Nasal Epithelial Cells Activated with Alternaria and House Dust Mite Induce Not Only Th2 but Also Th1 Immune Responses.

Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by mucosal inflammation. Airborne allergens are associated with upper and lower airway inflammatory disease. We investigated the effects of airborne allergen stimulation in the nasal epithelial cells and their effect on the peripheral blood mononuclear cells’ (PBMCs) Th immune polarization. Interleukin (IL)-10, IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) levels were determined using the enzyme-linked immunosorbent assay (ELISA) in nasal polyp tissues. Cultured primary nasal epithelial cells were stimulated with Alternaria alternata, Aspergillus fumigatus, Dermatophagoides pteronyssinus (DP), and Dermatophagoides farina (DF) for 48 hours. IL-6, IL-25, IL-33, and TSLP production were measured by ELISA, and the nuclear factor-κB (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) expression were determined by western blot analyses. PBMCs were cultured with nasal epithelial cell-conditioned media (NECM), and IL-5, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were measured. Innate lymphoid type2 cells (ILC2) were analyzed with flowcytometry. IL-25, IL-33, and TSLP levels were significantly higher in eosinophilic nasal polyps. Alternaria, DP, and DF enhanced IL-33 and TSLP production from the nasal epithelial cells through the NF-κB, AP-1, and MAPK pathway. NECM induced IL-5, IFN-γ, and TNF-α production from PBMCs, without increasing ILC2 expression. Alternaria and house dust mites enhanced the chemical mediator production from nasal epithelial cells and these allergens may induce not only Th2 inflammatory responses but also Th1 inflammatory responses in the nasal mucosa.


Production of Chemical Mediators from Nasal Epithelial Cells by Airborne Allergens
To determine the adequate stimulation time, nasal epithelial cells were cultured with 50 ug/ml and 100 ug/ml of Alternaria alternata for 6, 24, and 48 h. IL-33 and TSLP production significantly increased after 24 and 48 h of stimulation, but not IL-25 ( Figure 2). Without stimulation, IL-33 tended to decrease over time. Then, we stimulated nasal epithelial cells with airborne allergens for 48 h for further studies. Kinetic studies to determine the optimal stimulation interval. IL-33, TSLP, and IL-25 production were significantly increased after 24 and 48 h stimulation of the nasal epithelial cells with 50 and 100 ug/ml of Alternaria. Negative control (NC); 100 ug/ml of Alternaria (Alt100); 50 ug/ml of Alternaria (Alt50); * p < 0.05 compared with NC.

Production of Chemical Mediators from Nasal Epithelial Cells by Airborne Allergens
To determine the adequate stimulation time, nasal epithelial cells were cultured with 50 ug/ml and 100 ug/ml of Alternaria alternata for 6, 24, and 48 h. IL-33 and TSLP production significantly increased after 24 and 48 h of stimulation, but not IL-25 ( Figure 2). Without stimulation, IL-33 tended to decrease over time. Then, we stimulated nasal epithelial cells with airborne allergens for 48 h for further studies. Nasal epithelial cells were activated with 50 and 100 ug/ml of Alternaria, Aspergillus, DP, and DF for 48 h. IL-25 production was not increased by airborne allergens. Alternaria and house dust mites (DP and DF) enhanced IL-33, TSLP, and IL-6 production from nasal epithelial cells. However, Aspergillus only influenced the IL-6 production ( Figure 3). Therefore, Alternaria, DP, and DF were used for further experiments. Nasal epithelial cells were activated with 50 and 100 ug/mL of Alternaria, Aspergillus, DP, and DF for 48 h. IL-25 production was not increased by airborne allergens. Alternaria and house dust mites (DP and DF) enhanced IL-33, TSLP, and IL-6 production from nasal epithelial cells. However, Aspergillus only influenced the IL-6 production ( Figure 3). Therefore, Alternaria, DP, and DF were used for further experiments.

Expression of Transcription Factors from Nasal Epithelial Cells by Airborne Allergens
We determined the expression of NF-κB, activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) transcription factor with Alternaria, DP, and DF, because of these allergens' results in cytokine IL-33 and TSLP production, which promote a Th2 immune response. Alternaria enhanced phosphorylated NF-κB and phosphorylated C-Jun expressions, while DP and DF enhanced NF-κB, phosphorylated NF-κB, phosphorylated C-Jun, and p38 from nasal epithelial cells. However, they did not influence extracellular signal-regulated kinase (ERK), phosphorylated ERK, jun kinase enzyme (JNK), and phosphorylated JNK expression ( Figure 4).

Expression of Transcription Factors from Nasal Epithelial Cells by Airborne Allergens
We determined the expression of NF-κB, activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) transcription factor with Alternaria, DP, and DF, because of these allergens' results in cytokine IL-33 and TSLP production, which promote a Th2 immune response. Alternaria enhanced phosphorylated NF-κB and phosphorylated C-Jun expressions, while DP and DF enhanced NF-κB, phosphorylated NF-κB, phosphorylated C-Jun, and p38 from nasal epithelial cells. However, they did not influence extracellular signal-regulated kinase (ERK), phosphorylated ERK, jun kinase enzyme (JNK), and phosphorylated JNK expression ( Figure 4).

Expression of Transcription Factors from Nasal Epithelial Cells by Airborne Allergens
We determined the expression of NF-κB, activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) transcription factor with Alternaria, DP, and DF, because of these allergens' results in cytokine IL-33 and TSLP production, which promote a Th2 immune response. Alternaria enhanced phosphorylated NF-κB and phosphorylated C-Jun expressions, while DP and DF enhanced NF-κB, phosphorylated NF-κB, phosphorylated C-Jun, and p38 from nasal epithelial cells. However, they did not influence extracellular signal-regulated kinase (ERK), phosphorylated ERK, jun kinase enzyme (JNK), and phosphorylated JNK expression ( Figure 4).

Effects of Transcription Factor Inhibitors on Chemical Mediator Production from Nasal Epithelial Cells
Because Alternaria, DP, and DF enhanced the IL-33 and TSLP production by increasing the NF-κB, C-Jun, and p38 expression, activation of transcription factors was blocked along with their inhibitors. Alternaria-induced IL-33 and TSLP productions were significantly inhibited by the NF-κB, AP-1, and MAPK inhibitors. DP-induced IL-33 was also significantly inhibited by these three inhibitors. However, DF induced IL-33 production was not suppressed by the MAPK inhibitor. DP and DF-induced TSLP productions were significantly inhibited by the AP-1 and MAPK inhibitors ( Figure 5).

Effects of Transcription Factor Inhibitors on Chemical Mediator Production from Nasal Epithelial Cells
Because Alternaria, DP, and DF enhanced the IL-33 and TSLP production by increasing the NF-κB, C-Jun, and p38 expression, activation of transcription factors was blocked along with their inhibitors. Alternaria-induced IL-33 and TSLP productions were significantly inhibited by the NF-κB, AP-1, and MAPK inhibitors. DP-induced IL-33 was also significantly inhibited by these three inhibitors. However, DF induced IL-33 production was not suppressed by the MAPK inhibitor. DP and DF-induced TSLP productions were significantly inhibited by the AP-1 and MAPK inhibitors ( Figure 5).
After 72 h of treatment with NECM, PBMCs were stained to identify ILC2 expression. Lymphocytes were identified from whole PBMCs, and Lin-cells were gated and further assessed for co-expression of CD127 and CRTH2. ILC2s were identified as Lin-CRTH2+CD127+ lymphocytes. The number of ILC2s was not significantly increased in PBMCs after the NECM treatment, compared with the negative control. Figure 6 shows the representative flowcytometric finding of PBMCs for ILC2.  Effects of transcription factor inhibitors on the IL-33 and TSLP production from nasal epithelial cells. Alternaria induced IL-33 and TSLP production were significantly inhibited by BAY 11-8082 (NF-κB inhibitor), curcumin (activator protein 1 (AP-1) inhibitor), and SB 203580 (mitogen-activated protein kinase (MAPK) inhibitor). DP and DF-induced IL-33 and TSLP production was also suppressed with transcription factor inhibitors. Negative control (NC); not treated (NT), 100; 100 ug/mL, * p < 0.05 compared with NT.
After 72 h of treatment with NECM, PBMCs were stained to identify ILC2 expression. Lymphocytes were identified from whole PBMCs, and Lin-cells were gated and further assessed for co-expression of CD127 and CRTH2. ILC2s were identified as Lin-CRTH2+CD127+ lymphocytes. The number of ILC2s was not significantly increased in PBMCs after the NECM treatment, compared with the negative control. Figure 6 shows the representative flowcytometric finding of PBMCs for ILC2.
After 72 h of treatment with NECM, PBMCs were stained to identify ILC2 expression. Lymphocytes were identified from whole PBMCs, and Lin-cells were gated and further assessed for co-expression of CD127 and CRTH2. ILC2s were identified as Lin-CRTH2+CD127+ lymphocytes. The number of ILC2s was not significantly increased in PBMCs after the NECM treatment, compared with the negative control. Figure 6 shows the representative flowcytometric finding of PBMCs for ILC2.

Discussion
CRS is phenotypically divided into CRSwNP and CRSsNP. Th2 inflammation is commonly associated with CRSwNP, whereas Th1 inflammation is associated with CRSsNP. However, CRS had a combination of Th1, Th2, and other inflammatory patterns with mixed endotypes [15]. ILC2s are increased in CRSwNP or eosinophilic CRS and epithelial cell-derived IL-25, IL-33, and TSLP are the key cytokines to induce Th2 cytokine production by ILC2s [16,17]. Although Alternaria, DP, and DF enhanced the IL-33 and TSLP production from nasal epithelial cells, their conditioned media induced the Th1, Th2, and pro-inflammatory cytokine production from PBMCs.
IL-25, IL-33, and TSLP were increased in the tissue sample of CRS and these mediators were more strongly expressed in ENP, which is consistent with other studies [14,18]. TSLP was also significantly higher in NENP, compared with control tissues. Among the three cytokines, IL-33 was most strongly expressed in sinonasal mucosa. CRS and nasal polyps are developed with the predisposition of allergic airway inflammation [19]. Because airborne allergens have been known to induce the chemical mediator production from nasal epithelial cells, we stimulated nasal epithelia cells with two different fungi and house dust mites. Alternaria, DP, and DF induced IL-33 and TSLP production from nasal epithelial cells but not IL-25. Although IL-25, IL-33, and TSLP are expressed in the epithelial area of sinonasal tissues, previous studies have shown that IL-25 is mainly produced by Th2 cells and mast cells [20,21]. These findings suggest that airborne allergens may increase tissue IL-25 levels through the interaction of epithelial cells and other IL-25 producing inflammatory cells.
Toll-like receptor (TLR) 1 is associated with the production of IL-25 and IL-33, while TLR2 is associated with TSLP production, and IL-33 is induced by treatment with the TLR2, TLR4, and TLR9 ligands [22][23][24]. Alternaria has been known to induce chemical mediator production in nasal epithelial cells through the TLR2, TLR3, or TLR4 pathways [25]. House dust mites (HDM) can activate respiratory epithelial cells through TLR 2 and TLR4 [23]. Alternaria and HDM can activate respiratory epithelial cells through the NF-κB, AP-1, and MAPK pathways [26,27]. In this study, Alternaria and HDM induced the IL-33 production and TSLP interaction through the NF-κB, AP-1, and MAPK pathways. NF-κB, AP-1, and MAPK inhibitors suppressed the production of IL-33 and TSLP from nasal epithelial cells. These transcription factor inhibitors could be used as therapeutic or preventive agents for CRS. Moreover, epithelial cell membrane TLRs may be associated with IL-33 and TSLP production from nasal epithelial cells.
NECM produced by Alternaria and HDM induced IL-5, TNF-α, and INF-γ from PBMCs. In this study, IL-25, IL-33, and TSLP in NECM were produced by nasal epithelial cells and they can also produce several other chemical mediators by Alternaria and HDM, such as granulocyte macrophage colony stimulating factor (GM-CSF), IL-1β, IL-8, etc. [22,25]. The activation of nasal epithelial cells may induce not only Th2 inflammation but also Th1 inflammation in nasal mucosa through the interaction between structural and inflammatory cells. In CRS with or without nasal polyps, although CRSwNP has Th2 dominant and CRSsNP has Th1 dominant endotype characteristics; approximately 30% of CRS had mixed endotypes [15]. In the nasal mucosa, which contains epithelial cells, structural cells, and other inflammatory cells, exogenous pathogenic stimulation simultaneously triggers not only epithelial cells but also structural cells and the produced chemical mediators may have autocrine or paracrine effects, which enhance the inflammatory reaction in the nasal mucosa. Because Alternaria and HDM enhanced IL-33 and TSLP production in nasal epithelial cells, NECM is expected to increase Th2 cytokines and ILC2 in PBMCs. However, NECM induced Th1 and Th2 cytokine production, but did not influence ILC2 expression. Although Alternaria and HDM induced IL-33 and TSLP production from nasal epithelial cells, the concentration of these two chemical mediators in cultured media was much lower than that of nasal polyp tissues. The cellular proportion of ILC2 in PBMCs is very low and was less than 0.1% in patients with allergic rhinitis. The concentration of epithelial cell-derived chemical mediators can insufficiently polarize PBMCs to ILC2, or the polarized ILC2 number might not be enough to detect with the flowcytometric technique. We used two different allergens, fungi and HDM. Although these allergens can activate nasal epithelial cells, antigens contain several different protein and enzymatic allergens, which interact with nasal epithelial cells through various immune receptors, such as TLR, protease activated receptors, G-protein-coupled receptors, etc., and induce the production of chemical mediators through different transcription pathways. Although IL-33 and TSLP productions were enhanced by Alternaria and HDM, their exact immunopathologic mechanism may be different. In this study, we used normal nasal epithelial cells, which could not represent the CRS or nasal polyp epithelial cells. If we used pathologic mucosal epithelial cells, they might show different or stronger immune responses against airborne allergens.

Chemical Mediators in CRSwNP
The chemical mediators were evaluated from 38 subjects. A total of 30 nasal polyps were obtained from 14 patients with eosinophilic NP (ENP) and 16 with non-eosinophilic NP (NENP). Eight uncinate process mucosa were used as the control tissue, which was obtained from patients with a medial blowout fracture or endoscopic dacryocystostomy. CRSwNP was confirmed by computed tomography and endoscopic examinations in accordance with the "European position paper on rhinosinusitis and nasal polyps 2020" guidelines [19]. All patients were aged above 18 years. Patients were excluded if they had received systemic or topical steroids, and had taken antibiotics or antihistamines during the four weeks that preceded the study. This study was approved by the institutional review board of Daegu Catholic University Medical Center, and all patients signed a consent form that outlined the objectives of the study (CR-17-152, 13 December 2017).
Freshly obtained tissue specimens were homogenized and centrifuged at 3000 rpm for 10 minutes at 4 • C. After centrifugation, 500 µL aliquots of the supernatant were assayed for IL-10, IL-25, IL-33, and TSLP using commercially available enzyme linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). The chemical mediator data representing protein concentrations was calculated based on 40 µg of protein.

Stimulation of Primary Nasal Epithelial Cells with Airborne Allergens
Primary nasal epithelial cells were isolated from the inferior turbinate of 10 subjects (6 men and 4 women; 45.2 ± 11.8 years) during the septal surgery. Subjects were excluded if they had an active and fixed with 4% paraformaldehyde. ILC2s were identified as Lin-CRTH2+CD127+ lymphocytes with a flowcytometer machine (Beckman Coulter, Hercules, CA, USA).

Statistical Analysis
All measured parameters are expressed as mean ± standard deviation. Clinicopathological differences between eosinophilic and non-eosinophilic CRS were determined using two sample t-tests and a chi-squared test. A one-way analysis of variance followed by a Tukey's test was performed for normally distributed data, and the Kruskal-Wallis test with post-hoc Bonferroni-Dunn test was performed for non-normally distributed data. The analysis was conducted with the Statistical Package of Social Sciences software version 21 (SPSS Inc., Chicago, IL, USA). Results with p < 0.05 were regarded as statistically significant.

Conclusions
The results of this study demonstrated that Alternaria, and HDM induced IL-33 and TSLP production from nasal epithelial cells through the NF-κB, AP-1, and MAPK pathways. Alternaria-induced IL-33 and TSLP productions were significantly suppressed by NF-κB, AP-1, and MAPK inhibitors, however HDM-induced TSLP production was not suppressed by the NF-κB inhibitor. These findings suggest that Alternaria and HDM may induce the production of chemical mediators in a different immune pathway.
NECM produced by nasal epithelial cells activated with Alternaria and HDM enhanced the Th1, Th2, and pro-inflammatory cytokine production from PBMCs. Although we focused on IL-33 and TSLP production from nasal epithelial cells, many other chemical mediators could be produced by Alternaria and HDM. These finding suggest that although Alternaria and HDM are commonly known for being part of a Th2 inflammation associated with an allergen, these allergens could induce Th2 and other inflammation responses in the nasal mucosa.

Conflicts of Interest:
All authors have no conflicts of interest to disclose.