Eosinophilic chronic rhinosinusitis (ECRS), a subgroup of chronic rhinosinusitis with nasal polyps is characterized by ethmoid-predominant sinusitis with eosinophilic inflammation [1
]. ECRS is a refractory eosinophilic airway disease because ECRS patients have a much higher incidence (about 50%) of bronchial asthma [3
]. In general, conventional ECRS treatments, including intranasal corticosteroids and oral corticosteroids, do not control the disease adequately. Recently, we reported the usefulness of inhaled corticosteroid (ICS) exhalation through the nose (ETN) treatment for ECRS patients with bronchial asthma [4
]. ICS-ETN treatment reduces operative interventions by half [6
] and reduces recurrence rates within three years, after endoscopic sinus surgery (ESS), by half in patients with severe ECRS (unpublished data, in preparation). However, about 30% of these patients experienced recurrence even after ESS. Bronchial asthma is a known risk factor of relapse after ESS [7
], suggesting that severe ECRS with asthma is much more difficult to control because of reduced corticosteroid sensitivity [8
Nuclear translocation of the glucocorticoid receptor (GR) is a critical process that mediates corticosteroids’ biological actions [9
]. In patients with refractory airway inflammation, including severe asthma, the reduced nuclear translocation of GR is an important molecular mechanisms of corticosteroid resistance. GR-Ser226
phosphorylation induces this resistance due to the reduced activity and expression of phosphatases, such as protein phosphatase 2A (PP2A) [10
]. Interestingly, a recent report has shown that PP2A expression is lower in nasal polyps from ECRS patients compared with controls [13
]. In addition, there are reports of reduced corticosteroid responses in nasal polyp epithelial cells from patients with refractory chronic rhinosinusitis [8
]. However, the local response to corticosteroids in patients with ECRS and the underlying mechanisms have not been elucidated fully.
In this study, we focused on corticosteroid sensitivity in nasal bronchial cells from patients with ECRS and investigated the molecular mechanisms of steroid resistance.
2. Materials and Methods
2.1. Cell Preparation
Tissue specimens were obtained from nasal polyps in the ethmoid sinuses of patients with ECRS and uncinate process tissues of patients with allergic rhinitis or ECRS during surgery. Nasal epithelial cells were collected from healthy volunteers and patients with allergic rhinitis, ECRS, or chronic rhinosinusitis (non-ECRS), by brushing the inferior turbinate (middle meatus side) with a cervical cytology brush. The desquamated epithelium from the tissue samples and nasal epithelial cells was incubated until EpCAM (Millipore, Temecula, CA, USA) confirmed a monolayer of epithelial-like cells. The human bronchial epithelial cell line BEAS-2B was obtained from the European Collection of Authenticated Cell Culture (Salisbury, UK). Eosinophils (purity > 98%) were isolated from the peripheral blood of healthy volunteers with mild eosinophilia (approximately 4%–8% of total white blood cells) by negative selection using a MACS system in conjunction with the Eosinophil Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Table S1
shows the characteristics of subjects. This study was approved by the local ethics committee of Kansai Medical University (approval number: KanIRin1313), and written informed consent was obtained from each patient or volunteer.
2.2. Corticosteroid Sensitivity
Nasal epithelial cells were treated with dexamethasone for 45 min, followed by TNFα (10 ng/mL) stimulation overnight. The ability of dexamethasone to inhibit TNFα-induced CXCL8 release was determined in cell medium by sandwich ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). IC50 of dexamethasone on CXCL8 production (Dex-IC50), calculated using Prism® 6.0 statistical software (GraphPad, San Diego, CA, USA), was used as a marker for corticosteroid sensitivity. In addition, BEAS-2B cells were treated overnight with FITC-conjugated dexamethasone (Molecular Probes, Eugene, OR; 10−6 M). The nuclear fraction was prepared by 10 min incubation with hypotonic buffer (Epigentec, Farmingdale, NY, USA), followed by pulse vortexing. Dexamethasone’s ability to translocate into the nucleus was evaluated by FITC-dexamethasone detected in the nuclei using a fluorescent plate reader.
2.3. Quantitative RT-PCR
Total RNA extraction and reverse transcription were performed using a PureLink RNA Micro kit (Invitrogen, Carlsbad, CA, USA) and a PrimeScript RT MasterMix (Perfect Real Time; Takara Bio, Shiga, Japan). Gene transcript levels of protein phosphatase 2 catalytic subunit alpha isozyme (PPP2CA
), protein tyrosine phosphatase-RR (PTP-RR
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH
) were quantified by real-time PCR using a Rotor-Gene SYBR Green PCR kit (Qiagen, Hilden, Germany) on a Rotor-Gene Q HRM (Corbett Research, Cambridge, UK). Table S2
shows detail of the amplification primers.
2.4. Immunofluorescence Staining
After treatment with dexamethasone (10−7 M) for 1 h, the cells were fixed with 4% formaldehyde for 20 min, permeabilized, and blocked. The cells were then incubated with the primary antibodies (mouse monoclonal antibody to GR; Abcam, Cambridge, UK and rabbit polyclonal antibody to PP2A; GeneTex, Alton Pkwy Irvine, CA, USA), followed by the fluorescently labeled secondary antibodies (Alexa 488 donkey anti-mouse and APC donkey anti-rabbit; Jackson Immuno Research, West Grove, PA, USA). Control antibodies and Hoechst (Invitrogen, Paisley, UK) were included in each experiment. GR’s ability to translocate into the nucleus was evaluated using a Carl Zeiss LSM700 confocal microscope.
2.5. In-Cell Western Assay
BEAS-2B cells fixed with 4% formaldehyde for 20 min were permeabilized and blocked. Cells were incubated with primary antibodies (mouse monoclonal antibody to phospho-PP2A; Santa Cruz Biotechnology, Dallas, TX and rabbit polyclonal antibody to PP2A; GeneTex) and the fluorescently-labeled secondary antibodies (IRDye 800CW goat anti-mouse and IRDye 680RD goat anti-rabbit; LI-COR Bioscience, Lincoln, NE, USA). Ratio of fluorescence intensity of phospho-PP2A to that of PP2A was analyzed by Odyssey infrared imaging system (LI-COR Bioscience) according to the manufacturer’s instructions.
2.6. Cell lysis, Immunoprecipitation, and Western Blotting
Cell protein extracts were prepared using modified RIPA buffer (50 mM Tris HCL pH 7.4, 1.0% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl with freshly added complete protease inhibitor), as described previously [10
]. Phosphatase inhibitor was used as needed. Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Immunoprecipitation was conducted with anti-GR antibody (Cell Signaling Technology, Danvers, MA, USA) or anti-PP2A antibody (Santa Cruz Biotechnology). Protein extracts or immunoprecipitates were separated by SDS-PAGE (Bio-Rad) and detected by Western Blot analysis using Odyssey infrared imaging system (LI-COR Bioscience) according to the manufacturer’s instructions. The mouse monoclonal antibody to PP2A and GR (Santa Cruz Biotechnology), the rabbit polyclonal PTP-RR antibody (Aviva Systems Biology, San Diego, CA, USA), and the rabbit monoclonal mucin 1 antibody (Abcam) were used for primary antibodies. β-actin expression was used as a control for protein loading as needed.
2.7. Phosphatase Activity
Phosphatase activity in immunopurified PP2A was assayed using SensoLyteTM
MFP Protein Phosphatase Assay Kit (AnaSpec, San Jose, CA, USA) as previously described [10
2.8. RNA Interference
PP2ACα siRNAs and non-silencing scrambled control siRNA were purchased from QIAGEN (Crawley, UK). The siRNA sequences (0.6 μM) were transfected using Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s specifications.
Transfections were done by Xfect Transfection Reagent (Takara Bio), accoding to the manufacturer’s specifications. A total of 2 μg of DNA/plasmids (Thermo Fisher Scientific, Tokyo, Japan) containing PP2Acα gene were transfected to BEAS-2B cells.
2.10. Statistical Analysis
Comparisons of two groups of data were performed using Mann-Whitney U test or paired t-test. Correlation coefficients were calculated with the use of Spearman’s rank method. Other data were analyzed by ANOVA with post hoc test adjusted for multiple comparisons (Dunn’s test or Newman-Keuls test), as appropriate. Differences were considered statistically significant if p value was <0.05. Descriptive statistics were expressed as means ± SEM.
In this study, we demonstrated reduced local responses to corticosteroids in ECRS patients with bronchial asthma, particularly at eosinophilic inflammatory sites (eosinophil-rich areas). Impaired PP2A in these areas resulted in reduced GR nuclear translocation and corticosteroid insensitivity. In addition, epithelial cells in these areas enhanced the production of ILC2-stimulating cytokines, which served to induce type 2 allergic responses [15
] and the expression of several chemokines involved in eosinophilic inflammation [16
]. Among these chemokines, CCL4 was found to be markedly elevated. CCL4 is also released, not only from macrophages and mononuclear cells, but also activated eosinophils [16
]. Since CCR5, a specific receptor for CCL4 [20
], is expressed on eosinophils, T cells, and ILC2 [21
], these cells could be further recruited in eosinophil-rich areas and cooperatively enhance type 2 allergic responses.
We recently showed that eosinophils are activated in eosinophil-rich nasal polyps compared with peripheral blood eosinophils in the same patients [22
]. Several granule proteins, such as EPX and EDN, degranulate from activated eosinophils [23
]. In addition, our results demonstrated that PP2A phosphorylation levels were elevated in epithelial cells after EPX stimulation (Figure 2
A) but not in those under co-incubation with non-activated eosinophils (Figure S1
). The PP2A catalytic subunit, a regulator of PP2A complexes and activity [24
], is phosphorylated at the Tyr307
], which reduces PP2A activity [26
]. Thus, at local sites where eosinophils are activated, impaired PP2A may induce corticosteroid insensitivity. As one of the mechanisms for PP2A hyperphosphorylation in eosinophilic airway inflammation, we confirmed that PTP-RR, a regulator of PP2A-Tyr307
], was reduced under stimulation with EPX and in nasal epithelial cells from ECRS patients with severe asthma (Figure S2
). Furthermore, PTP-RR expression in nasal epithelial cells was positively correlated with PP2A expression, suggesting that PTP-RR may regulate PP2A-dependant local responses to corticosteroids.
Regarding the therapeutic targets of local corticosteroid insensitivity, restoration of PP2A function may be a candidate. Increased PP2A expression with plasmids, or LABA’s activation of PP2A, may enhance responses to corticosteroids, as we have previously shown in mononuclear cells [10
]. In same cases of refractory eosinophilic airway inflammation, increased drug delivery to the local inflammatory site with a novel method using inhaled ICS/LABA ETN has been shown to be effective [5
]. Our findings are supported by recent reports showing that LABA ameliorates eosinophilic inflammation via PP2A activation [28
Milara et al. reported that responses to corticosteroids were reduced concomitant with the downregulation of the mucin 1protein in nasal polyp epithelial cells from patients with refractory chronic rhinosinusitis [8
]. Mucin 1 may contribute to corticosteroid-induced MAP kinase phosphatase 1 (MKP1) upregulation [8
], inhibiting MAPK activity and GR-Ser226
phosphorylation, which is associated with reduced GR nuclear translocation [10
]. Although the interaction between mucin 1 and PP2A has not been elucidated, we confirmed mucin 1 and PP2A are located in the same complex from GR-immunoprecipitates (Figure S3
). Further study is warranted to investigate mucin 1’s role in the PP2A-dependent regulation of corticosteroid sensitivity.