Budesonide repairs decreased barrier integrity of eosinophilic nasal polyp epithelial cells caused by PM2.5

Abstract Background Eosinophilic chronic rhinitis with nasal polyps (eos‐CRSwNP) is a subtype of nasal polyps (NPs) characterized by severe type‐2 inflammation and defective epithelial barrier function. The epithelial barrier plays important roles in the pathogenesis of NPs and type‐2 inflammation. Particular matter 2.5 (PM2.5) are fine particles with a diameter less than 2.5 μm, containing a mixture of different components. Here, we investigated the impact of PM2.5 on the barrier function of the eos‐CRSwNP epithelium and explored the reparative function of budesonide. Methods Samples from noninflammatory nasal mucosa and eos‐CRSwNP were collected to establish an in vitro air–liquid interface cultured model. The cells were exposed to PM2.5 at 50 or 100 µg/ml intermittently for 72 h, with or without budesonide pretreatment. Barrier function and tight junction (TJ) expression were reflected by measuring transepithelial resistance (TER), paracellular flux permeability of fluorescein isothiocyanate‐labeled 4‐kDa dextran, quantitative real‐time polymerase chain reaction (qPCR), and immunofluorescence staining of TJ proteins. Cytokine expression was measured by qPCR and enzyme‐linked immunosorbent assay or Luminex. Results PM2.5 increased paracellular flux and downregulated TJ protein expression (zona occuldens‐1, occludin, and claudin‐1), but did not change TER. These changes could be partially restored by budesonide treatment. Interleukin (IL)‐8, IL‐10, IL‐1α, and tissue inhibitor of metalloproteinase (TIMP)‐1 concentrations were significantly increased in the culture medium of cells exposed to PM2.5, and budesonide significantly reduced the changes in IL‐8, IL‐1α, and TIMP‐1. Conclusion PM2.5 impaired the barrier function of eos‐CRSwNP epithelial cells and increased the permeability of large molecules. PM2.5 also increased the secretion of pro‐inflammatory cytokines by nasal epithelial cells. Budesonide could partially repair the damage, suggesting potential applications in clinical practice.


| INTRODUCTION
According to the World Health Organization, 1 air pollution is a major health threat worldwide. Particular matter 2.5 (PM 2.5 ) are fine particles containing a range of different components, including acids (such as nitrate and sulfates), organics, metals, and soil or dust particles. 2 Many studies have demonstrated that PM 2.5 is closely related to human diseases and mortality, causing systematic inflammation and affect the immune system. 3,4 As the first line of immunological defense, nasal epithelial cells provide a physical barrier to prevent the invasion of inhaled environmental pathogens, allergens, and other airborne irritants. 5 Apical junctional complexes, which consist of apical tight junctions (TJs) and underlying adherens junctions (AJs), are the most important part of the epithelial barrier.
The prevalence of the type-2 nasal inflammatory disease is rising rapidly. Previous studies have shown that dysregulation of the epithelial cell response or barrier function could drive chronic type-2 inflammation, 6 including many nasal inflammatory diseases. 7 Eosinophilic chronic rhinitis with nasal polyps (eos-CRSwNP) is a type-2 inflammatory disease characterized by a tendency for recurrence after surgery 8 and usually shows sensitivity to corticosteroid treatment. 9 Eos-CRSwNP patients are also more likely to have other allergic comorbidities and higher blood eosinophilia percentage. 10 Previous studies considered that PM 10 have more effect on the upper airway, recent studies began to raise attention on the effects of PM 2.5 . One recent epidemiological study in Europe 11 confirmed that among all the factors they assessed about air quality (PM 10 , PM 2.5 , PM corase , NO 2 , traffic load, and traffic intensity), PM 2.5 and PM 10 were the only two showed a clear relationship with severity of symptoms in the upper airway. Our previous study using noninflammatory human nasal epithelial cells demonstrated that PM 2.5 causes deficiencies in barrier integrity, whereas steroids fail to repair this damage. 12 However, whether PM 2.5 can exacerbate damage to inflammatory cells is still unclear.
Accordingly, in this study, we evaluated the impact of PM 2.5 on the air-liquid interface (ALI) of cultured eos-CRSwNP epithelial cells and further explored the underlying mechanisms and reparative effects of steroids.

| Patients
Patients who underwent nasal endoscopic surgery at Beijing Tongren Hospital from May to August 2018 were randomly enrolled in the study. The criteria of eos-CRSwNP subjects are listed as follows: (1) adults (18-70 years old) from Beijing or other cities in north China; (2) patients with endoscopically visible nasal polyp (NP); (3) patients with a history of smoking, taking oral/intranasal steroid, or systematic disease that might influence the results of this study were excluded; (4) all of the NP samples underwent histopathological inspection, and those with tissue eosinophils percentage less than 27% were excluded from this study.

| Transepithelial electrical resistance and paracellular flux measurement in ALI-cultured cells
After 21 days of ALI culture for cell differentiation, when the transepithelial electrical resistance (TER) reached a plateau of more than 300 Ω*cm, 2 the cultures were ready to be used for further experiments. The TER was measured using a Millicell-ERS Volt Ohm Meter (Millipore). Similar to our previous study, the cultures were exposed to 50 or 100 μg/ml PM 2.5 or culture medium as a control for 8 h each day in the apical chamber. The cells were then washed three times with PBS. To investigate the effects of budesonide, 1 μM budesonide (Sigma-Aldrich) was added to the apical chamber at 1 h before PM 2.5 exposure.
TER was measured at 0, 24, 48, and 72 h in triplicate after initiation of exposure. After 72-h TER measurement, the paracellular permeability of the cell layer was evaluated by adding 2 mg/ml fluorescein isothiocyanate (FITC)-labeled 4-kDa dextran (Sigma-Aldrich) to the apical layer of the ALI cells. After 12 h, the concentration of FITC in the basolateral medium was measured using an enzyme-linked immunosorbent assay (ELISA) reader (Mithra LB 940; Berthold Technologies) at 480 nm in duplicate. System (Applied Biosystems). Details of the primers used in this study are described in Table S1.

| Immunofluorescence staining
At 72 h after PM 2.5 exposure, ALI cultures were fixed in a 1:1 mixture of methanol-acetone for 10 min at 4°C. The cells were then washed in PBS and blocked with 5% skim milk. The methods for staining, examining, and analyzing were the same as described in our previous study. 12

| Statistical analysis
All data were analyzed using GraphPad Prism 8 software (GraphPad Software). Data are presented as median and interquartile range unless otherwise noted. Wilcoxon matched-pairs signed-rank tests were used to analyze differences between two paired groups, and Mann-Whitney U-tests were used to analyze two unmatched groups.
For the comparison of multiple groups, Friedman test was used, and posttest was performed to analyze the results from matched groups.
Results with p values of less than 0.05 were considered statistically significant.

| PM 2.5 caused impairment of barrier integrity in eos-CRSwNP epithelial cells
Compared with that of noninflammatory nasal epithelial cells, the TER baseline of eos-CRSwNP epithelial cells was lower after 21 days of ALI culture ( Figure S1A). Additionally, the FITC-labeled 4-kDa dextran concentration in the culture medium of eos-CRSwNP epithelial cells tended to be higher than that in the culture medium of noninflammatory nasal epithelial cells ( Figure S2).
Similar to its toxicity in normal nasal epithelial cells, concentrations of 50 and 100 μg/ml PM 2.5 resulted in the death of less than 10% eos-CRSwNP epithelial cells ( Figure S3); therefore, these concentrations were used for treatment of well-differentiated ALI cultures.
In the continuous monitoring of TER, the relative TER (ratio at each time point of TER to that at 0 h) did not change significantly ( Figure 1A) in each group. However, at 72 h after exposure, the relative paracellular flux, which was expressed as a ratio of FITClabeled 4-kDa dextran concentration in each group to that of culture medium group, showed a significant, concentrationdependent increase (50 μg/ml PM 2  Figure 1E).

| Impairment of barrier integrity caused by PM 2.5 could be partially repaired by budesonide
Although budesonide pretreatment did not influence the relative TER ( Figure 1B-D Figure 1E).  Figure S2).

| Changes in barrier integrity caused by PM 2.5 were different between noninflammatory nasal epithelial cells and eos-CRSwNP epithelial cells
Following budesonide pretreatment, the changes in TER caused by 100 μg/ml PM 2.5 showed much more difference between those two cell types ( Figure

| Effects of PM 2.5 on the expression of TJrelated mRNAs and proteins in eos-CRSwNP epithelial cells
In eos-CRSwNP epithelial cells exposed to 100 μg/ml PM 2.5 , the expression of claudin-1 was significantly decreased ( The expression levels of claudin-1, ZO-1, and occludin were then evaluated by confocal fluorescence microscopy and semiquantified using ImageJ software. After exposure to PM 2.5 , the signals for TJrelated proteins were weaker and ruptured. The budesonide pretreatment partially enhanced the expression of TJ-related proteins ( Figure 3).

| Effects of PM 2.5 on cytokine secretion by eos-CRSwNP epithelial cells
At the mRNA level, the expression levels of IL-8, IL-1α, and TIMP were significantly increased in both the 50 and 100 μg/ml PM 2.5 groups. Moreover, IL-1α was upregulated in a concentrationdependent manner. The expression of TSLP mRNA was significantly decreased in the 100 μg/ml PM 2.5 group, and IL-10 and matrix metalloproteinase-9 (MMP-9) tended to decrease, but the difference was not significant (Figure 4).
The detected concentrations of IL-25 and IL-33 were lower than the limit of detection for the Luminex kits used. The results of ELISA and Luminex analyses showed that in the cell culture medium, the concentrations of IL-8, IL-10, IL-1α, and TIMP-1 increased significantly in both the 50 and 100 μg/ml PM 2.5 groups. However, TSLP and MMP-9 expression levels did not change ( Figure 5).
As shown in Figure 6, when the cells were pretreated with budesonide 1 h prior to 100 μg/ml PM 2.5 exposure, IL-8, IL-10, IL-1α, TSLP, and TIMP-1 levels decreased significantly in the culture medium.
MMP-9 concentrations did not show a clear tendency. For noninflammatory human nasal epithelial cells exposed to PM 2.5 , budesonide pretreatment failed to induce any significant changes ( Figure S4).

| DISCUSSION
Epithelial cells are vital in both immune response regulation and host defense, functioning to mediate innate immunity and play important roles in adaptive immunity. 6,14-16 The impaired barrier function of the nasal epithelial has been shown to be related to type-2 inflammatory diseases, for example, nasal allergic disease and polyps. 17,18 However, the relationship between the increasing rate of type-2 inflammation F I G U R E 3 Expression of TJ-related proteins exposed to PM 2.5 and budesonide. (A) Representative images of immunofluorescence staining for claudin-1, occludin, and ZO-1 in ALI-cultured eos-CRSwNP epithelial cells (400� magnification). (B) Fluorescence intensities were evaluated using ImageJ software. *p < 0.05, **p < 0.01. ALI, air-liquid interface; eos-CRSwNP, eosinophilic chronic rhinitis with nasal polyps; PM 2.5 , particular matter 2.5; TJ, tight junction; ZO, zona occludens and deteriorating air pollution is still unclear. In this study, we used eos-CRSwNP epithelial cells as a model of type-2 inflammatory cells to explore the effects of PM 2.5 on the barrier function of epithelial cells and its relationship to adaptive immunity. We also investigated a mechanism to repair the damage.
The components of PM 2.5 vary in different locations, but the key components remain the same. 19,20 Here, we used PM 2.5 collected in Beijing and processed with a standard procedure used in many studies to ensure that the sample was representative. 21,22 CRSwNP is a heterogeneous disease that can be divided into four inflammatory patterns (Th1/Th2/Th17/Th22). 23 Similar to allergic rhinitis, eos-CRSwNP is a type-2 inflammatory disease 14,24 that is relatively difficult to cure. Therefore, it is reasonable to use epithelial cells from eos-CRSwNP to establish a relatively homogeneous type-2 inflammatory model in vitro. have been shown to regulate the leak pathway 25 but not the pore pathway. In this study, PM 2.5 increased paracellular flux but did not change TER, after 72 h of intermittent exposure, implying that PM 2.5 may have more influence on the leak pathway.
Our previous study showed that PM 2.5 affected both TER and paracellular flux in noninflammatory ALI-cultured cells. 12 The different results may support that eos-CRSwNP epithelial cells and noninflammatory cells exhibit different intrinsic characteristics, especially for the steroids treatment. Moreover, the TER baseline of eos-CRSwNP epithelial cells was already significantly lower than that of noninflammatory cells, suggesting that PM 2.5 may not further decrease TER for the barrier-defected eos-CRSwNP epithelial cells.
Budesonide did not cause any significant change in TER but was capable of repairing barrier function according to paracellular flux.
Although budesonide has been shown to strengthen TJs, its effects on TER typically do not reflect a treatment that was just applied In accordance with previous studies, glucocorticosteroid can increase the expression of occludin,35 In this study, although budesonide did not completely alleviate the effects of PM 2.5 on paracellular flux and TJ proteins, it partially reversed the trend, in contrast to the results in noninflammatory cells. Thus, steroid treatment may be essential for patients with eos-CRSwNPs and other nasal type-2 inflammatory diseases to prevent damage caused by PM 2.5 .
As a bridge between innate and adaptive immunity, cytokines secreted by epithelial cells play important roles in the initiation of inflammation. 36 IL-1α is a major initiation factor of many inflammatory processes and is referred to as an "alarmin" and a critical dangerassociated molecular pattern. 37 It was reported that IL-1α can promote the secretion of Th1-type cytokines, such as IL-8, 38 and stimulate the production of pro-Th2 cytokines, such as IL-25, IL-33, and granulocyte macrophage colony-stimulating factor. 6 Although rarely reported in the respiratory system, previous studies have shown that PM 2.5 can promote the secretion of IL-1α in skin and blood. [39][40][41] In this study, we could not exclude the possibility that IL-1α may be the initiation promotor of IL-8, and further studies are needed to explore these mechanisms.
IL-10 is an anti-inflammatory cytokine that is secreted by respiratory epithelial cells. 42,43 IL-10 can directly suppress the antigen-presenting function of antigen-presenting cells and inhibit the secretion of pro-inflammatory cytokines, including IL-1α. 44 Our previous in vivo study showed that IL-10 is decreased in nasal secretions after PM 2.5 exposure but increased in the supernatants of peripheral blood mononuclear cells after incubation with PM 2.5 . 45 In this study, we assumed that elevated levels of IL-10 may be a selfprotective reaction in epithelial cells, supporting the self-regulatory function of the epithelial cells without interfering with adaptive immune cells.
Similar to our results observed in noninflammatory cells, 12 the secretion of TIMP-1, an inhibitor of MMP-9, was increased in type-2 inflammatory cells upon exposure to PM 2.5 . However, we did not observe significant changes for TSLP, potentially because of the higher baseline concentration in type-2 inflammatory cells. 46 For the noninflammatory epithelial cells, budesonide could not repaire the barrier disruption caused by PM 2.5 , which is consistent with our previous results. 12 In this study, we also found budesonide could not alter the cytokine production of noninflammatory cells exposed to PM 2.5 , but significantly reversed the changes of eos-CRSwNP epithelial cells. Thus, we assume that the influence of F I G U R E 5 Cytokine concentrations in the culture medium after exposure to PM 2.5 . After intermittent treatment with 50 or 100 μg/ml PM 2.5 for 72 h, levels of IL-8, IL-10, IL-1α, TIMP-1, TSLP, and MMP-9 in the culture medium were evaluated. *p < 0.05, **p < 0.01. IL, interleukin; MMP, matrix metalloproteinase; PM 2.5 , particular matter 2.5; TIMP, tissue inhibitor of metalloproteinase; TSLP, thymic stromal lymphopoietin MA ET AL. CRSwNP epithelial cells exposed to PM 2.5 is interesting and calls for further exploration.

| CONCLUSION
In summary, our study demonstrated that PM 2.5 could further impair the barrier function of eos-CRSwNP epithelial cells and increase the permeability of the cells to large molecules. PM 2.5 could increase the secretion of both type-1 and type-2 cytokines from nasal epithelial cells in situ and promote remolding. Finally, although corticosteroid has been already widely used for CRSwNP, our finding that budesonide partially reversed these changes in barrier function and cytokine levels caused by PM 2.5 implies its role in preventing disease progression in high PM 2.5 situation.

ACKNOWLEDGMENTS
We thank the Institute for Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention for providing the PM 2.5 samples.

CONFLICT OF INTEREST
The authors have no conflict of interest.

AUTHOR CONTRIBUTIONS
Siyuan Ma and Mu Xian performed the experiments, analyzed the data, and prepared the manuscript; Yang Wang participated in the experiments and data collection; Chengshuo Wang and Luo Zhang were responsible for the overall study design, data analysis, and manuscript revision.

F I G U R E 6
Effects of budesonide on cytokine concentrations in the culture medium. Cells were pretreated with or without budesonide before exposure to 100 μg/ml PM 2.5 , and the concentrations of IL-8, IL-1α, TIMP-1, IL-10, TSLP, and MMP-9 in the culture medium were evaluated. *p < 0.05. IL, interleukin; MMP, matrix metalloproteinase; PM 2.5 , particular matter 2.5; TIMP, tissue inhibitor of metalloproteinase; TSLP, thymic stromal lymphopoietin