with AR and 23 HCs. The PBMCs were stimulated with LPS (100 ng/ml) in the presence of Brefeldin A (1:1,000) for 5 h, then stained with PerCP-A-conjugated anti-human CD14 and Pacific Blue™-conjugated anti-human IL-1β prior to flow cytometric analysis. Density of the distribution of the cells is represented by color; red is high density, yellow is medium density and blue is low density. (B) Percentage statistic data of flow cytometric and (C) mean fluorescence intensity statistic data of flow cytometry. *P<0.05 with comparisons shown by lines. PBMC, peripheral blood mononuclear cells; LPS, lipopolysaccharides; AR, allergic rhinitis; HC, healthy controls; NS, not significant.

Mitochondrial ROS and NLRP3 are required for IL-1β synthesis in monocytes/macrophages and PBMCs in patients with AR

Studies have demonstrated that ROS derived from mitochondria have been involved in NLRP3 inflammasome activation (27,28), which could be indirectly implicated in IL-1β production. Therefore, the present study investigated whether inflammasome stimuli enhance mitochondrial ROS generation in CD14+ cell fractions of PBMCs, and whether this was greater in patients with AR, compared with in healthy controls. As presented in Fig. 3A, LPS-primed PBMCs were pretreated with ATP alone or ATP plus Mito-TEMPO, which inhibits mitochondrial ROS production, and the results revealed that the generation of mitochondrial ROS in LPS-primed PBMCs stimulated with ATP was higher in cells from patients with AR than in cells from healthy controls and ATP-induced IL-1β secretion in LPS-primed PBMCs were inhibited in Mito-TEMPO groups. Subsequently, it was investigated whether mitochondrial ROS are required for IL-1β secretion in PBMCs. As shown in Fig. 3B, the mitochondria-targeting antioxidant Mito-TEMPO was used, which could inhibit mitochondrial ROS production to pre-treat PBMCs. Mito-TEMPO was shown to significantly inhibit ATP-induced IL-1β secretion in LPS-primed PBMCs in the examined groups (n=11). These results suggest that the monocyte/macrophage fractions of PBMCs from patients with AR exhibit raised mitochondrial ROS levels.

Figure 3. Upregulated NLRP3 inflammasome activation in patients with AR are mediated by mitochondrial reactive oxygen species. (A) PBMCs isolated from patients with AR (n=11) and HCs (n=11) were primed with LPS (100 ng/ml) for 5 h and then stimulated with ATP (1 mmol/l) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 µmol/l). The cells were then stained with MitoSOX, gated for the CD14+ population, and analyzed by flow cytometry. Representative images (left) and quantitative analysis of mean fluorescence intensities (right) are shown. Data are expressed as the means ± SEM. (B) PBMCs from AR (n=11) and HCs (n=11) were primed with LPS (100 ng/ml, for 5 h), and then stimulated with ATP (1 mmol/l) for 1 h in the absence or presence of Mito-TEMPO (mit; 200 µmol/l). ELISA analysis of IL-1β. Data are expressed as the means ± SEM. *P<0.05 vs. HCs. AR, allergic rhinitis; PBMCs, peripheral blood mononuclear cells; LPS, lipopolysaccharides; HC, healthy controls; ELISA, enzyme-linked immunosorbent assay.

Levels of IL-1β and IL-17 are increased in patients with AR and are positively correlated with each other

Previous studies in patients with AR indicated that IL-17 is also likely to be involved in the pathogenesis of AR (29–33). Potential differences in the basal level of IL-1β in patients with AR and healthy controls, whilst simultaneously detecting the serum IL-17 levels in patients with AR were investigated. As shown in Fig. 4, patients with AR had increased serum levels of IL-1β and IL-17 (Fig. 4A) when compared with the healthy controls. Serum IL-1β and IL-17 were also identified to be positively correlated with one other (Fig. 4B). These data suggest that IL-1β may be involved in the development of AR by inducing the production of IL-17.

Figure 4. Levels of IL-1β and IL-17 were increased in patients with AR and are positively correlated with each other. (A) Sera collected from the peripheral blood of patients with AR (n=45) and HCs (n=23). IL-1β and IL-17 production in the sera was measured by an ELISA. (B) Correlation between IL-1β and IL-17 in the sera. Data are expressed as the means ± SEM. *P<0.05 vs. HCs. AR, allergic rhinitis; LPS, lipopolysaccharide; HC, healthy controls; ELISA, enzyme-linked immunosorbent assay.

Discussion

AR affects up to 40% of the global population, and its incidence is increasing (34–36). The symptoms of AR are inconvenient for the sufferer, and AR is a strong risk factor for other chronic respiratory diseases, including chronic rhinosinusitis and asthma. Characteristics of AR include an inflammatory reaction sustained by T-helper 2 cell (Th2) polarization (37). The Th bias is evidenced by the PBMCs of patients with AR. Th2 cells preferentially produce the cytokine IL-4 that promotes IgE production and inhibits Th1 (38,39). The dichotomy in function between Th1 and Th2 cells has been modified by the discovery of another T-lymphocyte subset, namely Th17 cells (40). IL-17 is one of the cytokines produced by Th17 cells. IL-17 could recruit macrophages and had also been identified as a survival factor for airway macrophages (41). Serum levels of IL-17 are elevated in a number of disorders, including rheumatoid arthritis (42), acute hepatic injury (43) and AR (31). Conversely, specific studies regarding serum IL-17 levels in patients with AR have presented conflicting results, which may be explained by varying clinical characteristics of patients and the type of sensitization used between studies (32,44,45).

In the present study, patients with AR were identified as having higher serum IL-17 levels compared with the healthy controls. In obesity-associated asthma, IL-17A was hypothesized to cause airway hyper-reactivity (46). Furthermore, IL-17 serves a critical role in, and is associated with, the clinical severity of AR (31,32). IL-17 producing T cells are associated with polysensitization in patients with AR, though not with bronchial hyper-responsiveness (47).

The results of the present study revealed that NLRP3 and IL-1β expression in PBMCs from patients with AR were elevated compared with the healthy controls. There was also an upregulated maturation of IL-1β activation in monocytes/macrophages and PMBCs in patients with AR. A previous study demonstrated that nitric oxide could sustain IL-1β expression in human dendritic cells by enhancing their capacity to induce IL-17-production (48). T-regulatory cells highly expressed IL-1R1, and IL-1β could induce prominent activation of p38 and c-Jun N-terminal kinases, which are involved in IL-17 production (49). IL-1β directly causes airway hyper-reactivity by inducing IL-17A production in obesity-associated asthma (46). This was corroborated by the results of the present study, which identified a positive correlation between serum IL-1β and IL-17 levels in patients with AR. Therefore, it is hypothesized that the high levels of serum IL-1β may have a role in the pathogenesis of AR through inducing IL-17-production.

There is evidence to suggest that an excess of ROS serve an important role in the pathogenesis of airway inflammation (50–53). Furthermore, previous studies have suggested that abnormalities in mitochondria are associated with the development of asthma (54,55) and that mitochondrial ROS serve critical roles in the pathogenesis of allergic airway inflammation through modulation of NLRP3 inflammasome activation (56). However, the association between mitochondrial ROS generation and AR is not well understood. In the present study, monocyte/macrophage fractions of PBMCs from patients with AR demonstrated increased production of mitochondrial ROS prior to and following treatment with NLRP3 inflammasome stimuli, suggesting that the elevated production of mitochondrial ROS affects NLRP3 inflammasome activation in patients with AR. Following inhibition of mitochondrial ROS by Mito-TEMPO, significantly decreased IL-1β secretion from LPS-primed PBMCs from patients with AR was observed, suggesting that mitochondrial ROS are responsible for NLRP3 inflammasome activation in AR. Mitochondrial ROS production increases regularly due to defective mitochondrial homeostasis in macrophages, which render mitochondria more susceptible to damage by inflammasome stimuli (28). Furthermore, mitochondrial membrane permeability transition and mitochondrial ROS generation are required for the activation of caspase-1 and IL-β secretion (28). Therefore, mitochondria ROS serve an important role in the upregulation of inflammatory responses via acting as signal-transduction molecules (57).

Although the data of the present study suggest that activation of the NLRP3 inflammasome is associated with AR pathogenesis, it has its limitations. At present, the roles of inflammatory cytokines in AR primarily focus on nasal local inflammation in patients with AR, which is highly significant compared with systemic inflammation. The level of systemic inflammation and local inflammation is not consistent between a number of diseases. Therefore, further experiments are required to verify the exact role of IL-1β in AR. However, despite these limitations, the findings of the present study may provide novel avenues for anti-AR therapies, as well as for a variety of inflammatory diseases.

Acknowledgements

Not applicable.

Funding

National Natural Science Foundation of China (grant no. 3150050081), China Postdoctoral Science Foundation (grant no. 2015M572411), Natural Science Foundation of Guangdong Province, China (grant no. 2015A030313312), and Foundation Sciences Jinan University (grant no. 11615479).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HJ and HY initially proposed the study. Wrote the protocol: QS, ZL, DL and GC. Wrote the manuscripts: QS. Collected the samples and data: QS, ZL, DL, GC, QW. Analyzed the data: QS, SL, HJ. Reviewed drafts of the paper: QS, GC, SL, HY.

Ethics approval and consent to participate

The present study was approved by Research Ethics Committee of the First Affiliated Hospital of Jinan University [approval no. 2016 (022)]. All patients provided written informed consent for participation in the present study.

Consent for publication

Informed consent was obtained from all patients included in the present study for the publication of the associated data and the accompanying images.

Competing interests

The authors declare that they have no compeing interests.

References