Symbiotic microbiome Staphylococcus aureus from human nasal mucus modulates IL-33-mediated type 2 immune responses in allergic nasal mucosa

Abstract

Background: The host-microbial commensalism can shape the innate immune responses in respiratory mucosa and nasal microbiome also modulates front-line immune mechanism in the nasal mucosa. Inhaled allergens encounter the host immune system first in the nasal mucosa, and microbial characteristics of nasal mucus directly impact the mechanisms of initial allergic responses in nasal epithelium. However, the roles of the nasal microbiome in allergic nasal mucosa remain uncertain. We sought to determine the distribution of nasal microbiomes in allergic nasal mucosa and elucidate the interplay between nasal microbiome Staphylococcus species and Th2 cytokines in allergic rhinitis (AR) models.

Results: Staphylococcus aureus (AR-SA) and S. epidermidis (AR-SE) were isolated from the nasal mucosa of patients with AR. The influence of nasal microbiome Staphylococcus species on allergic nasal mucosa was also tested with in vitro and in vivo AR models. Pyrosequencing data showed that colonization by S. epidermidis and S. aureus was more dominant in nasal mucus of AR subjects. The mRNA and protein levels of IL-33 and TSLP were significantly higher in AR nasal epithelial (ARNE) cells which were cultured from nasal mucosa of AR subjects, and exposure of ARNE cells to AR-SA reduced IL-33 mRNA and secreted protein levels. Particularly, ovalbumin-driven AR mice inoculated with AR-SA by intranasal delivery exhibited significantly reduced IL-33 in their nasal mucosa. In the context of these results, allergic symptoms and Th2 cytokine levels were significantly downregulated after intranasal inoculation of AR-SA in vivo AR mice.

Conclusion: Colonization by Staphylococcus species was more dominant in allergic nasal mucosa, and nasal commensal S. aureus from subjects with AR mediates anti-allergic effects by modulating IL-33-dependent Th2 inflammation. The results demonstrate the role of host-bacterial commensalism in shaping human allergic inflammation.

Keywords: Allergic rhinitis; Interleukin-33; Nasal microbiome; Staphylococcus aureus; Symbiosis.

Fig. 1

Distribution of bacterial colonies in nasal mucus of subjects with allergic rhinitis (AR). a Bacterial species cultured from mucus samples obtained from middle turbinates of subjects with allergic rhinitis (AR) (N = 17) were identified via 16S rRNA gene sequencing. Distribution of the 112 identified bacterial species is presented in the graph. b The relative abundance of nasal commensal, such as S. epidermidis, S. aureus, E. aerogenes and others, in each AR subjects. The bar graph presents the relative abundance of nasal commensal organisms of 17 allergic rhinitis subjects at the species level

Fig. 2

Susceptibility of nasal commensal Staphylococcus species to allergic rhinitis nasal epithelial (ARNE) cells. ARNE cells from four AR subjects were inoculated with nasal commensal, S. aureus and S. epidermidis at a multiplicity of infection (MOI) of 0.25. The mRNA levels of femA genes specific for S. aureus (a) and S. epidermidis (b), normalized to cellular GAPDH transcript levels, were monitored by real-time PCR for 48 h post-infection. Colony counts of supernatants in cultured ARNE cells inoculated with nasal commensal S. aureus (c) and S. epidermidis (d) from allergic rhinitis subjects were performed. Results are presented as mean ± standard deviation (SD) from five independent experiments. *p < 0.05 compared with control ARNE cells

Fig. 3

Staphylococcus species suppress expression of interleukin-33 (IL-33). ARNE cells were inoculated with nasal commensal S. aureus (AR-SA) and S. epidermidis (AR-SE) from subjects with AR at a MOI of 0.25. Levels of mRNAs encoding epithelial cell-derived Th2 cytokines including IL-33 and thymic stromal lymphopoietin (TSLP) were monitored by real-time PCR (IL-33 mRNA in AR-SA-inoculated ARNE cells (a) and AR-SE-inoculated ARNE cells (b), TSLP mRNA in AR-SA-inoculated ARNE cells (c) and AR-SE-inoculated ARNE cells (d). IL-33 mRNA and protein levels of normal human nasal epithelial (NHNE, white bar) and ARNE cells (Black bar) inoculated with AR-SA were analyzed by real-time PCR (e) and Western blot (f), respectively. Secreted IL-33 protein levels of normal human nasal epithelial (NHNE, white bar) and ARNE cells (Black bar) inoculated with AR-SA were analyzed by ELISA (g) Results are presented as mean ± standard deviation (SD) from five independent experiments and western blot results is also representative of five independent experiments. *p < 0.05 compared with control ARNE cells (a, b, c, and d). *p < 0.05 comparing levels between NHNE and ARNE cells (e, g). The original, unprocessed versions of full-length gel/blot images are included in the online data supplement (Additional file 3: supplementary figure)

Fig. 4

Experimental protocol and comparison of nasal symptoms, serum immunoglobulin E (IgE) levels, and eosinophilic infiltration levels. a The experimental protocol for development of allergic asthmatic 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. aureus (AR-SA) (3.2 × 10⁶ CFU/30 μl. PBS) was inoculated at indicated time points (day 19, 20). b mRNA levels of femA gene, specific for S. aureus and normalized to cellular GAPDH transcript levels, were monitored by real-time PCR. c Colony counts of nasal lavage fluid were performed. Frequencies of sneezing (d) and rubbing (e) events were assessed over a 15 min period after OVA provocation. Serum levels of total IgE (f) and OVA-specific IgE (g) were significantly lower in SA-OVA mice than in WT-OVA mice (N = 5). Histologic findings in nasal mucosa of each group (× 400 magnification) with Sirius red staining for eosinophils (h) and periodic acid-Schiff (PAS) staining for secretory cells (i). Results are presented as mean ± standard deviation (SD) (N = 5) and histologic finding is representative of nose sections from five mice. *p < 0.05

Fig. 5

Comparison of cytokine mRNA levels in nasal mucosa. Wild type mice (PBS/PBS) and AR mice (OVA/OVA) were inoculated with human nasal S. aureus (3.2 × 10⁶ CFU/30 μl PBS) at indicated time points (day 19, 20) and mRNA levels of interleukin (IL)-4 (a), IL-5 (b), IL-13 (c), and IL-33 (d) were measured by real-time PCR. e Secreted IL-33 protein levels secreted from nasal mucosa were measured by ELISA using nasal lavage fluid. Results are presented as mean ± standard deviation (SD) (N = 5). *p < 0.05