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Water-soluble chitosan inhibits nerve growth factor and attenuates allergic inflammation in mite allergen–induced allergic rhinitis  Pei-Chi Chen, MSc,

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Presentation on theme: "Water-soluble chitosan inhibits nerve growth factor and attenuates allergic inflammation in mite allergen–induced allergic rhinitis  Pei-Chi Chen, MSc,"— Presentation transcript:

1 Water-soluble chitosan inhibits nerve growth factor and attenuates allergic inflammation in mite allergen–induced allergic rhinitis  Pei-Chi Chen, MSc, Miao-Hsi Hsieh, PhD, Wen-Shuo Kuo, PhD, Hui-Fan Kao, RN, PhD, Chih-Li Hsu, PhD, Jiu-Yao Wang, MD, DPhil  Journal of Allergy and Clinical Immunology  Volume 140, Issue 4, Pages e8 (October 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 EWSC improves nasal pathological function and airway inflammationin Der p–induced AR. A, Total IgE and Der p–specific IgE concentrations of serum were measured through ELISA. B, Respiration rate and airway resistance were measured using the Buxco noninvasive airway-resistant measurement system. C, Thickness of the nasal RE (OE: olfactory epithelium). The positive-stained eosinophils (black arrows) (D) and degranulated mast cells (red arrows) (E) were counted in 400× microscopic fields per group, and the average numbers were calculated. F, Draining lymph nodes of the nasal mucosa were isolated, and TH2 and ILC2 cells were analyzed using flow cytometry and TH2+IL4+, TH2+IL5+, and TH2+IL13+ cell percentage. G, ILC2+IL4+, ILC2+IL5+, and ILC2+IL13+ cell percentages in the draining lymph nodes of the nasal mucosa. H, Sections of the nasal cavity were immunostained with RORα antibodies and reacted with DAB (scale bars = 100 μm). Data are expressed as mean ± SEM (n ≥ 8). *P < .05; **P < .01 compared with the AR group, in Student t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Der p fails to promote allergen-induced inflammation in NGF-deficient mice. A, Thickness of the nasal respiratory epithelium. The positive-stained eosinophils (black arrows) (B) and degranulated mast cells (red arrows) (C) were counted in 400× microscopic fields per group, and the average numbers were calculated (scale bars: 50 μm or 100 μm). D, Draining lymph nodes of the nasal mucosa were isolated, and TH2 and ILC2 cells were analyzed using flow cytometry and TH2+IL4+, TH2+IL5+, and TH2+IL13+ cell percentages. E, ILC2+IL4+, ILC2+IL5+, and ILC2+IL13+ cell percentages in the draining lymph nodes of the nasal mucosa. Data are expressed as mean ± SEM (n ≥ 8). *P < .05; **P < .05, compared with the AR group in Student t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 EWSC inhibits allergen-induced NGF production and airway inflammation. A, Experimental schema. Mice were sensitized with PBS or Der p on days 0 and 7. After the second sensitization, mice were challenged intranasally with PBS or Der p without anesthesia from days 14 to 20. Selected groups of mice were intranasally administered with 1% EWSC at 6 hours before the Der p challenge without anesthesia. TNF-α (B), IL-4 (C), IL-5 (D), IL-13 (E), BDNF (F), NGF (G), VIP (H), and IL-6 (I) production in NLFs. The levels were determined through ELISA. Data are expressed as mean ± SEM (n ≥ 12). BDNF, Brain-derived neurotrophic factor. *P < .05; **P < .01, compared with the AR group in Student t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 EWSC inhibits allergen-induced NGF production and airway inflammation. Sections of the nasal cavity were immunostained with T-bet (A), GATA3 (B), NGF (C), p75NTR (D), TrkA (E), and TRPV-1 (F) antibodies and reacted with DAB (scale bars = 100 μm). The positive-stained T-bet and GATA3 cells were counted in 400× microscopic fields per group, and the average numbers were calculated. The NGF, TrkA, p75NTR, and TRPV-1 in the immunohistochemically stained nasal sections were quantified by Image J. OE, Olfactory epithelium p75NTR, p75 neurotrophin receptor. Data are expressed as mean ± SEM (n ≥ 6). *P < .05; **P < .01 compared with the AR group in Student t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 EWSC inhibits NGF expression in vitro. A, Primary lung cells were incubated for 6 hours with EWSC at different concentrations (0, 8, 40, 200 μg/mL) before TNF-α (20 ng/mL) stimulation. NGF levels in the culture supernatants were measured through ELISA. B, RPMI-2650 cells were incubated for 6 hours with EWSC at different concentrations (10, 100, 200 mg/mL) before Der p (100 μg/mL). Thereafter, whole-cell extracts were used for Western blotting with anti-NGF. Protein expression levels were detected using densitometry and were depicted as proportional lines and bar graphs. Data are expressed as mean ± SEM (n ≥ 3). *P < .05 compared with the nontreatment group in Student t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Der p fails to promote allergen-induced inflammation in NGF-deficient mice. A, Experimental schema. Mice were sensitized and challenged intranasally with Der p allergen daily for 5 days per week for 3 consecutive weeks. B, Total IgE concentrations of serum were measured through ELISA. C, Sections of the nasal cavity were immunostained with NGF antibodies and reacted with DAB. D, NGF production in NLFs was determined through ELISA. i.n., Intranasal. Data are expressed as mean ± SEM (n ≥ 12). *P < .05; **P < .05 compared with the AR group in Student t test (scale bars: 100 μm). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Gating strategy of isolated TH2 and ILC2 cells in the draining lymph nodes of the nasal mucosa. A, Draining lymph nodes of the nasal mucosa were isolated, and TH2 cells were analyzed using flow cytometry. TH2s were identified as CD45+CD4+GATA3+ through the gating strategy. B, Draining lymph nodes of the nasal mucosa were isolated, and ILC2 cells were analyzed using flow cytometry. ILC2s were identified as CD45+Lineage−GATA3+CD127+ST2+ through the gating strategy. ILC2s were negative for TrkA expression. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 Model of NGF involvement in type 2 inflammation and the EWSC inhibition mechanism. During allergen exposure, the nasal mucosa was activated by dendritic cells and epithelial cells. Dendritic cells polarize precursor TH0 cells into TH2 cells, whereas epithelial cells produce NGF and thymic stromal lymphopoietin, which activates nociceptors to release VIP and stimulates ILC2 cell activation, respectively. Furthermore, VIP also stimulates ILC2 and newly differentiated TH2 cells via the VPAC2 receptor. Type 2 cytokines, including IL-4, IL-5, andIL-13, are released by ILC2 and TH2 cells and promote eosinophilia and degranulated mast cell infiltration in the nasal mucosa, IgE secretion by B cells, mucus production by goblet cells, and nasal hyperresponsiveness. Furthermore, NGF- and TH2-related cytokines create an amplification loop resulting in broader allergic inflammation in the upper-airway epithelium. EWSC attenuates allergen-induced nasal AHR and inflammation through the inhibition of NGF and its amplification loop of type 2 immune response. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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