Introduction: Inhalation of fungal allergens induces airway epithelial damage following airway inflammation and excessive mucus secretion, which can lead to severe asthma with fungal sensitization (SAFS). Comprehensive gene expression analysis in Alternaria-exposed mouse airways, a model of SAFS, has not been conducted. Methods: BALB/c mice received intranasal administration of Alternaria extract or phosphate-buffered saline twice a week for 6 weeks. Lung sections and bronchoalveolar lavage fluid were obtained to assess airway inflammation. RNA-Seq in the central airway was performed, and gene ontology (GO) analysis and gene set enrichment analysis (GSEA) were conducted for pathway analyses. An in vitro experiment using human airway epithelial cell 16HBE14o- was performed to validate the RNA-Seq findings. Results: Eosinophilic airway inflammation with mucus overproduction and airway remodeling was observed in mice exposed to Alternaria. RNA-Seq analysis revealed 403 upregulated and 108 downregulated genes in airways of Alternaria-exposed mice. In GO analysis, the functions of immunoglobulin (Ig) receptor binding, Ig production, inflammatory response, and T-cell activation were upregulated, while those of keratinization and defense response to other organisms were downregulated. GSEA revealed positive enrichment in T-cell receptor complex, immunological synapse, antigen binding, mast cell activation, and Ig receptor binding, and negative enrichment in keratinization and cornification in Alternaria-exposed mice relative to control. Alternaria exposure to 16HBE14o- cells validated the downregulation of epithelial keratinization-related genes, including SPRR1A, SPRR1B, and KRT6B. Conclusion: RNA-Seq analysis showed that Alternaria exposure induced inflammatory response and impaired defense mechanisms in mice airway epithelium, which might be therapeutic targets for SAFS.

Asthma is characterized by chronic airway inflammation, accompanied by sensitivity to airborne allergens [1]. Sensitization to fungi could be a potential risk factor for poor asthma control [2] and severe asthma, which is referred to as severe asthma with fungal sensitization (SAFS) [3]. A population-based study in France showed that a positive skin prick test for a fungal allergen of Alternaria alternata was an independent risk factor for severe asthma [4]. Children with SAFS had higher serum IgE levels, earlier onset disease, and more severe disease than those without SAFS [5]. The precise mechanism of SAFS is still under investigation. An experimental study using knockout mice showed that IL-33 is a key molecule for the development of fungus-induced eosinophilic airway inflammation [6]. Innate lymphoid cell type 2 was also involved in Alternaria-induced airway inflammation in a mouse model of allergic asthma [7]. These findings indicate that the IL-33/ST2 axis mediated by innate lymphoid cell in the airway epithelium is assumed to play pivotal roles in a study using an Alternaria-induced allergic asthma mouse model [5].

Alternaria alternata is a major fungal allergen that is involved in the pathogenesis of allergic asthma. The protease activity of Alternaria causes airway epithelial damage and acts as an eosinophil chemoattractant, resulting in prominent eosinophilic airway inflammation. Exposure of the airway to Alternaria results in innate and adaptive immunity responses [8]. The mouse model created by intranasal administration of Alternaria is often used as an allergic asthma model.

Alternaria can induce IL-33 expression in the airway epithelium, which leads to airway inflammation, mucus secretion, and impaired lung function [9]. An in vitro study using 16HBE14o- cells revealed that Alternaria exposure activated protease-activated receptor 2 (PAR-2) through its serine protease activity [10]. In another in vitro study using BEAS-2B cells, Alternaria exposure induced GM-CSF, IL-6, and IL-8 production through PAR-2 activation [11]. A recent study using knockout mice of Ormdl3, known as a genome-wide susceptible gene of childhood asthma, showed that ORMDL3 is involved in Alternaria-induced airway inflammation [12]. The airway epithelium acts as a barrier against external pathogens to prevent aberrant allergic inflammation. Disrupted epithelial barrier function, such as keratinization, might result in the development of allergic diseases. Mutations in the filaggrin gene, which is an epithelial barrier protein, are involved in allergic diseases, including asthma [13, 14]. However, the association between epithelial barrier function and the pathogenesis of SAFS is not yet clear. Alternaria inhalation induces a wide variety of inflammatory and immunological changes in airways by disrupting airway epithelial cells; however, a study of unbiased comprehensive gene expression analysis by RNA-Seq has not yet been conducted. We hypothesized that chronic Alternaria inhalation would induce distinct gene expression patterns in the airway epithelium, and the findings might contribute to elucidating the pathogenic pathways of SAFS.

Development of Animal Model for SAFS

Female 7-week-old BALB/c mice (n = 3) received an intranasal instillation of 20 μL of 50 μg Alternaria extract (#10117, ITEA Inc., Tokyo, Japan), which was re-suspended in filtered phosphate-buffered saline (PBS) as a model of SAFS. Control mice (n = 3) received 20 μL sterile PBS. Each mouse received intranasal administration of Alternaria extract or PBS twice a week (Monday and Thursday) for 6 weeks. All mice were sacrificed for evaluation 24 h after the last exposure. The animal protocol was approved by the Institutional Animal Care and Use Committee of Showa University (approval number: 09073).

Lung Immunohistochemistry and BALF Cytology

The lungs of the mice were fixed with 4% paraformaldehyde and embedded in paraffin. They were sectioned for subsequent staining with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson’s trichrome, and immunohistochemistry. Immunostaining was performed using siglec-F (BD Pharmingen, 552125, 1:12.5) as an eosinophil-specific marker for mice, with antigen retrieval using citric acid buffer at 105°C for 10 min. The samples were incubated with secondary antibody (DAKO, Envision K4063 or Abcam, ab97057) followed by visualization with 3, 3′-diaminobenzidine and counter-staining with H&E. Epithelial thickness, represented as the depth from the mucosal surface to the basement membrane, was measured at 6 points per bronchus for 4 bronchi. Mucus area and connective tissue assessed by PAS and Masson’s trichrome staining, respectively, were also measured for independent 4 independent bronchi. Masson’s trichrome staining is a 3-color staining method used for the detection of collagen fibers from surrounding tissue. Airway remodeling accompanied by excessive collagen deposition could be assessed using Masson’s trichrome staining to measure the blue-stained area. Bronchoalveolar lavage fluid (BALF) was also obtained by intratracheal instillation of 1 mL normal saline before mice were sacrificed. Cells in the BALF were stained with cytospin preparation and counted to determine cell differentiation in BALF. Images were obtained and analyzed using a BZ-X800 microscope (Keyence, Osaka, Japan).

RNA-Seq Analysis

For RNA-Seq analysis, the trachea and central brochi were resected and immediately immersed in RNA later (Ambion, Austin, TX, USA) after sacrifice. Total RNA was extracted using an RNeasy-Mini spin column (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. RNA concentration and quality were analyzed using a Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA), 28S/18S rRNA ratio by agarose gel electrophoresis, and RNA integrity number using TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed using a NovaSeq 6000 (Illumina, San Diego, CA, USA). The reference genome sequence for mapping was GRCm38.p6. The expression in each sample was statistically analyzed and normalized using transcripts per million algorithms. Sequence data are available in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA011150. As a comprehensive unbiased approach, a heatmap with hierarchical clustering, gene ontology (GO) analysis, and gene set enrichment analysis (GSEA) were utilized to identify biological processes whose gene sets were differentially expressed due to Alternaria exposure. GO analysis was performed using Metascape software (http://metascape.org).

Additionally, as a hypothesis-driven approach, some genes recently highlighted for their involvement in the pathogenesis of SAFS were also analyzed, and their expression levels were compared between control and Alternaria-exposed mice. The analyzed genes included Il-33, St2 (Il1rl1), Ormdl3, and Par-2 (F2rl1), which have recently been recognized as key molecules for the development of SAFS [6, 7, 9‒12].

In vitro Validation Using Human Airway Epithelial Cells with Alternaria Exposure

In vitro experiments, using human airway epithelial cells were performed to validate the findings obtained by the RNA-Seq results. The human airway epithelial cell line 16HBE14o- was purchased from Merck (Darmstadt, Germany). Cells were seeded onto 24-well plates coated with a mixture of collagen (30 μg/mL), fibronectin (10 μg/mL), and bovine serum albumin (100 μg/mL). After confluence under submerged conditions, PBS-diluted Alternaria extract at concentrations of 0.1 μg/mL and 1 μg/mL was applied to the media. After 24 h, the cells were harvested and processed for RNA purification using acid guanidinium thiocyanate-phenol-chloroform extraction. The quality of total RNA was assessed using the methods described above. cDNA was synthesized and RT-PCR was conducted using SYBR Green I Mastermix with a BIO-RAD CFX96 System (Bio-Rad Laboratories, Hercules, CA, USA). Because of the study design of the unbiased approach using RNA-Seq, we focused on the top-rated downregulated genes of airway epithelial cells, including SPRR1A, SPRR1B, and KRT6B, which are described in the Results section, for the validation analysis. The sequences of the primer sets for SPRR1A, SPRR1B, KRT6B, and GAPDH are listed in Table 1. GAPDH was used as the housekeeping gene. The relative expression of each gene was evaluated using the delta-delta CT calculation [15].

Table 1.

Primer sequences for quantitative RT-PCR used in the in vitro experiment

 Primer sequences for quantitative RT-PCR used in the in vitro experiment
 Primer sequences for quantitative RT-PCR used in the in vitro experiment

Statistics

The data are expressed as the mean ± standard error (SE) or median with interquartile range. Intergroup comparisons between control and Alternaria-exposed mice groups were made using the unpaired Student’s ttest. Intergroup comparisons in the in vitro experiment were performed using the Wilcoxon rank sum test. Statistical significance was set at p < 0.05. Statistical analysis was performed using JMP Pro software (version 15, SAS Institute, Cary, NC, USA). To identify differentially expressed genes between control and Alternaria-exposed mice in RNA-Seq analysis, low-expression genes with transcripts per million ≤1 were excluded, and data were normalized with the trimmed mean of M values normalization followed by Fisher’s exact test to calculate the p value. For hierarchical clustering and the heatmap, genes with adjusted p values <0.05, and log2-transformed fold-change equal to or >0.5 were identified as differentially expressed genes.

Alternaria Inhalation Induced Prominent Eosinophilic Airway Inflammation with Airway Remodeling

Alternaria inhalation for 6 weeks resulted in prominent airway inflammation, as shown by the increased accumulation of inflammatory cells in BALF compared to that in the control (Fig. 1a). For cell differentiation in BALF, eosinophils were significantly increased in Alternaria-exposed mice compared to control mice (Fig. 1b). The percentage of macrophages in BALF from Alternaria-exposed mice was lower than that from control mice (Fig. 1c). There were no differences in neutrophil or lymphocyte differentiation between the 2 groups (Fig. 1d, e). H & E staining of lung sections revealed that lungs from Alternaria-exposed mice had thicker airway membranes than those from control mice (Fig. 2a). PAS staining of the lungs showed that Alternaria-exposed mice had more mucus production in large airways than control mice (Fig. 2b). Moreover, Masson’s trichrome staining showed more prominent airway remodeling in Alternaria-exposed mice than that in control mice (Fig. 2c). Immunohistological staining of siglec-F revealed that there were more siglec-F positive cells in the airways of Alternaria-exposed mice than control mice (Fig. 2d). The results indicated that Alternaria inhalation in mice induced prominent eosinophilic airway inflammation with mucus overproduction and airway remodeling, which represents a severe eosinophilic asthma phenotype model.

Fig. 1.

Alternaria exposure induced eosinophilic-airway inflammation in BALF from a mouse model of allergic asthma. Total cell numbers (a) and eosinophils (b), macrophages (c), neutrophils (d), and lymphocytes (e) differentiation in BALF after 6-week exposure to Alternaria extract or PBS. Each bar represents mean ± SE. Statistical analysis was performed by Student’s t test. PBS, phosphate-buffered saline; SE, standard error; BALF, bronchoalveolar lavage fluid.

Fig. 1.

Alternaria exposure induced eosinophilic-airway inflammation in BALF from a mouse model of allergic asthma. Total cell numbers (a) and eosinophils (b), macrophages (c), neutrophils (d), and lymphocytes (e) differentiation in BALF after 6-week exposure to Alternaria extract or PBS. Each bar represents mean ± SE. Statistical analysis was performed by Student’s t test. PBS, phosphate-buffered saline; SE, standard error; BALF, bronchoalveolar lavage fluid.

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Fig. 2.

Alternaria exposure induced eosinophilic airway inflammation, mucus hypersecretion, and remodeling in mice airways. Comparisons between control and Alternaria-exposed mice in basement membrane thickness in H & E staining (a), airway mucus area in PAS staining (b), and airway remodeling in Masson’s trichrome staining (c). Immunohistological staining of siglec-F showed more siglec-F positive cells in airways of Alternaria-exposed mice than in those of the control mice (d). Each bar represents mean ± SE. Statistical analysis was performed by Student’s t test. H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; SE, standard error.

Fig. 2.

Alternaria exposure induced eosinophilic airway inflammation, mucus hypersecretion, and remodeling in mice airways. Comparisons between control and Alternaria-exposed mice in basement membrane thickness in H & E staining (a), airway mucus area in PAS staining (b), and airway remodeling in Masson’s trichrome staining (c). Immunohistological staining of siglec-F showed more siglec-F positive cells in airways of Alternaria-exposed mice than in those of the control mice (d). Each bar represents mean ± SE. Statistical analysis was performed by Student’s t test. H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; SE, standard error.

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RNA-Seq Revealed Upregulation of Immunoglobulin-Related Genes and Downregulation of Keratinization Genes in Alternaria-Exposed Mice Airways

Next, comprehensive gene expression analysis using RNA-Seq was performed in the airways of Alternaria-exposed mice. Between 40 and 48 million reads were obtained for each sample. Quality control ensured that the Q30 (%) was >94% for each sample. Uniquely mapped reads were above 87% (average: 88.2%), and multiple mapped reads were 9.3% (average: 8.1%). In a heatmap with hierarchical clustering, we identified 403 upregulated and 108 downregulated genes after 6-week Alternaria exposure compared to control mice (Fig. 3a). GO analysis revealed significantly upregulated gene functions, including immunoglobulin (Ig) receptor binding, Ig production, inflammatory response, and T-cell activation (Fig. 3b; Table 2).

Table 2.

A list of DEGs in representative GO terms identified by GO analysis

 A list of DEGs in representative GO terms identified by GO analysis
 A list of DEGs in representative GO terms identified by GO analysis
Fig. 3.

Alternaria exposure induced the distinct gene expression pattern in mice airways using RNA-Seq analysis. Heatmap with hierarchical clustering showed that Alternaria-exposed mice had 403 upregulated and 108 downregulated genes compared to control mice (a). GO analyses showed significantly upregulated gene functions (b) and downregulated gene functions (c) after Alternaria exposure. GSEA revealed pathways with positive enrichment (d–g) and negative enrichment (h–k) in Alternaria exposure mice relative to control mice. GO, gene ontology; GSEA, gene set enrichment analysis.

Fig. 3.

Alternaria exposure induced the distinct gene expression pattern in mice airways using RNA-Seq analysis. Heatmap with hierarchical clustering showed that Alternaria-exposed mice had 403 upregulated and 108 downregulated genes compared to control mice (a). GO analyses showed significantly upregulated gene functions (b) and downregulated gene functions (c) after Alternaria exposure. GSEA revealed pathways with positive enrichment (d–g) and negative enrichment (h–k) in Alternaria exposure mice relative to control mice. GO, gene ontology; GSEA, gene set enrichment analysis.

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In GO of “immunoglobulin receptor binding,” “immunoglobulin production,” and “immunoglobulin binding,” genes of receptors and Ig fragments were upregulated, including Fcgr3, Ighg1, Jchain, Trbc1, Trbc2, Igha, Ighe, Igkv, and Fcer1a. In GO of inflammatory response, Adam8, Timp1, and Mmp12 were included, which suggested that production and degradation of the extracellular matrix was upregulated by Alternaria exposure in mouse airways. Eosinophil migration was also upregulated, as shown by the upregulated genes of Ccr3, Ccl11, Ccl6, Ccl7, Ccl8, and Ccl9. Interestingly, the GO function of serine-type peptidase activity was also upregulated with genes including Ctsh (cathepsin H), Klk1 (Kallikrein 1), Cma1 (Chymase 1), Mcpt1 (Mast cell tryptase 1), Mcpt2, Mcpt4, Tpsb2 (Tryptase beta 2), and Tmprss2 in mouse airways followed by Alternaria exposure, which itself has several kinds of proteases. Additionally, GO of the leukotriene metabolic process was upregulated as shown by Alox5, Alox5ap, and Fcer1a. The most downregulated gene function was keratinization with genes including Krt16, Krt6b, Sprr1a, Sprr1b, and Sprr2b (Fig. 3c; Table 2). Other downregulated gene functions were cellular responses to interferon-beta and defense response to other organisms.

GSEA revealed positive enrichment of several pathways, including T-cell receptor complex, immunological synapse, antigen binding, mast cell activation, and Ig receptor binding (Fig. 3d–g). GSEA also revealed negative enrichment of several pathways of keratinization, cornification, regulation of water loss via skin, and desmosome in Alternaria-exposure mice relative to control mice (Fig. 3h–k).

As a hypothesis-driven approach, expression levels of Il-33, St2, Ormdl3, and Par-2 were compared between control and Alternaria-exposed mice using the RNA-Seq data set. St2 expression was significantly increased in Alternaria-exposed mice compared to that in control mice (Table 3). In contrast, the expression of Il-33, a ligand of ST2, was marginally decreased in Alternaria-exposed mice compared to the control, without statistical significance. There were no differences in the expression of Ormdl3 or Par-2 between the 2 groups. In vitro Alternaria exposure validated downregulation of GO function of keratinization in human airway epithelial cells.

Table 3.

The comparisons of hypothesis-driven gene expression between control and Alternaria-exposed mice using the RNA-Seq data set

 The comparisons of hypothesis-driven gene expression between control and Alternaria-exposed mice using the RNA-Seq data set
 The comparisons of hypothesis-driven gene expression between control and Alternaria-exposed mice using the RNA-Seq data set

In vitro Alternaria Exposure Validated Downregulation of GO Function of Keratinization in Human Airway Epithelial Cells

A validation experiment using human airway epithelial cells was performed to assess the effect of Alternaria on airway epithelial cells. In GO analysis using RNA-Seq data, the most downregulated gene function upon Alternaria exposure was keratinization; therefore, we focused on the top-rated downregulated genes of airway epithelial cells, including SPRR1A, SPRR1B, and KRT6B, for the validation analysis. Alternaria exposure at the dose of 1 μg/mL resulted in significant downregulation among genes of keratinization including SPRR1A, SPRR1B, and KRT6B in human airway epithelial cells 16HBE14o- compared to the control, and the downregulation of the genes was not observed at the dose of 0.1 μg/mL Alternaria extract (Fig. 4a–c). These results were consistent with the RNA-Seq results and the downregulation of GO function of keratinization in Alternaria-exposed mice airways.

Fig. 4.

Alternaria exposure in vitro downregulated gene expressions of SPRR1A (a), SPRR1B (b), and KRT6B (c) in airway epithelial cells of 16HBE14o- (n = 4 in independent experiments). Each bar represents the median with IQR. Statistical analysis was performed by the Wilcoxon rank sum test. IQR, interquartile range.

Fig. 4.

Alternaria exposure in vitro downregulated gene expressions of SPRR1A (a), SPRR1B (b), and KRT6B (c) in airway epithelial cells of 16HBE14o- (n = 4 in independent experiments). Each bar represents the median with IQR. Statistical analysis was performed by the Wilcoxon rank sum test. IQR, interquartile range.

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This comprehensive gene expression analysis using a mouse model of SAFS revealed that Alternaria exposure to airways induced prominent eosinophilic airway inflammation accompanied by upregulation of inflammatory response with Ig production and binding, and downregulation of epithelial defense genes and keratinization. Alternaria is a major allergen that induces prominent airway inflammation and may be a factor in the severe asthma phenotype. The protease activity of Alternaria damages the airway epithelium and results in the development of inflammatory responses [9, 11]. In this study, 6-week Alternaria exposure resulted in prominent airway inflammation in mice, as shown by increased eosinophils in the BALF. In lung histology, Alternaria exposure resulted in airway membrane thickening and mucus hyperproduction with excessive collagen deposition, which is a major characteristic of airway remodeling in severe asthma. Severe airflow obstruction and poor asthma control are often observed in patients with SAFS. In the pathology of SAFS, chronic airway inflammation and airway remodeling should be induced by sustained exposure to fungal allergens. The 6-week Alternaria-exposed mouse model used in this study would be consistent with the phenotype of severe asthma, which is associated with inhaled fungal allergens.

This comprehensive gene expression analysis using RNA-Seq in Alternaria-exposed asthma mouse model showed upregulation of gene sets of Ig receptors and binding. This overexpression of Ig due to Alternaria exposure was characterized by local immunoglobulins, including IgG, IgA, and IgE. Alternaria exhibits several protease activities, and airway exposure to Alternaria induces airway epithelial damage, which disrupts airway epithelium integrity. Fungal components, such as chitin, β-D-glucan, and glycosidase are recognized by receptors of airway epithelial cells and dendritic cells, including toll-like receptors and proteinase-activated receptors [16]. Matsuwaki et al. [11] reported that Alternaria induced the production of inflammatory cytokines in BEAS-2B and Calu-3 cells, including GM-CSF, IL-6, and IL-8, which were inhibited by PAR-2 antagonist or aspartate protease inhibitors. Activated dendritic cells contribute to the differentiation of CD4+ T-cells into TH2 cells. TH2 cells produce a set of Th2 cytokines, including IL-4, IL-5, and IL-13, which can activate eosinophils [17]. The Th2 cytokines could also contribute to class switching from IgG to IgE produced by B cells [18]. Then, exposure of the airway to Alternaria could result in upregulation of Ig production and eosinophil accumulation in mouse airways.

Our study revealed that Alternaria exposure also led to airway epithelium-induced gene expression of serine proteases, such as Ctsh, Klk1, Cma1, Tpsb2, and Tmprss2. These proteases from damaged airway epithelium could aggravate airway epithelial damage caused by Alternaria exposure. Ctsh encodes cathepsin H, which is a member of the cathepsin family, which is a lysosomal acidic enzyme [19]. Mcpt1, Mcpt2, Mcpt4, and Tpsb2 encode a tryptase, which is a serine-type proteinase released from activated mast cells as well as club cells in the airways [20, 21]. Tmprss2 encodes a transmembrane serine protease, which is expressed on airway epithelial cells and utilized for coronavirus entry into the cells [22]. Human airway trypsin-like protease (HAT; aka TMPRSS11d) increases mucin gene expression in airway epithelial cells [23]. As shown by the increased protease gene expression among our Alternaria-exposed mice, internal protease activity as well as fugus-derived external protease might contribute to airway inflammation with mucus overproduction. In our study, Ig production, including IgE, was upregulated in the Alternaria-exposed mice. In asthmatic airways, IgE induces degranulation of local mast cells and activation of basophils, which aggravates airway inflammation by releasing various cytokines, chemokines, and inflammatory mediators [24]. A retrospective study of an Australian severe asthma cohort revealed that omalizumab, a humanized anti-IgE monoclonal antibody, significantly improved asthma control and exacerbation frequency, and reduced the dosage of regular-use oral corticosteroids used by SAFS patients compared to that of severe asthma patients without fungal sensitization [25]. Moreover, in vitro analysis using human basophils from patients with allergic bronchopulmonary aspergillosis demonstrated that omalizumab treatment decreased sensitivity to Aspergillus fumigatus, surface-bound IgE, and FcεRI levels compared to those of control subjects [26]. Blocking IgE signaling in Alternaria-induced asthma might be a potential therapeutic approach to reduce excessive airway inflammation. The development of an anti-IgE treatment in an Alternaria-induced allergic mouse model is warranted to improve SAFS treatment options.

Airway epithelial cells act as the first barrier against external pathogens to maintain airway homeostasis. Airway epithelial cells express keratin proteins, which contribute to airway epithelial differentiation and development [27]. In this study, some keratinization-related proteins were downregulated after Alternaria exposure. In addition to keratin genes, small proline-rich proteins, including Sprr1a and Sprr1b, which constitute cornified cell envelope precursors, were downregulated in Alternaria-exposed mice airways, which indicated that the expression of cytoskeleton proteins was disrupted by Alternaria exposure to mouse airways. Krt6b is a type II cytokeratin involved in epithelial cell differentiation [28]. Sprr1a and Sprr1b are precursors of cornified envelopes clustered in the epidermal differentiation complex [29]. Downregulation of KRT6B, SPRR1A, and SPRR1B in airway epithelial cells after Alternaria exposure observed in this study might suggest defective airway epithelial differentiation in Alternaria-exposed airways. Restoration of airway integrity in terms of cytoskeleton production might help suppress aberrant inflammation caused by external pathogens containing protease activity.

As a hypothesis-driven approach, the IL-33/ST2 axis is considered to play a pivotal role in fungal allergen-induced airway inflammation through innate immunity. In accordance with this hypothesis, our results showed that St2 gene expression was upregulated in Alternaria-exposed mice compared to that in control mice. On the other hand, Il-33 expression was slightly lower in Alternaria-exposed mice than that in control mice. Because our allergic asthma model is chronic due to 6-week Alternaria exposure, internal IL-33 expression might be modified by excessive airway inflammation with airway epithelial cell disruption. Ormdl3 and Par-2 were also analyzed in this study; however, no difference was observed between control and Alternaria-exposed mice. A possible explanation for this dissociation from previous reports might be the use of comprehensive gene expression analysis as opposed to using a protein-level approach. Moreover, chronic Alternaria exposure (not acute exposure) might not result in gene expression differences in Ormdl3 or Par-2 in airway epithelium. Protein expression analyses and time-course experiments should be considered for further analysis to reveal the role of ORMDL3 or PAR-2 in chronic fungal allergic asthma models.

In the validation analysis, keratinization genes, including SPRR1A, SPRR1B, and KRT6B, were downregulated upon Alternaria exposure in a human airway epithelial cell monolayer culture. It has been reported that SPRR1A is also expressed in airways with basal cells and airway epithelial cells, which might implicate squamous cell metaplasia upon airway inflammation [30]. In another study, SPRR2 was upregulated in an allergic asthma mouse model using ovalbumin or Aspergillus fumigatus in the airway epithelium [31]. However, this study was an acute model of allergic airway inflammation, different from our chronic asthma mouse model using 6-week Alternaria exposure. Long-term exposure to Alternaria might result in impaired expression of keratinization genes, including SPRR1A, SPRR1B, and KRT6B. Our unbiased comprehensive gene expression analysis highlighted the impaired expression of airway epithelial keratinization genes. As a next step, gain-of-function or loss-of-function studies should be considered to elucidate the roles of these keratinization genes in the pathogenesis of SAFS.

There are several limitations to this study. First, RNA-Seq samples included various cell types in the bronchial tissue. The results of RNA-Seq reflect gene expression signatures not only of airway epithelial cells, but also of fibrotic cells, smooth muscle cells, and immune cells. However, increased gene functions of Ig production, T-cell activation, and mast cell tryptase suggested that B cells, T-cells, and mast cells play major roles in Alternaria-induced airway inflammation. Additionally, downregulation of airway epithelial gene functions, including keratinization and defense response to other organisms, might indicate that airway epithelial cells also play pivotal roles in protecting the host from external fungal allergens. Second, a validation experiment was conducted using a monolayer culture of airway epithelial cells. Further studies will be needed to validate the effect of Alternaria exposure to inflammatory cells and Ig production and binding using primary human airway epithelial cells. In conclusion, RNA-Seq analysis using airways of allergic asthma mouse model revealed that Alternaria exposure induces airway inflammation with overproduction of Ig and impaired defense mechanisms in the mouse airway epithelium. Controlling of abnormal airway inflammation and restoration of the epithelial barrier may be treatment strategies for SAFS.

We thank Megumi Matsuda and Teru Haba for their technical assistance. Gene expression analysis using RNA-Seq was performed by Genble Inc. (Fukuoka, Japan).

Experiments using mice were performed in accordance with the institutional guidelines and regulations of Showa University for the care and use of laboratory animals. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Showa University, approval number 09073.

All authors have stated explicitly that there are no conflicts of interest to disclose in connection with the article.

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number: 18K16160 and 21K16308 to H.I.).

H. Inoue, S.S., A.T., and H.S. contributed to the conception and design of the study. H. Inoue, K.A., H. Ikeda, H.S., Y.F., Y.M., T.H., S.O., and M.Y. contributed to perform experiments. T.U., Y.U., and T.K. contributed to data acquisition. H. Inoue and K.H. contributed to statistical analyses. The manuscript was written by H. Inoue, and all authors read it and approved it for submission.

The RNA-Seq data that support the findings of this study are available in the DDBJ at https://www.ddbj.nig.ac.jp/, reference number DRA011150. These data were derived from the following resources available in the public domain: https://ddbj.nig.ac.jp/DRASearch/submission?acc=DRA011150.

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Edited by: H.-U. Simon, Bern.