Solitary chemosensory cells amplify eosinophilic inflammation via PAR-2 activation in house dust mite-sensitized allergic rhinitis

Abstract

Background

Recent studies have shown that solitary chemosensory cells (SCCs) are rare nasal epithelial cells effectively responding to pathogens and are the predominant epithelial sources of IL-25. We investigated the roles of SCCs in nasal eosinophilic inflammation in house dust mite (HDM)-sensitized allergic rhinitis (AR).

Methods

Epithelial phenotypes were detected in nasal mucosal specimens from control participants and HDM-AR patients. Differentiated human nasal epithelial cells (hNECs) were exposed to HDM extract Dermatophagoides pteronissinus (Derp), with or without a Protease-activated receptor (PAR-2) antagonist, and epithelial phenotypes were characterized. IL-25 receptor (IL-17RA and IL-17RB) expression in eosinophils was analyzed in human nasal mucosal cells (hNMCs). Finally, the expression of PAR-2 and POU domain class 2 transcription factor 3 (POU2F3) in SCCs within human nasal mucosal epithelium was evaluated.

Results

POU2F3⁺ SCCs and MUC5AC⁺ goblet cells were enriched in the nasal mucosa, along with elevated IL-25 levels in the nasal secretions of HDM-AR patients. Exposure of hNECs to Derp increased SCCs expansion and mucin production. Moreover, IL-25 protein potently promoted mucin production in hNECs and IL-25 receptor expression in eosinophils of hNMCs. PAR-2 expression was significantly enhanced in Derp-exposed hNECs, and inhibiting PAR-2 activation reversed the Derp-induced effects in hNECs and hNMCs. Finally, PAR-2 expression levels significantly increased in nasal epithelium from HDM-AR patients, correlating with POU2F3⁺ SCCs density and nasal eosinophil percentage.

Conclusions

These findings demonstrated that SCCs could sense HDM and produce IL-25, triggering mucin overproduction and eosinophilic IL-25 receptor expression via PAR-2 activation, which suggesting that SCCs might be powerful immunomodulators of nasal eosinophilic inflammation in HDM-AR.

Introduction

Allergic rhinitis (AR) is a chronic inflammatory disease of the upper airway characterized by sneezing, nasal congestion, nasal itching, and rhinorrhea.¹ Its prevalence ranges from 5% to 50% globally, posing increasing health challenges.^2,3 Inhaled allergens, particularly house dust mites (HDM), drive the development and exacerbation of AR.¹ HDM, which is common in perennial AR, can cause severe disease manifestations through its protease activity.^4,5 The airway mucosal epithelium acts as an immunological barrier against inhaled allergens and maintains mucosal homeostasis.⁶ Epithelial cells recognize HDM and initiate an innate immune response by releasing alarmins, including IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 to prime the type 2 adaptive immune response 7, 8, 9, 10. However, the mechanisms of innate recognition of HDM by epithelial cells and the resulting cellular changes contributing to AR inflammation are not fully understood.

A rare population of epithelial cells with a well-developed apical microvillus apparatus has been identified for sentinel functions in the intestine.¹¹ These cells, which depend on the transcription factor POU2F3 for differentiation, are known as “solitary chemosensory cells (SCCs)” in the upper airways and “tuft cells” in the lungs and intestines.^12,13 Tuft cells orchestrate type 2 immunity by releasing multiple effector molecules, such as IL-25 and cysteinyl leukotrienes, in the gut 14, 15, 16 and lungs.¹⁷ Kotas et al found increased SCC-related transcripts in chronic rhinosinusitis with nasal polyps (CRSwNP), correlating with IL-13 signatures and the Lund-Mackay score.¹⁸ SCCs are the predominant epithelial source of IL-25,¹⁹ a key cytokine that initiates type 2 inflammation in the upper airway²⁰ and induces IL-5 and IL-13 expression in the peripheral blood of patients with HDM-AR.²¹ These studies suggest that nasal SCCs may play a pivotal role in mucosal inflammation in HDM-AR.

Protease-activated receptors (PARs), a family of 7-transmembrane G protein-coupled receptors, are activated by exogenous proteases from aeroallergens,²² including HDM.²³ PAR-2 plays a critical role in sensing airborne allergens, such as fungi and cockroaches, and mediates allergen sensitization and pro-inflammatory cytokine production.^24,25 Studies have shown a significant increase in PAR-2 in the bronchial epithelium of patients with asthma,²⁶ and PAR-2 blockade has been found to prevent airway remodeling and allergic inflammation in cockroach extract-induced asthmatic mice.^27,28 Moreover, PAR-2 activation in human small airway epithelial cells contributes to eosinophilic recruitment,²⁹ highlighting its crucial role in allergic respiratory inflammation. Although the roles of SCCs and PAR-2 in airway have been separately studied as mentioned above, little is known about their interaction mechanisms. Moreover, the roles of epithelial PAR-2 on SCCs and the downstream inflammatory response in HDM-AR remain unclear.

Here, we explored the cellular alterations and roles of nasal SCCs in the mucosal inflammation of HDM-AR using the HDM-exposed models of differentiated human nasal epithelial cells and human nasal mucosal cells. Our findings reveal a functional role of SCCs in sensing HDM allergens and amplifying nasal chronic eosinophilic inflammation in the nasal mucosa via PAR-2 activation in AR, which may provide novel therapeutic target for treating patients with HDM-AR.

Material and methods

Human biospecimen collection

All biospecimens were obtained from Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Nasal swab samples were obtained from outpatients with HDM-AR (n = 17) and healthy volunteers (n = 17) using sterile 100 mm flocked swabs (Copan Diagnostics, Murrieta, CA, USA) on the surface of the inferior nasal turbinate. Nasal secretions were collected from HDM-AR patients (n = 15) and healthy volunteers (n = 15) using polyurethane sponges (Huayi Foam, Changzhou, China) against the middle meatus for 5 min. Peripheral blood samples were collected from outpatients with HDM-AR (n = 5). Biopsy specimens of the nasal mucosa were obtained from CRSwNP patients with HDM-AR (n = 8) and CRSwNP patients without AR (n = 22) who underwent endoscopic surgery. The posterior end of the inferior turbinate was collected from control participants (n = 8) without sinusitis or AR who underwent septal surgery. The diagnosis of HDM-AR was confirmed by symptoms, including nasal congestion, rhinorrhea, sneezing, and nasal itching, and positive skin prick test results for HDM. Clinical characteristics and sampling numbers are detailed in Table E1, Table E2 and Table E3. Patients were free of systemic disease and had not receive any antibiotic or glucocorticoid for at least 4 weeks prior to the study. This study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology [NO. 2024-0036-01]. All participants provided written informed consent.

Table 1.

Clinical characteristics of control participants and HDM-AR patients.

	Control participants (n = 32)	HDM-AR patients (n = 32)
Clinical data
Sex (male/female)	13/19	11/21
Age (y), mean ± SD	27.00 ± 8.68	34.81 ± 13.87
Asthma (no.)	0	3
Smoker (no.)	3	7
Positive HDM-specific SPT results (no.)	0	32
Methodologies used
Nasal swab sampling (no.)	17	17
Nasal secretions measurements (no.)	15	15

HDM, house dust mite; SPT, skin prick test

Table 2.

Clinical characteristics of control participants, non-AR and HDM-AR patients.

	Control participants (n = 8)	Non-AR patients (n = 22)	HDM-AR patients (n = 8)
Clinical data
Sex (male/female)	6/2	12/10	6/2
Age (y), mean ± SD	27.63 ± 9.38	38.73 ± 14.93	35.75 ± 16.28
Asthma (no.)	0	1	2
Smoker (no.)	0	6	3
CRSwNP	0	22	8
Positive HDM-specific SPT results (no.)	0	0	8
Methodologies used
ALI epithelial culture (no.)	0	9	0
Nasal mucosal cell stimulation assays (no.)	0	5	0
Tissue staining (no.)	8	11	8

HDM, house dust mite; SPT, skin prick test; ALI, air-liquid interface

Table 3.

Clinical characteristics of HDM-AR patients who provided peripheral blood samples.

	HDM-AR patients (n = 5)
Clinical data
Sex (male/female)	2/3
Age (y), mean ± SD	27.8 ± 3.43
Asthma (no.)	0
Smoker (no.)	0
Positive HDM-specific SPT results (no.)	5

HDM, house dust mite; SPT, skin prick test

Human nasal epithelial cell culture

Fresh nasal polyp specimens were cut and digested to obtain human nasal mucosal cells (hNMCs). hNMCs were seeded and cultured in an epithelial growth medium (PneumaCult-Ex; STEMCELL Technologies, Vancouver, Canada) and then transferred to an air-liquid interface (ALI) system (approximately 1 × 10⁵ cells per well). After 28 days in the ALI system, fully differentiated human nasal epithelial cells (hNECs) in epithelial differentiation medium were used for in vitro stimulation studies including HDM extract Derp (D. pteronyssinus; 200 μg/mL) (Geer Laboratories, Lenoir, NC), PAR-2 antagonist AZ3451 (50 μM) (Selleck, Houston, TX) or human recombinant IL-25 protein (1, 10, 100 ng/ml) (BioLegend, San Diego, CA) treatment for 48 h. Detailed methods are described in the Supplementary Methods section.

Nasal swab processing and measurement

Nasal swabs were processed to obtain nasal mucosal epithelial cell suspensions. The cells were subsequently used for RNA extraction or cytological staining to detect POU2F3 and MUC5AC proteins. Detailed methods are provided in the Supplementary Methods section.

Immunofluorescent staining and evaluation

POU2F3, MUC5AC and βIV-Tubulin proteins were detected in differentiated hNECs by using immunofluorescent staining. Detaied methods are described in the Supplementary Methods section.

Cellular stimulation experiment

hNMCs obtained from fresh nasal tissue were seeded in epithelial growth medium in 6-well culture plates and treated with human recombinant IL-25 protein (100 ng/ml), Derp (200 μg/mL) with or without PAR-2 antagonist AZ3451 (50 μM) for 48 h.

Eosinophils were purified from human peripheral blood by EasySep Direct Human eosinophil sorting kit (STEMCELL Technologies, Vancouver, Canada) following the manufacturer's instructions and seeded in 24-well culture plates and treated with human recombinant IL-25 protein (100 ng/ml) for 2 h.

Hematoxylin-eosin staining and immunohistochemistry staining

Nasal tissue sections were stained with hematoxylin-eosin to assess eosinophil infiltration. POU2F3 and PAR-2 proteins were detected in nasal mucosa by using immunohistochemistry staining. Detailed methods are described in the Supplementary Methods section.

RNA isolation, quantitative RT-PCR and RNA-seq experiment

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). For quantitative RT-PCR, RNA was reverse transcribed to cDNA with a First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). cDNA was then detected using ChamQ SYBR qPCR Master Mix (Vazyme). For bulk RNA-seq, Total RNA from the differentiated hNECs from the nontreated group and the Derp-exposed group was sequenced and performed the differentially expressed genes (DEGs) and enrichment analysis. Detailed methods are included in the Supplementary Methods section.

ELISA assays

Human IL-25 protein levels in nasal secretions and supernatants for hNECs collected from the apical chambers of the ALI culture system were measured with an ELISA kit (BosterBio, Pleasanton, CA, USA) following the manufacturer's instructions.

Western blotting

Cells were isolated by using RIPA buffer (Thermo Fisher Scientific). Details of western blotting analysis for POU2F3, DCAMKL1 and IL-25 proteins are shown in the Supplementary Methods section.

Flow cytometry

Untreated and IL-25-treated hNMCs and purified eosinophils were harvested, stained and washed twice with cold phosphate buffer solution. Flow cytometric analysis was performed to detect IL-25 receptor expression status of the eosinophils by using the CD45⁺CD16⁻Siglec-8⁺IL-17RA⁺IL-17RB⁺ panel³⁰ in hNMCs and the IL-17RA⁺IL-17RB⁺ panel in human eosinophils. Antibodies used in the flow cytometry assays are listed in Table 4. Detailed methods are described in the Supplementary Methods section.

Table 4.

Antibody information.

Target antigen	Species	Clone	Conjugate	Manufacturer
CD45	Mouse	2D1	APC-Cy7	BD Pharmingen
CD16	Mouse	3G8	FITC	Biolegend
Slglec-8	Mouse	7C9	APC	Biolegend
IL-17RA	Mouse	W15177A	PE/Cyanine7	Biolegend
IL-17RB	Mouse	170220	PE	R&D systems

Statistical analysis

Data analysis utilized GraphPad Prism 9.0 software (GraphPad Software, La Jolla, CA, USA). Comparison between the 2 groups performed with Mann-Whitney U test or Student's t-test, while correlation analysis employed the Spearman r test. P value < 0.05 indicated statistically significant.

Results

Hyperplasia of SCCs and goblet cells in HDM-AR nasal mucosa versus control mucosa

To investigate the epithelial phenotype in the nasal mucosa from patients with HDM-AR (n = 17) and control participants (n = 17), we collected nasal swab samples for RT-qPCR and immunofluorescence staining. Clinical characteristics are listed in Table 1. Our results indicated significantly elevated expression levels of SCC signature genes (POU2F3 and doublecortin-like kinase 1 [DCAMKL1]) and the mucin marker gene (MUC5AC) in HDM-AR-derived cells compared to cells from the control group (Fig. 1A). Higher percentages of POU2F3⁺ SCCs and MUC5AC⁺ goblet cells were observed in patients with HDM-AR group compared to control group (Fig. 1B and C). ELISA assays revealed that IL-25 protein levels were significantly higher in nasal secretions from patients with HDM-AR than in those from control participants (Fig. 1D). Collectively, these findings reveal a notable phenotype of SCC and goblet cell hyperplasia in the nasal epithelium of HDM-AR patients.

Fig. 1.

Aberrant SCC and goblet cell hyperplasia phenotype in nasal mucosa from patients with HDM-AR (A). POU2F3, DCAMKL1 and MUC5AC mRNA levels in nasal swab specimens from normal control (NC) (n = 17) and HDM-AR (n = 17) donors. (B–C). POU2F3 and MUC5AC proteins were detected and analyzed in total nasal mucosal cells obtained by nasal swabs from NC (n = 17) and HDM-AR (n = 17) donors (scale bar = 50 μm). (D). Release of IL-25 protein in nasal secretions from NC (n = 15) and HDM-AR (n = 15) donors. Data are presented as medians with interquartile ranges. The Mann-Whitney U test was used in (A–D)

Transcriptional profiles of Derp-stimulated hNECs and control hNECs

To further evaluate the influence of HDM on nasal epithelial cell transcriptional profiles, we stimulated differentiated hNECs with or without Derp, a major HDM component. Total RNA from both groups underwent microarray analysis.

The individual samples were separated into 2 distinct groups the Derp group and non-treated (NT) group. A Venn diagram showed the number of genes specifically expressed in the Derp group (n = 1789) and NT group (n = 1916) (Fig. 2A). A Heatmap displayed differentially expressed genes (DEGs) in the hNECs from the Derp group and NT group. (Fig. 2B). Analysis of DEGs revealed 496 upregulated and 260 downregulated genes in hNECs from the Derp group compared to the NT group (Fig. 2C). DEGs were enriched in biological processes related to inflammation regulation (such as, cytokine activity and regulation of leukocyte migration) (Fig. 2D). Moreover, KEGG analysis identified pathways involving cytokine-cytokine receptor interaction, IL-17 signaling, and NF-κB signaling in Derp-stimulated hNECs (Fig. 2E). Genes related to the WNT pathway (WNT7A), type I IFN signaling (eg, IFIT1, IFIT2, IFI6, STAT1), and anti-inflammation (eg, CD300A, LGALS1) were decreased, whereas genes linked to the NOTCH pathway (NOTCH4), SCC differentiation (POU2F3), mucin production (MUC2) and pro-inflammatory response (eg, ALOX15B, ALOXE3, CCL17, CCL20) were significantly increased in Derp-stimulated hNECs compared to control hNECs (Fig. 2F). Taken together, our results indicate that HDM induces SCC signature gene expression and promotes mucin overproduction and pro-inflammatory phenotypes in hNECs.

Fig. 2.

Bulk RNA-seq analysis of Derp-treated and nontreated hNECs. (A). Venn diagram from Derp group and nontreated (NT) group. (B–C). Heatmap and Volcano plot show differentially expressed genes (DEGs) in the hNECs from the Derp group and NT group. (D–E). Top 15 enriched biological processes (D) and top 15 enriched pathways (E) of DEGs in hNECs from the Derp group versus the NT group. (F). Heatmap shows key genes involved in epithelial differentiation and inflammation regulation in hNECs from the Derp group versus the NT group. The experiments were independently performed in hNECs from 3 different donors

Derp induces SCCs expansion and mucin overproduction in hNECs

We then validated the phenotypes of SCCs and goblet cells induced by Derp stimulation in the ALI culture system. mRNA levels of POU2F3, DCAMKL1, and MUC5AC were significantly elevated in Derp-stimulated hNECs compared to control hNECs (Fig. 3A). POU2F3⁺ SCCs and MUC5AC⁺ goblet cells were also increased in Derp-treated hNECs (Fig. 3B and C). Moreover, the total expression levels of SCC marker proteins, including POU2F3, DCAMKL1, and IL-25, were higher in Derp-stimulated hNECs compared to control hNECs (Fig. 3D and E), along with IL-25 protein being more abundant in the supernatants of Derp-stimulated hNECs (Fig. 3F). These results confirm SCC enrichment and mucin overproduction in Derp-treated hNECs, which is consistent with the findings from RNA-seq analysis.

Fig. 3.

Derp induces SCC expansion and IL-25 overproduction in cultured hNECs. (A). POU2F3, DCAMKL1 and MUC5AC mRNA levels were detected in Derp and NT group of hNECs. (B–C). POU2F3 and MUC5AC proteins were detected and compared in hNECs from the Derp group and NT group by immunofluorescence staining (scale bar = 50 μm). (D–E). POU2F3, DCAMKL1 and IL-25 proteins were detected and compared in hNECs from the Derp and NT group by Western blot analysis. (F). Release of IL-25 protein in hNEC supernatants from the Derp and NT group. The experiments were performed in hNECs from 3 different donors. Data are presented as mean ± SD. The paired Student t-test was performed in (A), (E) and (F); the unpaired Student t-test was used for comparison analysis in (C)

IL-25 directly promotes mucin production in hNECs and IL-25 receptor expression of eosinophils in hNMCs

Previous studies have established the key role of IL-25 in triggering type 2 inflammation in the intestinal mucosa and lungs.^14,17 Since the nasal mucosa in AR is characterized by mucus overproduction and eosinophilic infiltration, we further investigated the involvement of IL-25, the signature cytokine of SCCs,¹⁹ in nasal mucosal inflammation. The results showed that human IL-25 protein promoted MUC5AC production in hNECs in a dose-dependent manner (Fig. 4A and B), indicating that SCCs could directly act on neighboring goblet cells. These findings are consistent with the results observed in HDM-AR nasal epithelium (Fig. 1) and Derp-treated hNECs (Fig. 3).

Fig. 4.

IL-25 triggers mucin production in hNECs and promoted the expression of IL-25 receptors in eosinophils in hNMCs. (A–B). MUC5AC protein was detected and anaylzed in human recombinant IL-25 protein -treated hNECs (n = 3) versus NT group (n = 3) by immunofluorescence staining (scale bar = 50 μm). (C). Proportion of CD45⁺CD16⁻Siglec-8⁺IL-17RA⁺IL-17RB⁺ eosinophils in human IL-25-treated hNMCs (n = 5) or NT group (n = 5) measured by flow cytometry. Data are presented as mean ± SD. The unpaired Student t-test was performed in (B). The paired Student t-test was used in (C). ns, not significant

In the present study, the expression of IL-25 receptors, IL-17RA and IL-17RB, was significantly upregulated in the eosinophil subpopulation of IL-25-treated hNMCs compared to control hNMCs (Fig. 4C). Furthermore, IL-25 could significantly enhance IL-17RA and IL-17RB expression on eosinophils purified from peripheral blood of HDM-AR patients (Supplemental Fig. 1). These results demonstrate the direct effects of IL-25 on eosinophils, crucially promoting their infiltration into human nasal mucosa, suggesting SCCs contribute to inflammatory response in human nasal mucosa.

Inhibition of PAR-2 activation attenuates SCC expansion and mucin production induced by Derp in hNECs

PAR-2, a member of the protease-activated receptor family, is vital for protease allergen recognition and the development of allergic inflammation in the airway.²² Our findings showed that Derp enhanced PAR-2 expression (both at mRNA and protein levels) in cultured hNECs (Fig. 5A and B).

Fig. 5.

Derp promotes SCC expansion and mucin overproduction in hNECs in a PAR-2-dependent way. (A). PAR-2 mRNA level in Derp-treated and nontreated (NT) hNECs. (B). PAR-2 protein was analyzed in hNECs from Derp group versus NT group by Western-blot analysis. (C). POU2F3, DCAMKL1 and MUC5AC mRNA levels in NT, Derp-treated and Derp + AZ3451 (AZ)-treated hNECs. (D). POU2F3 and MUC5AC proteins detected and compared in hNECs from NT, Derp and Derp + AZ groups by immunofluorescence staining (scale bar = 50 μm). (E–F). POU2F3, DCAMKL1 and IL-25 proteins were detected and compared in hNECs from NT, Derp and Derp + AZ groups by Western-blot analysis. (G). Release of IL-25 protein in hNEC supernatants from NT, Derp and Derp + AZ groups. The experiments were performed in hNECs from 3 different donors. Data are presented as mean ± SD. The paired Student t-test was performed in (A), (B), (C), (F) and (G); the unpaired Student t-test was used in (D). ns, not significant

We then explored whether PAR-2 had an impact on SCC phenotypes and inflammation status in the nasal mucosa of HDM-AR using ALI-cultured hNECs. We found that the PAR-2 antagonist (AZ3451, AZ) could notably reduce mRNA expression levels of POU2F3 and MUC5AC, which DCAMKL1 also showed a downward tendency. (Fig. 5C). Immunofluorescence staining showed that AZ treatment in hNECs could significantly reverse the increase in POU2F3⁺SCC and MUC5AC⁺goblet cell proportions induced by Derp stimulation (Fig. 5D). In addition, the results demonstreted that the total expression levels of POU2F3, DCAMKL1, and IL-25 protein in Derp-stimulated hNECs were dependent on PAR-2 activation (Fig. 5E and F). AZ treatment also reduced IL-25 secretion levels in hNEC supernatants triggered by Derp exposure (Fig. 5G), suggesting that Derp-induced SCC expansion and mucin production are dependent on PAR-2 activation.

Inhibition of PAR-2 activation reverses the increase of IL-25 secretion and IL-25 receptor expression of eosinophils in hNMCs

Since IL-25 triggers eosinophilic inflammation response upon Derp exposure,^17,30 we explored whether PAR-2 antagonism could reverse this effect by suppressing SCC-derived IL-25. We found that IL-25 secretion significantly decreased in Derp + AZ-treated hNMCs compared to Derp-treated hNMCs (Fig. 6A). Furthermore, the expression of IL-25 receptors on eosinophils was defined as CD45⁺CD16⁻Siglec-8⁺IL-17RA⁺IL-17RB⁺, and it was also decreased in Derp + AZ-treated hNMCs versus Derp-treated hNMCs (Fig. 6B). Hence, PAR-2 activation is crucial for promoting allergic eosinophilic inflammation by mediating SCC-derived IL-25 secretion in the nasal mucosa.

Fig. 6.

HDM stimulated the secretion of IL-25 and promoted the expression of IL-25 receptors in eosinophils in hNMCs. (A). Release of IL-25 protein was detected by ELISA assay in hNMC supernatants from NT (n = 5), Derp (n = 5) and Derp + AZ (n = 5) groups. (B). Proportion of CD45⁺CD16⁻Siglec-8⁺IL-17RA⁺IL-17RB⁺ eosinophils in hNMCs from NT (n = 5), Derp (n = 5) and Derp + AZ (n = 5) groups by flow cytometry. Data are presented as mean ± SD. The paired Student t-test was used in (A) and (B). ns, not significant

PAR-2 overexpression is positively correlated with SCCs hyperplasia and eosinophil infiltration of HDM-AR nasal mucosa

To examine the roles of PAR-2 in abnormal SCC hyperplasia and eosinophil infiltration in the nasal mucosa of HDM-AR patients, we collected clinical nasal specimens for subsequent analysis. The results showed that PAR-2 mRNA levels were significantly upregulated in nasal swab specimens from patients with HDM-AR compared to control participants (Fig. 7A), and positively correlated with POU2F3 mRNA expression (r = 0.454, P = 0.007) (Fig. 7B). We then analyzed PAR-2 protein expression and POU2F3⁺ SCC distribution in nasal tissues from non-AR and HDM-AR participants. Hematoxylin-eosin staining revealed higher eosinophil percentages in the HDM-AR group compared to the non-AR groups, indicating a typical eosinophilic inflammatory phenotype in the nasal mucosa of HDM-AR patients (Fig. 7C). Additionally, immunohistochemical staining results showed that PAR-2 protein was universally expressed throughout the entire epithelial layer, while POU2F3 protein was diffusely located in the regions of differentiated epithelial cells (Fig. 7D). Both PAR-2 protein expression levels and POU2F3⁺ SCC density were significantly increased in the HDM-AR epithelium compared with those in the non-AR epithelium (Fig. 7E). Furthermore, PAR-2 protein levels positively correlated with POU2F3⁺ SCC density (r = 0.571, P < 0.001) and eosinophil percentage (r = 0.631, P < 0.001) in the nasal mucosa (Fig. 7F). These results suggest that increased epithelium-derived PAR-2 is essential for SCC expansion and local eosinophilic inflammation in the nasal mucosa of patients with HDM-AR.

Fig. 7.

The correlation of PAR-2 overexpression with SCC hyperplasia and eosinophil infiltration in HDM-AR nasal mucosa. (A–B). PAR-2 mRNA level (A) and the correlation of PAR-2 and POU2F3 mRNA levels (B) were analyzed in nasal swab specimens collected from normal control (NC, n = 17) and HDM-AR (n = 17) donors. (C–E). Comparison of eosinophil percentage in nasal tissue (C) and PAR-2 and POU2F3 proteins in human nasal epithelium (D–E) among NC (n = 8), non-AR (n = 12) and HDM-AR (n = 7) groups (scale bar = 50 μm). (F). The correlation of epithelial PAR-2 expression levels with distribution density of POU2F3⁺ SCCs and eosinophil percentage of total submucosal cells in nasal tissue collected from NC (n = 8), non-AR (n = 12) and HDM-AR (n = 7) donors. Data are presented as medians with interquartile ranges. The Mann-Whitney U test was performed in (A), (C) and (E); the Spearman r test was used in (B) and (F)

Discussion

SCCs, which are first well-understood for driving type 2 immunity in the intestinal mucosa, are considered epithelial sentinels against pathogens. Our study highlights their crucial roles in mucosal eosinophilic inflammation in HDM-AR. Based on nasal specimen analysis and in vitro cell experiments, the present study demonstrated that SCCs were enriched in HDM-exposed nasal epithelium and triggered IL-25-mediated mucin production and eosinophilic infiltration. Moreover, PAR-2 overexpression in the nasal epithelium of HDM-AR directly promoted SCC expansion and mucosal eosinophilic immunity. These findings reveal the significance of SCCs in epithelium-immune interactions during the mucosal inflammation of HDM-AR.

Derp, the major component of HDM, is commonly used to construct in vitro HDM-AR models. Typically, these models use monolayer primary nasal epithelial cells,^31,32 which limits the detection of changes in differentiated cells. To addressed this limitation, we established an ALI model to investigate morphological and functional changes in nasal pseudostratified epithelium upon Derp exposure. Previous studies using similar stimulation models focused on epithelial barrier alterations or pro-inflammatory mediator responses, but without identifying epithelial cell phenotypes.^33,34 In the current model, we explored the cellular and molecular characteristics of epithelial cells upon Derp stimulation and investigated the role of the nasal epithelium in HDM-AR-induced allergic inflammation.

SCCs are enriched in the nasal epithelium of patients with CRSwNP³⁵ and allergic fungal rhinosinusitis (AFRS),³⁶ but their presence in HDM-AR patients has not yet been reported. Our results revealed a higher proportion of SCCs in the nasal mucosa of HDM-AR compared to control mucosa. Fungal extracts, a type of common indoor allergen, have been shown to induced SCC expansion in cultured epithelial cells of patients with AFRS.³⁶ Here, we found that POU2F3, the key transcription factor for SCC development, was upregulated in Derp-treated hNECs through RNA-seq analysis. We also validated SCC expansion by identifying specific markers, including POU2F3, DCAMKL1, and IL-25, in Derp-exposed hNECs. Thus, our results confirm SCC enrichment in nasal epithelium in response to HDM exposure.

Our results showed that goblet cell hyperplasia was detected in the nasal mucosa of HDM-AR patients, consistent with findings in an HDM-induced murine AR model.³⁰ Additionally, Derp stimulation significantly increased mucin production in hNECs, and exogenous IL-25, the signature cytokine of SCCs, mimicked this effect in a dose-dependent manner. These findings suggest that SCC-derived IL-25 contributes to mucin overproduction in HDM-AR.

Epithelium-derived IL-25 drives Th2-skewed mucosal inflammation in allergic airway diseases, such as asthma.³⁷ SCCs have been recognized as the primary source IL-25 in the intestine¹⁴ and nasal epithelium.¹⁹ Here, we found elevated IL-25 levels in nasal secretions from patients with HDM-AR than control group, along with increased IL-25 protein expression in Derp-treated hNECs, which were in consistent with previous findings.³⁸ Surprisingly, RNA-seq analysis showed no significant difference in IL-25 expression between control and HDM-exposed hNECs. This suggest that Derp might enhance IL-25 production mainly at the protein, but not at the transcriptional level. Previous studies have shown that IL-25 from SCCs activates ILC2-mediated allergic inflammation¹⁷ and induced eosinophilic recruitment in murine lung allergy models.³⁰ Interestingly, we tested the direct effect of IL-25 on sorted eosinophils and found that IL-25 directly triggered IL-25 receptor (IL-17RA/IL-17RB) expression in eosinophils, indicating that IL-25 likely acts as a potent upstream mediator of eosinophilic inflammatory responses in human nasal mucosa.

PAR-2 has been implicated in HDM-evoked murine allergic lung diseases and could initiate inflammatory signaling.^39,40 Only 1 study has shown increased PAR-2 expression in the nasal submucosal glands of patients with HDM-AR compared to the control group.⁴¹ Our data demonstrated elevated PAR-2 mRNA and protein expression levels in the nasal epithelium of patients with HDM-AR and Derp-exposed hNECs, which were accompanied by SCC expansion. These results suggest that PAR-2 is involved in SCC hyperplasia in the nasal mucosa of HDM-AR patients. Kouzaki et al reported that PAR-2 siRNA treatment significantly inhibited HDM-induced IL-25 production in human bronchial epithelial cells.24, 42 In our study, we investigated the role of PAR-2 in SCC expansion and eosinophilic inflammation in Derp-induced hNECs using a PAR-2 antagonist. We found that inhibition of PAR-2 activation significantly attenuated HDM-induced SCC expansion, mucin overproduction, and IL-25 receptor (IL-17RA/IL-17RB) expression in eosinophils in the nasal mucosa. These data demonstrate that the effects of HDM on SCC expansion and eosinophilic inflammation in the nasal mucosa are dependent on PAR-2 activation.

We fully acknowledge the limitations of drawing conclusions based on CRSwNP-derived samples. In future studies, we plan to collect nasal mucosal samples from volunteers with pure HDM-AR through other approaches, such as clinical trials, in order to directly validate and further explore the findings presented in this study. Another limitation of our research findings is that we assessed individual SCC signature gene expression in total hNECs using bulk RNA-seq rather than investigating specific transcriptional profiles of SCCs. This was primarily due to the absence of surface markers and the limited number of SCCs in the nasal epithelium. Single-cell RNA-seq could be used in the future to identify SCC molecular features and deepen our understanding of their functions. Additionally, the mechanisms underlying HDM-induced PAR-2 activation promoting SCC expansion remain unclear, necessitating further studies to explore the signaling pathways regulating the SCC phenotype through functional experiments.

Conclusion

Although this study has certain limitations,our results revealed that HDM allergen exposure in the nasal epithelium triggered PAR-2 activation, promoting SCC expansion and downstream IL-25 production and secretion. IL-25 could trigger mucin overproduction and promote IL-25 receptor expression in eosinophils, which may exacerbate allergic immunity in the nasal mucosa of HDM-AR patients. These findings suggest that SCCs play a pivotal role in epithelium-immune interactions and act as crucial upstream immunomodulators in local allergic inflammation. In the future, we can utilize single-cell RNA sequencing to identify the molecular characteristics of SCC and provide a novel target for therapeutic strategies in HDM-AR.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Author contributions

Pei Gao performed the research and analyzed the data. Liyue Li wrote the initial draft of the Manuscript. Yan Zou was involved in methodology. Jianxin Yue, Tao Zhou, Liuqing Zhou, Yue Zhou, Shan Chen, Yanyan Ding, Han Wu, Qing Cheng, Yanjun Wang recruited patients and collected samples. Jianjun Chen and Hongjun Xiao designed the study and revised the manuscript. All authors have reviewed and approved the final version of this manuscript.

Ethics approval

This study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology [NO. 2024-0036-01]. All participants provided written informed consent.

Authors’ consent for publication

All authors checked the final manuscript and approved it for publication.

Disclosure of the use of Generative AI and AI-assisted technologies

Nothing to disclose.

Funding

This study was supported by the National Natural Science Foundation of China (No. 82101229, 82371124 and 82201261), Key Research and Development Program of Hubei Province, China (No. 2021BCA144), National Key Research and Development Program of China (No. 2022YFC2504100), Natural Science Foundation General Program of Hubei Province, China (No. 2024AFB611).

Declaration of competing interest

The authors declare that they have no known conflict financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: