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Symbiotic microbiome Staphylococcus epidermidis restricts IL-33 production in allergic nasal epithelium via limiting the cellular necroptosis

BMC Microbiology volume 23, Article number: 154 (2023) Cite this article

A Correction to this article was published on 14 June 2023

This article has been updated

Abstract

Background

Allergic rhinitis (AR) is characterized by airway inflammation in nasal mucosa from inhaled allergens and interleukin (IL)-33 is the potent inducer of Th2 inflammation in allergic nasal epithelium. Staphylococcus epidermidis is one of the most abundant colonizers of the healthy human nasal mucosa and might impact the allergen-induced inflammatory responses in the nasal epithelium. Thus, we sought to characterize the mechanism of S. epidermidis regulating Th2 inflammation and IL-33 production in AR nasal mucosa.

Results

The AR symptoms were alleviated and eosinophilic infiltration, serum IgE levels, and Th2 cytokines were significantly decreased in OVA-sensitized AR mice in response to human nasal commensal S. epidermidis. The inoculation of S. epidermidis to normal human nasal epithelial cells reduced IL-33 and GATA3 transcriptions and also reduced IL-33 and GATA3 expression in AR nasal epithelial (ARNE) cells and the nasal mucosa of AR mice. Our data exhibited that the cellular necroptosis of ARNE cells might be involved in IL-33 production and inoculation of S. epidermidis decreased the phosphorylation of necroptosis enzymes in ARNE cells, which was related to the reduction of IL-33 production.

Conclusions

We present that human nasal commensal S. epidermidis reduces allergic inflammation by suppressing IL-33 production in nasal epithelium. Our findings indicate that S. epidermidis serves a role in blocking allergen-induced cellular necroptosis in allergic nasal epithelium which might be a key mechanism of reduction of IL-33 and Th2 inflammation.

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Background

Allergic rhinitis (AR) is initiated according to inhalant allergen exposure at the nasal epithelium and the inhaled allergen-provoked inflammatory process begins with increased secretion of epithelial cell-derived cytokines. This allergen-specific immune response is thought to arise from an imbalance in T helper (Th)1-Th2 immune regulation that results in increased levels of Th2 cytokines [1, 2]. Actually, nasal epithelial cells which are exposed to external allergens induce Th2 inflammatory responses and allergic inflammation occurs in the upper airway or AR subjects [3]. The allergen-mediated inflammatory immune response begins with increased secretion of epithelial cell-derived cytokines which provide critical signals to innate and adaptive cell populations related to Th2 inflammation [4, 5]. Interleukin (IL)-33, which is dominantly produced by the airway epithelium, is a key epithelial-derived cytokine involved in the pathogenesis of allergic airway diseases and provides an essential axis for rapid allergic immune responses [6]. Remarkable progress has recently been achieved to identify the biological function of IL-33 in the nasal epithelium of AR and the potential value of IL-33 as a novel therapeutic target of AR at the nasal epithelium level is strongly suggested [7,8,9,10]. Therefore, we sought to understand the mechanism by which IL-33 is secreted from epithelial cells to induce an allergic reaction.

As stated before, IL-33 production is stimulated by tissue damage and its various cleavage sites by caspases regulating extracellular activity [8, 10]. Recently, the necroptosis of epithelial cells has been suggested as a release mechanism of IL-33, which is driven not by apoptotic caspases but by receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase-like protein (MLKL)-dependent cell death pathway, control IL-33 release [11]. However, studies on whether inhibition of cellular necroptosis can reduce the secretion of IL-33 from the nasal epithelium and regulate AR inflammation are still lacking.

Studies of the nasal symbiotic microbiome and host-to-host communication incorporate environmental cues in the nasal mucosa that reflect the effects of immune responses and specific symbiotic microbiome-mediated defenses from external pathogens. [12]. In the nasal mucosa, inhaled allergens first encounter the host immune system, and the microbial properties of nasal mucus have a direct impact on the mechanism of the initial allergic reaction in the nasal epithelium [12,13,14]. Insights into the microbiota and dysbiosis of allergic nasal mucosa provide basic information regarding the relationship between susceptibility to allergens and allergic inflammation, including the stimulation of epithelial cell-derived Th2 cytokines such as IL-33. [6, 15]. Recent studies have determined how the nasal symbiotic microbiota modulates sensitization tolerance to inhaled allergens in asthma [16,17,18]. Intriguingly, our knowledge of microbial composition in allergic nasal mucus is limited, and a mechanistic consideration of immunomodulatory properties of the nasal microbiome to inhaled allergens has not been comprehensively performed in AR. Previously, we have shown that the composition of the nasal commensal microbiota differs between healthy volunteers and AR patients [6]. Staphylococcus epidermidis is the most dominant bacterial strain in both healthy volunteers and AR patients, and the distribution of S. epidermidis significantly is increased in AR patients compared to healthy volunteers [19]. We hypothesized that this alternated structure of nasal commensal microbiota in AR, which is known as dysbiosis, provides supporting information regarding susceptibility to inhaled allergens and their association with allergic inflammatory response, including Th2 cytokines induction through control of epithelial cell-derived cytokines such as IL-33.

The purpose of this study was to show the distinctive role of the nasal microbiome S. epidermidis, a major symbiont in healthy nasal mucus, and the potential impact on the reduction of Th2 inflammation through decreased cellular necroptosis and subsequent IL-33 release in nasal epithelium.

Results

Administration with human nasal commensal S. epidermidis suppresses allergic inflammation in vivo

First of all, we sought to explore whether the human nasal commensal S. epidermidis exhibited anti-allergic properties using an in vivo murine model of AR. BALB/C mice were sensitized and challenged with OVA (AR-OVA, N = 5) and they were also inoculated with human nasal commensal S. epidermidis for 2 days (day 19, 20) following nasal microbiota depletion (day 15, 16, 17) using 30 µl of an antibiotic cocktail (vancomycin, neomycin, ampicillin, and metronidazole) (Fig. 1A). Then, femA-SE mRNA expression and colony count of S. epidermidis were measured in vivo nasal mucosa to confirm effective colonization of the commensal S. epidermidis in WT and AR-OVA mice after administration. The results revealed that femA-SE mRNA expression and colony count from NAL fluid were significantly increased in WT (1.15 × 109 ± 0.71 × 109 CFU/mℓ) and AR-OVA mice (1.85 × 109 ± 3.54 × 108 CFU/mℓ) with the intranasal inoculation of S. epidermidis than in WT and AR-OVA mice without S. epidermidis inoculation (Fig. 1B and C). The difference in allergic symptoms of in vivo OVA-AR mice was observed depending on the inoculation of S. epidermidis (Fig. 2A and B). The scores for sneezing and rubbing were 2.20 ± 0.84 and 6.60 ± 3.21 in AR-OVA mice and were significantly reduced in AR-OVA mice with the S. epidermidis administration (0.60 ± 0.55, p = 0.009 and 1.60 ± 1.95, p = 0.022, respectively). Eosinophils and secretory cells of nasal mucosal tissue were detected by Sirius red and PAS staining respectively. The absolute eosinophil count in the submucosa of AR-OVA mice administrated with S. epidermidis was significantly lower than in AR-OVA mice (29.25 ± 4.79 and 11.50 ± 2.08, p = 0.002, Fig. 2C). Fewer PAS-stained secretory cells were observed in nasal mucosa of S. epidermidis-administrated AR-OVA mice (Fig. 2D). Serum levels of total IgE (Fig. 2E) and OVA-specific IgE (Fig. 2F) were significantly decreased in AR-OVA mice after inoculation with S. epidermidis, respectively. The real-time PCR data showed that intranasal inoculation of S. epidermidis markedly reduced mRNA levels of IL-4, IL-5, and IL-13 in the nasal mucosa of AR-OVA mice (Fig. 2G-I). These findings revealed that the human nasal commensal S. epidermidis suppresses allergic inflammation in AR in vivo model and reduces transcription of Th2 cytokines in the nasal mucosa.

Fig. 1
figure 1

Experimental protocol of intranasal inoculation withS. epidermidisin OVA-driven AR mouse model. (A) The experimental protocol for the development of AR mouse using BALB/C. Mice were sensitized by intraperitoneal injection of ovalbumin mixed with aluminum hydroxide on days 0, 7, and 14. Daily OVA intranasal challenge was performed from days 22 to 28 (OVA/OVA). Human nasal S. epidermidis (3.2 × 106 CFU/30 µl PBS) was inoculated at indicated time points (day 19, 20). (B) mRNA expression of femA gene, specific for S. epidermidis and normalized to cellular GAPDH transcript levels, were monitored by real-time PCR. (C) Colony counts of nasal lavage fluid were performed

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Fig. 2
figure 2

The alteration of allergic inflammation and transcription of Th2 cytokines in nasal mucosa of OVA-driven AR mouse in response toS. epidermidis. Human nasal S. epidermidis (3.2 × 106 CFU/30 µl PBS) was inoculated at indicated time points to the nose of OVA-sensitized BALB/C mice. Frequencies of sneezing (A) and rubbing (B) events were assessed over a 15 min period after OVA provocation. Histologic findings in nasal mucosa of each group (×400 magnification) with Sirius red staining for eosinophils (C) and Periodic Acid-Schiff (PAS) staining for secretory cells (D). Serum levels of total IgE (E) and OVA-specific IgE (F) were significantly lower in the nasal mucosa of AR-OVA with inoculation of S. epidermidis than in AR-OVA mice (N = 5). The mRNA expressions of IL-4 (G), IL-5 (H), IL-13 (I) were determined using real-time PCR. Results are presented as mean ± standard deviation (SD) (N = 5). *p < 0.05

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Nasal commensal S. epidermidis suppresses IL-33-dependent Th2 immune responses in allergic nasal epithelium

To determine the response of epithelial-derived Th2 cytokines in the allergic nasal epithelium, the human nasal S. epidermidis was inoculated into human ARNE cells for 2 dpi and the transcriptions of IL-33 and TSLP were measured using real-time PCR. The data showed that transcription of IL-33 was markedly attenuated after 1 dpi in ARNE cells after inoculation with S. epidermidis, which likely reduced GATA3 transcription (Fig. 3A and B). In addition, the IL-33 mRNA level (Fig. 3C) and secreted IL-33 and GATA3 protein concentrations (Fig. 3D and E) were significantly reduced in the nasal mucosa of AR-OVA mice in response to inoculation with human nasal S. epidermidis. However, TSLP mRNA expression and secreted protein concentration were slightly decreased in AR-OVA mice with the intranasal inoculation of S. epidermidis (p = 0.164 and p = 0.374, respectively) (supplementary Fig. S1A and B). These findings demonstrated that S. epidermidis-regulated anti-allergic immune responses might be involved in IL-33 downregulation of the nasal mucosa of in vivo AR model.

Fig. 3
figure 3

Human nasal commensalS. epidermidispromoted the reduction of IL-33 production in the nasal epithelium. ARNE cells from four AR subjects were inoculated with human nasal commensal, S. epidermidis at a multiplicity of infection (MOI) of 0.25. Levels of mRNAs encoding epithelial cell-derived cytokines including interleukin including IL-33 (A) and GATA3 (B) which is transcription factor involved in Th2 cell differentiation were monitored by real-time PCR. Results are presented as mean ± SD values from four independent experiments. *p < 0.05 vs. mock-infected ARNE cells. Wild type mice (PBS/PBS) and AR mice (OVA/OVA) were inoculated with human nasal S. epidermidis (3.2 × 106 CFU/30 µl PBS) at indicated time points. The mRNA expression of IL-33 (C) was measured by real-time PCR and IL-33 protein concentration (D) secreted from nasal mucosa were measured by ELISA using nasal lavage fluid. The mRNA expression of GATA3 (E) was also measured using real-time PCR. Results are presented as mean ± standard deviation (SD) (N = 5). *p < 0.05

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Nasal commensal S. epidermidis reduces IL-33 release by cellular necroptosis in ARNE cells

Lastly, we sought to understand whether S. epidermidis reduces IL-33 production by regulating necroptosis of nasal epithelial cells. First, we treated GSK’872 (RIPK3 inhibitor), GW80 (necroptosis inhibitor), and NS (MLKL inhibitor) to ARNE cells for suppression of cellular necroptosis and both transcription and protein expression of IL-33 were determined using real-time PCR, ELISA and western blot analysis. The results revealed that transcription of IL-33, extracellular secreted IL-33 concentration and intracellular IL-33 protein expression were markedly attenuated both after inoculation with S. epidermidis and after treatment of necroptosis inhibitors in ARNE cells (Fig. 4A-C).

Fig. 4
figure 4

S. epidermidisreduces IL-33 production via suppression of RIPK3/MLKL-dependent necroptosis and is independent of apoptosis in allergic nasal epithelium. ARNE cells from four AR subjects were inoculated with human nasal commensal, S. epidermidis at a multiplicity of infection (MOI) of 0.25. The mRNA expression of IL-33 (A) was measured by real-time PCR, IL-33 protein concentration (B) secreted from ARNE cells was measured by ELISA using supernatants, and intracellular IL-33 protein expression (C) was also measured. after treatment of necroptosis inhibitors. Key molecules of necroptosis including RIPK3 (cropped blot) and MLKL and apoptosis including caspase3 were monitored by Western blot analysis (D). The mRNA expression of RIPK3 (E), and MLKL (F), and caspase3 (G) was measured by real-time PCR. Results are presented as mean ± SD values from four independent experiments. *p < 0.05 vs. mock-infected ARNE cells

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The regulatory kinetics of the full IL-33, RIPK3, pMLKL, and CASP3 in response to S. epidermidis were studied in ARNE cells to assess the necroptosis pathway and both real-time PCR and western blot analysis were performed using cell lysates at the indicated time points after inoculation of S. epidermidis. The results of western blot analysis showed that full-length IL-33, total form of RIPK3 and phosphorylation of MLKL protein were reduced at 24 hpi and 48 hpi in S. epidermidis-inoculated ARNE cells and particularly, RIPK3 protein expression was significantly decreased from 8 hpi (Fig. 4D). The real-time PCR results revealed that the mRNA levels of RIPK3 (Fig. 4E) and MLKL (Fig. 4F) genes were significantly attenuated from 8 hpi and reduced expression were maintained until 48 hpi in S. epidermidis-inoculated ARNE cells. However, there was no significant change in CASP3 protein expression and mRNA levels after inoculation of S. epidermidis (Fig. 4D and G). The result elucidated that RIPK3/MLKL-dependent necroptosis pathway might be involved in the suppression of IL-33 expression and human nasal commensal S. epidermidis reduced IL-33 production from ARNE cells vis suppression of RIPK3/MLKL-dependent necroptosis.

Discussion

Our study revealed that human nasal commensal S epidermidis reduced the allergic inflammation in ARNE cells and an AR murine model through regulating IL-33 release from nasal epithelium. Following inoculation of S. epidermidis, the cellular necroptosis could be limited in AR nasal epithelium, and subsequent IL-33 production as an alarmin signaling was suppressed, resulting in the improvement of Th2 inflammation and AR-related pathologic findings. Our findings imply that intranasal inoculation of S. epidermidis might be a potent therapeutic option for controlling Th2 inflammation in nasal mucosa with distinctive mechanism from the existing medication of AR.

Recent studies have highlighted the interaction between the symbiotic microbiota and the host immune system, which play an important role in host immune homeostasis by evolutionary pressures on the host immune system in contrast to the pathogen [20,21,22]. There is also growing evidence that a microbiome community resides in the human nasal mucus and exhibits the potential crosstalk between the nasal microbiome and Th2 allergic inflammation in the upper airway [23, 24]. In agreement with our previous works, colonization by Staphylococcus species is highly increased in the nasal mucus of subjects with AR and particularly, inoculation of isolated nasal commensal S. aureus suppresses Th2 cytokine-dependent allergic inflammation by regulating epithelial cell-derived cytokines including IL-33, in in vitro and in vivo AR models [6]. We determined that additional studies whether the most dominant commensal S. epidermidis in healthy nasal mucus mediates anti-allergic immune responses to define the clear mechanism of host–bacterial commensalism. Actually, we found that inoculation of S. epidermidis was involved in the alteration of transcription profiles including T-cell activation and Th2 immune responses in NHNE cells. In particular, inoculation of human nasal commensal S. epidermidis to cultured ARNE cells and in vivo AR mice reduced allergic inflammation significantly by suppressing the transcription, translation and extracellular release of IL-33 from nasal epithelial cells.

It is noteworthy that IL-33 is a key molecule to induce allergic inflammation in the airway and known that functions as an alarm signal inducing Th2 cytokines [25]. Moreover, IL-33 acts as a proinflammatory cytokine in various immune responses by promoting local inflammatory responses through the recruitment of Th2 inflammatory effectors [25, 26]. There are three epithelial cell-derived cytokines such as IL-25, TSLP, and IL-33 known to mediate Th2 inflammation and IL-33 has been identified to initiate type 2 responses in asthma in response to the clinically relevant allergens including HDM [27]. Previously we also found that IL-33 expression was preferentially elevated in both primary cultured ARNE cells and nasal mucosa of OVA-driven AR mouse model [6, 15]. The current study showed that human nasal commensal S. epidermidis significantly attenuated IL-33 expression in both in vitro and in vivo AR models. These results indicate that S. epidermidis from healthy human nasal mucus provides a promising therapeutic route of AR by limiting IL-33 production in nasal epithelium and understanding of how IL-33 activity can be regulated by S. epidermidis will be necessary for controlling Th2 immune responses.

Up to date, several studies suggested that IL-33 might be released from the extracellular space following cellular damage or mechanical stress caused by allergens or infection [27, 28], and that IL-33 be released into plasma following necroptosis, which was characterized by regulated cell death pathway representing features of apoptosis and necrosis [29, 30]. We also found that inhaled allergens-caused cellular necroptosis might be required for IL-33 release in nasal epithelium. Inoculation of nasal commensal S. epidermidis could be involved in the restriction of cellular necroptosis of nasal epithelium. Our bulk-seq data revealed attenuation of RIPK3 gene expression in nasal epithelium following inoculation of S. epidermidis (supplementary Fig. S2 and Table S1). Both translation of RIPK3 and phosphorylation of MLKL were all significantly reduced in AR nasal epithelium in response to S. epidermidis independent of cellular apoptosis. Based on these findings, we estimate that inoculation of S. epidermidis results in reduced IL-33 production in the nasal epithelium and thus may impede Th2 inflammation in the nasal mucosa.

A major difficulty in understanding S. epidermidis-downregulated Th2 inflammation was that S. epidermidis did not limit the secretion of TSLP, which was also thought to be related to cellular damage of nasal epithelial cells as like IL-33. It can be inferred that TSLP has a distinctive mechanism of transcription and translation from IL-33, and its secretion is thought to be induced by a mechanism independent of cellular necroptosis upon exposure to antigen in nasal epithelium. Another limited point of this study is that we just nasally implanted human nasal commensal S. epidermidis in in vivo model and lacked data on interactions with other species of microbiota. Actually, S. epidermidis exists with other nasal microorganisms in nasal mucus and does not produce pathogenic determinates depending on sustain beneficial microbial environment [31]. Therefore, further experiments will be needed to demonstrate the alternative function of S. epidermidis in whole nasal commensal microbiota and the regulatory influence on additional Th2 cytokines. We acknowledge that limiting the production of IL-33 may not be the only way to regulate allergic inflammation in AR, our study focused to investigate the role and mechanism of S. epidermidis in suppressing IL-33 production in nasal epithelium. The balance between Th1 and Th2 immunity plays an important role in the development and regulation of allergic responses [32]. We used primary cultured human allergic rhinitis nasal epithelial cells, and there was a limitation in the experimental method to evaluate the immune response of lymphocytes such as T cells. Further investigation into the role of S. epidermidis in regulating Th1 cytokines and type 1 inflammatory responses in AR.

Conclusions

We described that the human nasal commensal S. epidermidis reduces IL-33-dependent Th2 inflammation, thereby preventing the pathologic findings of AR and inoculation of S. epidermidis was able to reduce IL-33 expression via RIPK3/pMLKL dependent necroptotic cell death. This intimate association of S. epidermidis and the amplification of cellular necroptosis-induced IL-33 secretion could potentially benefit the AR through the downregulation of IL-33 at the level of the nasal epithelium.

Materials and methods

Participant recruitment

We recruited 8 subjects referred to the Department of Otorhinolaryngology primarily for septoturbinoplasty in Seoul National University Hospital (Seoul, Korea) and Gyeongsang National University Hospital (Jinju, Korea) between September 2020 and January 2021. The Institutional Review Board of Seoul National University College of Medicine (No. 1709-049-883) and Gyeongsang National University Hospital (No. 2019-05-004) provided approval for this study and all participants provided written informed consent. All of them underwent a multi-allergen simultaneous test (MAST) to detect allergens and specific IgE levels to confirm whether subjects experienced atopic traits (Table 1). AR is defined based on the presence of allergen-specific IgE and documentation of the relationship between allergens and typical symptoms such as runny nose, stuffy nose, itching, and/or sneezing [5]. All participants did not have any history of smoking, asthma, other sinonasal diseases, or taking intranasal or systemic corticosteroids before sampling. Four subjects who did not have AR participated in nasal mucosal swabs to isolate nasal commensal S. epidermidis. In addition, intranasal mucosal brushing was done around the middle turbinate for the culture of normal (NHNE) and AR nasal epithelial cells (ARNE) in 4 healthy subjects and 4 AR patients during septoturbioplasty under general anesthesia.

Table 1 Demographics of AR patients included in this study
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Characterization and isolation of S. epidermidis in human nasal mucus

Mucus from the middle turbinate of healthy subjects was collected individually using sterile 3 M Quick swabs (3 M Microbiology Products, St. Paul, MN, USA) from four subjects using a rigid 0-degree endoscope in an operating room. For bacterial colony isolation, the mucus was plated on Lysogeny Broth (LB) plates. After two days of incubation, bacterial colonies were obtained from the LB plates, and the species of each colony was identified using GS-FLX 454 pyrosequencing and 16 S rRNA gene amplification, as described previously [19].

Cell culture and inoculation with human nasal commensal S. epidermidis

NHNE and ARNE cells were cultured from four healthy subjects and four AR patients (Table 1) using an air-liquid interface (ALI) method [33]. Human nasal commensal S. epidermidis was inoculated with NHNE and ARNE cells as previously described [19]. Briefly, the isolated commensal S. epidermidis was plated and grown overnight at 37 °C on LB plates. A single colony of S. epidermidis was grown overnight at 37 °C in 1 ml of LB liquid medium using an incubation shaker. The mixture of commensal S. epidermidis culture was diluted with the non-antibiotic feeding medium to adjust the amount to 300 µl and inoculated into the apical compartment of each NHNE and ARNE cell culture well. NHNE and ARNE cells were incubated for 0, 2, 8, 24, and 48 h at 37 °C in 5% CO2.

Bulk RNA sequencing (bulk-seq)

NovaSeq 6000 platforms (Illumina, San Diego, CA, USA), and preliminary sequencing results were converted to FASTQ files using the Cell Ranger pipeline (10× Genomics). We followed the 10× Genomics standard sequence protocol by trimming the barcode and unique molecular identifier end to 26 bp and the mRNA end to 98 bp. Then, the FASTQ files were aligned to the human reference genome (GRCh38). Subsequently, Cell Ranger was applied for preliminary data analysis to generate a file that contained a barcode table, a gene table, and a gene-expression matrix. We used WinSeurat version 2.1 (Ebiogen Inc., Seoul, Korea), based on Seurat version 3, for quality control, analysis, and exploration of bulk-seq data. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc.). Gene Ontology (GO) enrichment was evaluated using EnrichR analyses based on the cutoff criteria (adjusted p < 0.05, fold change ≥ or < 1.5 and normalized data (log2) ≥ or < 2.0). The original bulk-seq data of the NHNE cells with S. epidermidis inoculation are available (data accessible at NCBI Gene Expression Omnibus database, accession GSE167509).

Murine inoculation model with human nasal commensal S. epidermidis

Animal experiments were approved by the Institutional Animal Care and Use Committees of Gyeongsang National University (No. GNU-201,007-M0065) and the research methods were carried out by the approved guidelines. Five-week-old male wild-type (WT) BALB/C mice (Orient, Busan, Korea) were maintained under specific-pathogen-free conditions, and all mice were housed in a temperature-controlled environment with a 12-hour dark/light cycle. Mice were divided into four groups depending on S. epidermidis administration and OVA sensitization, which were the negative control group (WT), AR group (AR-OVA), S. epidermidis-administrated WT mice group, and S. epidermidis-administrated AR-OVA mice group. AR-OVA mice were sensitized and challenged with OVA as previously described [6]. Briefly, BALB/C mice were sensitized by intraperitoneal injection of 25 ug OVA mixed with 2mg alum on Day 0, 7, and 14 and then challenged by intranasal treatment of 100μg OVA for 7 consecutive days, from Day 22 to Day 28. At the end of the protocol, mice were euthanized by intramuscular injection of a high dose of a mixture of 10 mg/kg xylazine (Bayer, Puteaux, France) and 5 mg/kg ketamine (Merial, Lyon, France). If no heartbeat was detected, death was confirmed. When the mice were euthanized by injection, cervical dislocation was also performed to confirm that the mice were dead. Nasal symptoms such as sneezing and rubbing for 15 min were measured and total and OVA-specific IgE levels were analyzed using a serum sample. Nasal lavage (NAL) fluid was obtained from the nasal cavity by lavaging with 200 µl 0.5mM ethylene diamine tetraacetic acid (EDTA) in phosphate-buffered saline (PBS). The NAL fluid was used for absolute colony count and enzyme-linked immunosorbent assay for measuring secreted protein concentration. Nasal tissue was harvested for real-time PCR and histologic analysis.

Real-time PCR

Human nasal commensal S. epidermidis colonization was monitored using real-time PCR to assess factors that were essential for methicillin-resistant (femA) gene expression specific for S. epidermidis. The S. epidermidis mRNA was monitored using real-time PCR for the femA gene with forward and reverse primers and probe 5’-CAACTCGATGCAAATCAGCAA-3’, 5’-GAACCGCATAGCTCCCTGC-3’, and 5′-JOE-TACTACGCTGGTGGAACTTCAAATCGTTATCG-BHQ1-3′, respectively. Levels of transcripts encoding human IL-33 and GATA3 and mouse IL-4, IL-5, IL-13, GATA3, IL-33, TSLP, MLKL, and RIPK3 were determined using real-time PCR. All primers were purchased from Applied Biosystems (Foster City, CA, USA).

Western blot

Human primary IL-33 antibody was purchased from R&D Systems (AF3625; Minneapolis, MN, USA). Human primary antibodies for RIPK3 (PA5-19956), phospho-MLKL (Ser358, PA5-105678), total MLKL (PA5-34733), and CASP3 (9H19L2) were purchased from Invitrogen (Waltham, MA, USA).

Absolute colony count

Bacterial samples were serially diluted 10-fold with phosphate-buffered saline (PBS). Next, 10 µl of each diluted sample was plated onto an LB agar plate and incubated for 24 h at 37 °C. The number of S. epidermidis colonies growing on the agar surface was counted manually [6, 36]. Bacterial growth was reported based on the colony-forming units (CFUs) for each sample.

Serum levels of total and OVA-specific IgE

Serum samples were collected from mice at the time of sacrifice. Serum samples were stored at − 80 °C until further use. Serum levels of total IgE and OVA-specific IgE were measured by enzyme-linked immunosorbent assay (ELISA) as described previously [6]. The total IgE levels were analyzed by coating the plates overnight with anti-mouse IgE capture monoclonal antibody (mAb) (BD PharMingen, San Jose, CA, USA) at 4 °C. The plate was washed with PBST (PBS containing 0.05% Tween-20) three times and nonspecific antigen–antibody reactions were blocked by incubation with 300 µL of 3% bovine serum albumin (BSA) per well for 1 h at room temperature. Serum samples were added to the 96-well plates along with purified mouse IgE isotype (BD PharMingen) used as a standard, and the plates were incubated for 3 h at 4 °C. The total IgE was detected by adding anti-mouse IgE-horseradish peroxidase (HRP) (#1130-055, Southern Biotechnology, Birmingham, AL, USA) to the plates. The OVA-specific IgE was analyzed by coating the plates with 1 mg/mL OVA at 4 °C overnight. The plates were washed three times with PBST, and nonspecific antigen–antibody reactions were blocked by incubation with 300 µL of 3% BSA per well for 1 h at room temperature. Serum samples were added to the plates and incubated for 2 h at room temperature. The OVA-specific IgE were detected by incubating each plate with biotin rat anti-mouse IgE (BD PharMingen). The plates were then washed three times and incubated with Streptavidin-HRP (BD PharMingen) for 30 min at room temperature. The reactions were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate (Seracare life science, Milllford, MA, USA) and terminated by adding 1 M HCl. Optical density (OD) was measured in a microplate reader at 450 nm.

Histologic analysis

Mice heads were fixed in 10% formalin overnight at room temperature, decalcified, and embedded in paraffin blocks. Paraffin-embedded samples were cut into 4 μm-thick sections, deparaffinized in xylene and dehydrated through a series of graded ethanol solutions. Prepared slides of mice heads stained with hematoxylin and eosin (H&E) for inflammatory cell counting, Sirius red stain for eosinophil counting under a microscope (high power field, ×400 magnification) in 4 fields of the nasal septal mucosa, and Periodic Acid-Schiff (PAS) stain to indicate secretory cells of the nasal septal mucosa [6].

Quantification of secreted cytokines

Secreted human IL-33 (DY3625B) and mouse IL-33 (DY3626) were quantified using the DuoSet® ELISA kit from R&D Systems according to the manufacturer’s instructions.

Statistical analyses

The analyzed data were expressed as the mean ± standard deviation (SD) of 3 independent experiments, and at least 3 independent experiments were performed using primary cultured cells from each donor. We compared the groups with the Mann-Whitney U test to compare results between negative and positive controls, and treatment groups and positive control. We assessed the differences between treatment groups by one-way analysis of variance (ANOVA) accompanied with a post-hoc test. We considered that a p-value < 0.05 was statistically significant. We performed all statistical analyses were using SPSS 25.0 software (IBM, Chicago, IL, USA).

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Change history

Abbreviations

AR:

Allergic rhinitis

Th:

T helper

IL-33:

Interleukin-33

RIPK3:

Receptor-interacting protein kinase 3

MLKL:

Mixed lineage kinase-like protein

NHNE:

Normal human nasal epithelium

ARNE:

Allergic rhinitis nasal epithelium

TSLP:

Thymic stromal lymphopoietin

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Acknowledgements

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Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019M3C9A6091945 and 2022R1A2C2011867 awarded to HJK, 2019R1C1C1002064 and 2022R1C1C1004860 awarded to YJJ). This research was also supported by a grant from the Korean Health Technology Research and Development Project through the Korean Health Industry Development Institute, funded by the Ministry of Health and Welfare of the Republic of Korea (HI20C0546 awarded to HJK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Otorhinolaryngology, Gyeongsang National University Hospital, Jinju, Republic of Korea

    Yung Jin Jeon

  2. Department of Otorhinolaryngology, Seoul National University College of Medicine, Seoul, Republic of Korea

    Chan Hee Gil, Jina Won, Ara Jo & Hyun Jik Kim

  3. Sensory Organ Research Institute, Seoul National University Medical Research Center, Seoul, Republic of Korea

    Hyun Jik Kim

  4. Department of Otorhinolaryngology-Head and Neck Surgery, Seoul National University College of Medicine, 103, Daehak-ro, Jongno-gu, Seoul, 03080, Republic of Korea

    Hyun Jik Kim

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  1. Yung Jin Jeon
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  4. Ara Jo
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Contributions

Study conception and design: YJJ and HJK. Data acquisition: YJJ, CHG, JW, AJ, and HJK. Data analysis and interpretation: YJJ and HJK. Drafting the article or critically revising it for important intellectual content: YJJ and HJK. Final approval of the version to be submitted: HJK. All authors have contributed and approved the final manuscript.

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Correspondence to Hyun Jik Kim.

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Ethics approval and consent to participate

This study was approved and monitored by the Institutional Review Board (IRB) of Seoul National University College of Medicine (No. 1709-049-883) and Gyeongsang National University Hospital (No. 2019-05-004), and by the Institutional Animal Care and Use Committees of Seoul National University Hospital (No. 2016 − 1470). All research methods were carried out in accordance with approved guidelines. Participation was voluntary, and written informed consent was obtained from all subjects. All methods are reported in accordance with ARRIVE guidelines.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

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Jeon, Y.J., Gil, C.H., Won, J. et al. Symbiotic microbiome Staphylococcus epidermidis restricts IL-33 production in allergic nasal epithelium via limiting the cellular necroptosis. BMC Microbiol 23, 154 (2023). https://doi.org/10.1186/s12866-023-02898-7

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Keywords

BMC Microbiology

ISSN: 1471-2180

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