The 105th Annual Congress of the Taiwan Society of Otorhinolaryngology Head and Neck Surgery MyD88 Plays an Important Role in the Immune Response Toward House Dust Mite Allergen in Chronic rhinosinusitis Yi-Tsen Lin, MD Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan August 4, 2018

Allergy accelerates the progression of CRS Introduction Allergy accelerates the progression of CRS The impact of allergies is to accelerate the disease progression of chronic rhinosinusitis. The elevation in the serum-specific IgE was correlated with peripheral eosinophil percentage, especially in the CRSwNP patients.

House dust mite (HDM) allergen House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat Med. 2009 Apr;15(4):410-6. Nature Reviews Immunology 14, 546–558 (2014)

Tlr4 expression in specific cell compartments affects distinct arms of the airway immune response. Expression in hematopoietic cells  neutrophilic inflammation Expression in epithelial cells  eosinophilic inflammation Mucosal Immunol. 2015 Jul;8(4):863-73.

Nature Reviews Immunology 14, 546–558 (2014)

Hypothesis Epithelial cells orchestrate the innate immunity in response to house dust mite allergen. MyD88 and TRIF are critical molecules in innate immune signaling.

Patients Inclusion criteria: Exclusion criteria: Methods Patients with bilateral chronic rhinosinusitis Patients undergoing endoscopic sinus surgery Exclusion criteria: Patients <20 or >65 years of age Patients had systemic antibiotic and/or steroid treatment <1 month before the surgery Patients with cystic fibrosis or congenital mucocilliary problems Patients with immunodeficiency

Tissue samples Control tissues were obtained from patients without any sinonasal disease during other rhinologic surgeries, such as skull base, lacrimal duct, or orbital decompression surgery. We obtained uncinate process tissue and middle turbinate tissue from control subjects and patients with CRS. We also obtained nasal polyp tissue in patients with CRSwNP for air-liquid interface culture.

Tissue samples Each sample obtained was divided into 3 parts: Fixed in 10% formaldehyde and embedded in paraffin for histologic analyses IHC stain for IL-4R, IL-4, IL-13, MyD88, TLR4 Immediately frozen and stored at -80°C for subsequent isolation of mRNA and proteins qPCR for gene expression of IL-4R, MyD88 Homogenized and centrifuged; supernatants were separated and stored at -80°C for further analysis of cytokines and other inflammatory mediators.  ELISA for IL-4, IL-13

Air-liquid interface (ALI) culture Obtaining from nasal polyps of patients undergoing endoscopic sinus surgeries HDM allergen Cytokines Morphology Molecular pathways Functional changes

Patients Results Endoscopic Sinus Surgery Chronic rhinosinusitis Control (EEA, DCR) IHC qPCR ELISA Allergy (N=10) Non-allergy (N=21) EEA (N=5) DCR (N=1) Allergy (N=3) Non-allergy (N=4) ALI culture Cytokine stimulation

The expression of IL-4 receptor and MyD88 in the epithelium of polyps from HDM allergic and non-allergic CRSwNP cases IL-4R IL-4R MyD88 CRS/HDM allergy CRS/non-allergy

The gene expression of IL-4 receptor and MyD88 in the epithelium from HDM allergic and non-allergic CRS cases Uncinate Middle turbinate

Changes of cytokines in the the epithelium from HDM allergic and non-allergic CRS cases

Summary-1 We demonstrated the expression of IL-4, IL-13, IL-4R, and MyD88 and the cellular origins in sinonasal tissues and nasal polyps of human subjects. Investigation of the mechanisms of IL-4/IL-13 on the HDM induced TLR4-MyD88 signal transduction pathway was warranted.

Changes of cytokines and chemokines in LPS- and HDM-stimulated nasal epithelial cells We stimulated ALI cultured nasal epithelial cells with different dosages of house dust mite. We performed protein arrays study to evaluate the chemokine and cytokine expression in various time after stimulation with HDM and LPS. But no obvious change was shown.

Patients list Experiment Age Gender Diagnosis Allergy to Der p   Experiment Age Gender Diagnosis Allergy to Der p Eosinophil LMS SNOT22 1 ALI + cytokine 37 M CRSwNP, bil 22.4(Class=4) 1.6% 17 2 65 5.29(Class=3) 1.4% 10 27 3 57 0.03(Class=0) 8.7% 23 26 4 52 F 0.12(Class=0) 8% 21 56 5 44 0.00(Class=0) 0% 24 40 6 68 0.32(Class=0) 1% 18 22 7 59 0.46(Class=1) 2.3% 63 Here are the patient lists of our study.

Changes of cytokines and chemokines in HDM-stimulated nasal epithelial cells in exposure to distinct cytokines. 加上no HDM

Changes of expression of MyD88 and TRIF in HDM-stimulated nasal epithelial cells in exposure to distinct cytokines.

Changes of cytokines in the culture medium in HDM-stimulated nasal epithelial cells in exposure to distinct cytokines.

Summary-2 IL-4 and IL-13 may potentiate the epithelial reactions to house dust mite in MyD88-denpendant pathway. The up-regulation of MyD88 can only be observed in nasal epithelial cells from HDM allergy patients.

Discussion “Hygiene theory” Our findings were compatible with the research about hygiene theory. There was an interesting study published in NEJM last year. “Hygiene theory”

The exposure to natural dust has the protection effect in MyD88 dependent pathway.

“Monoclonal antibody tx for CRS” Another application of our study is that we started to treat chronic rhinosinusitis with monoclonal antibody

, but not every patients have good response to each monoclonal antibody. Probably we should select the patients to receive distinct monoclonal antibody treatment.

Cross-talk between epithelial cells and immunocytes Conclusion LPS IL-1α TH2 IL-4 IL-13 IL-4 IL-13 The epithelial cells need to be trained and in the right environment to initiate the airway allergy inflammation. The positive feedback between the epithelial cells and immunocytes will magnify the reactions to allergen. In the presence of IL-4 and IL-13, the MyD88 was upregulated. We will do further study on how the cytokine wake up the MyD88 pathway. It will help us to understand the mechanism of allerginicity. And we would like to know whether this group of patient, chronic rhinosinusitis with hust dust mite allergy, is a good candidate for anti-IL4 monoclonal antibody treatment. MyD88 TRIF “Allergenicity” Cross-talk between epithelial cells and immunocytes “Monoclonal Ab Tx”

Thank you for your attention Acknowledgement Prof. Te-Huei Yeh Department of Otolaryngology, National Taiwan University Hospital, Taiwan Prof. Bor-Luen Chiang Department of Medical Research, Thank you for your attention