ࡱ > q s p R gt bjbj@@ .z *g*g( 8 | ^ T , * * * * * * * $ Z. 1 N * A * H >, 8 8 8 S$ 8 * 8 8 g P,P^ 3 ?$ T, 0 , ; , ^1 ^ f3 g g \ ^1 B3 $ ! | 8 * * 4 , f3 X \ : Symbiotic microbiome Staphylococcus aureus from human nasal mucus modulates IL-33-mediated type 2 immune responses in allergic nasal mucosa Yung Jin Jeon, MD, Chan Hee Gil, Jina Won, Ara Jo, Hyun Jik Kim, MD, PhD Materials and methods Participant recruitment and Sample collection We recruited 17 patients referred to the Department of Otorhinolaryngology primarily for septal surgery in Seoul National University Hospital (Seoul, Republic of Korea) between October 2017 and September 2018, and in Gyeongsang National University Hospital (Jinju, Republic of Korea) between June 2019 and October 2019. 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). All subjects who participated in sampling of nasal mucosa provided written informed consent. Septal deviation was diagnosed with intranasal endoscope and paranasal sinus computed tomography (PNS-CT), and the subjects did not show any clinical or imaging findings about sinusitis. To confirm allergic rhinitis (AR), they underwent an allergic skin test (AST) or a multi-allergen simultaneous test (MAST) for the detection of allergens and specific IgEs. Twenty subjects were classified into healthy subjects and fifteen were diagnosed with AR. The mean age of the subjects was 35.2 7.1 years and there was no significant difference between AR and healthy subjects. 11 3 s i z e d n a s a l m u c o s a w a s o b t a i n e d f r o m t h e m i d d l e t u r b i n a t e o f s u b j e c t s u n d e r g e n e r a l a n e s t h e s i a . C e l l c u l t u r e H u m a n n a s a l e p i t h e l i a l c e l l s w e r e c u l t u r e d a s d e s c r i b e d p r e v i o u s l y [ 2 1 ] . B r i e f l y , p a s s a g e - 2 N H N E c e l l s ( 1 1 0 5 c e l l s / c u l t u r e ) w e r e s e e d e d i n 0 . 5 3 o f c u l t u r e m e d i u m o n T r a n s w e l l c l e a r c u l t u r e i n s e r t s ( 2 4 . 5 3, w i t h a 0 . 4 5 3 p o r e s i z e ; C o s t a r C o . , C a m b r i d g e , M A , U S A ) . C e l l s w e r e c u l t u r e d i n a 1 : 1 m i x t u r e o f b a s a l e p i t h e l i a l g r o w t h m e d i u m a n d D u l b e c c o s M o d i f i e d E a g l e s M e d i u m ( D M E M ) c o n t a i n i n g p r e v i o u s ly described supplements. Cultures were grown submerged for the first nine days. The culture medium was changed on day 1, and every other day thereafter. An airliquid interface (ALI) was created on day 9 by removing the apical medium and feeding the cultures from the basal compartment only. The culture medium was changed daily after the initiation of the ALI. We added antibiotics (such as 1% penicillin and streptomycin) into the media for subculture, and we also added the antifungal agent Fu n g i z o n e ( 1 3/ 1 0 0 0 3 m e d i a ; L i f e t e c h n o l o g i e s , G r a n d i s l a n d , N Y ) a f t e r f i l t e r i n g t h e m e d i a . D u r i n g t h e l a s t s e v e n d a y s , a b a s a l c o m p a r t m e n t f e e d i n g m e d i u m w i t h o u t a n t i b i o t i c s o r a n t i f u n g a l s w a s u s e d f o r t h e i n c u b a t i o n o f N H N E c e l l s w i t h S t a p h y l o c o c c u s s p e c i e s. All experiments described here used cultured nasal epithelial cells taken 14 days after the creation of the ALI. Isolated Staphylococcus aureus strain and Staphylococcus epidermidis strain from a patient with allergic rhinitis (AR-SA and AR-SE) were used to induce acute bacterial infection in NHNE and ARNE cells. The AR-SA and AR-SE strains were maintained in a -80C deep freezer until required, and was plated and grown overnight at 37C on LuriaBertani (LB) agar plates (Difco LB agar, Miller base; Bect o n D i c k i n s o n a n d C o m p a n y , F r a n k l i n L a k e s , N J , U S A ) . A s i n g l e c o l o n y o f A R - S A a n d A R - S E w a s g r o w n i n 1 3 o f L B m e d i u m ( D i f c o "! L B b r o t h , M i l l e r b a s e ; B e c t o n D i c k i n s o n a n d C o m p a n y , F r a n k l i n L a k e s , N J , U S A ) f o r 2 4 h , i n a 3 7 C i n c u b a t i o n s h a k e r . 1 0 3 o f f r e s h L B m e d i u m w a s a d d e d t o 1 0 0 3 o f t h e A R - S A a n d A R - S E c u l t u r e . T h i s m i x t u r e w a s g r o w n f o r 2 h t o r e a c h l o g p h a s e . P a s s a g e - 2 f u l l y d i f f e r e n t i a t e d N H N E c e l l s w e r e e i t h e r m o c k - i n f e c t e d ( P B S ) o r i n o c u l a t e d w i t h A R - S A a n d A R - S E s t r a i n s t o a p i c a l s i d e o f A L I a t a m u l t iplicity of infection (MOI) of 0.25. After inoculation, the cells were incubated at 37C in 5% CO2. At the designated times post-inoculation, the cell lysate and culture supernatant were collected. The AR-SA and AR-SE cultures were diluted with the non-antibiotic feeding medium to adjust the amount to 300 3 a n d i n o c u l a t e d t o t h e a p i c a l c o m p a r t m e n t o f e a c h N H N E a n d A R N E c e l l c u l t u r e w e l l s . N H N E a n d A R N E c e l l s w e r e i n f e c t e d w i t h A R - S A a n d A R - S E c u l t u r e s f o r 0 , 2 , 8 , 2 4 , a n d 4 8 h r s . M u r i n e i n o c u l a t i o n m o d e l A n i m a l e x p e r i m e n t s w e r e a p p r o v e d b y t h e I n s t i t u t i o n a l Animal Care and Use Committees of Seoul National University Hospital (No 2016-1470) and the research methods were carried out in accordance with the approved guidelines. Four-week-old female wildtype (WT) BALB/c mice (Orient, Gyeonggi, Republic of 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. For infections, AR-SA and AR-SE (3.2x106 C F U i n 3 0 3 P B S ) w e r e i n f e c t e d i n t o W T m i c e b y i n t r a n a s a l d e l i v e r y . M i c e w e r e e u t h a n i z e d b y c e r v i c a l d i s l o c a t i o n o r b y i n t r a m u s c u l a r i n j e c t i o n o f h i g h d o s e o f a m i x t u r e o f 1 0 m g / k g x y l a z i n e ( B a y e r , P u t e a u x , F r a n c e ) a n d 5 m g / k g k e t a m i n e ( M e r i a l , L y o n , F r a n c e ) according to the reviewed protocol. Death was verified when no heartbeat was detected. When mice were euthanized by injection, a cervical dislocation was also performed to ensure that the mice were dead. After euthanizing the mice, nasal lavage (NAL) fluid was obtained from the nasal cavity by lavaging with 1000 l 0 . 5 m M e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) i n p h o s p h a t e - b u f f e r e d s a l i n e ( P B S ) . T h e N A L f l u i d w a s u s e d f o r e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ( E L I S A ) f o r m e a s u r i n g s e c r e t e d p r o t e i n l e v e l s . M o u s e n a s a l t i s s u e w a s a l s o h a r v e s t e d f o r r e a l - t i m e P C R ( R T - P C R), Ovalbumin sensitization and nasal challenge The mice were divided into four groups. The negative control group (WT-PBS) was sensitized and challenged with phosphate-buffered saline (PBS), the positive control group (WT-OVA) was sensitized and challenged with ovalbumin (OVA), the SA-PBS group consisted of AR-SA infected mice sensitized and challenged with PBS, and the SA-OVA group consisted of AR-SA infected mice sensitized and challenged with OVA. The schedule for allergen sensitization and intranasal c h a l l e n g e i s s u m m a r i z e d i n f i g u r e 1 A . B r i e f l y , t h e W T - O V A a n d S A - O V A g r o u p s w e r e s e n s i t i z e d b y i n t r a p e r i t o n e a l i n j e c t i o n o f 2 5 3 O V A m i x e d w i t h 2 3 a l u m o n d a y s 0 , 7 , a n d 1 4 a n d t h e n c h a l l e n g e d b y i n t r a n a s a l t r e a t m e n t o f 1 0 0 3 O V A f o r 7 c o n s e c u t i v e d a y s , f r o m d a y s 2 2 t o d a y 2 8 . T h e W T - P B S a n d S A - P B S g r o u p s w e r e i n j e c t e d i n t r a p e r i t o n e a l l y a n d c h a l l e n g e d i n t r a n a s a l l y w i t h P B S f o l l o w i n g t h e s a m e s c h e d u l e . S e r u m l e v e l s o f t o t a l a n d O V A - s p e c i f i c I g E M o u s e s e r u m s a m p l e s w e r e s t o r e d a t - 7 0 ! b e f o r e u s i n g f o r m e a s u r e m ents of total IgE and OVA specific IgE, as described previously [22]. Briefly, total serum IgE was measured by a standard enzyme-linked immunosorbent assay (ELISA) using an anti-mouse IgE capture monoclonal antibody (BD Pharmingen, San Diego, CA, USA) and horseradish peroxidase (HRP)-conjugated anti-mouse IgE (Southern Biotechnology, Birmingham, AL, USA). To detect OVA-specific IgE, 96-well immune p l a t e s w e r e c o a t e d w i t h 1 0 0 3/ 3 o f O V A i n c a r b o n a t e b i c a r b o n a t e b u f f e r . 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