Short ragweed pollen triggers allergic inflammation through Toll-like receptor 4– dependent thymic stromal lymphopoietin/OX40 ligand/OX40 signaling pathways

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Short ragweed pollen triggers allergic inflammation through Toll-like receptor 4– dependent thymic stromal lymphopoietin/OX40 ligand/OX40 signaling pathways De-Quan Li, MD, PhD, Lili Zhang, MD, Stephen C. Pflugfelder, MD, Cintia S. De Paiva, MD, Xiaobo Zhang, MD, Guiqiu Zhao, MD, PhD, Xiaofen Zheng, MD, Zhitao Su, MD, Yangluowa Qu, MS Journal of Allergy and Clinical Immunology Volume 128, Issue 6, Pages 1318-1325.e2 (December 2011) DOI: 10.1016/j.jaci.2011.06.041 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Stimulated expression of TSLP signaling molecules and TH2 cytokines in BALB/c mice with SRW pollen–induced EAC. The mRNA expression of TSLP and its downstream signals in DCs (TSLPR, OX40L, and CD11c) and T cells (OX40 and CD4) and the expression of TH1 (IFN-γ) and TH2 cytokines (IL-4, IL-5, and IL-13) by corneal epithelium (A), conjunctiva (B), and CLNs (C) were evaluated by using RT-qPCR in BALB/c mice with EAC, with PBS-treated mice as controls. Results shown are means ± SDs of 4 independent experiments. ∗P < .05 and ∗∗P < .01 (n = 4). ND, Not detectable. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Stimulated production of TSLP signaling proteins and TH2-dominant inflammation in an SRW-induced EAC model requires TLR4 and MyD88. A and C, Immunohistochemical staining of TSLP signaling molecules and TH2 cytokines on the cornea and conjunctiva (Conj) of wild-type and Tlr4-d BALB/c mice (Fig 3, A) and C57BL/6-based wild-type MyD88+/+ and MyD88−/− mice (Fig 3, C) challenged with SRW pollen, with PBS-treated mice as controls. B and D, Immunofluorescent staining of TSLP-activated signals, TSLPR, OX40L, and OX40 in CLNs of different strains, as described above. Bar = 20 μm. Arrows, Red or red-brown positive staining signals. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Stimulated expression of TSLP signaling molecules and TH2 cytokines in an SRW-induced EAC model requires TLR4 and MyD88. The mRNA expression of TSLP signaling molecules and TH2 cytokines by corneal epithelium, conjunctiva, and CLNs in an EAC model with wild-type and Tlr4-d BALB/c mice (A) and with C57BL/6-based MyD88+/+ and MyD88−/− mice (B) sensitized and topically challenged by SRW pollen is shown, with PBS-treated mice as controls. Results shown are means ± SDs. ∗P < .05 and ∗∗P < .01 (n = 4) compared with PBS-treated control animals; ▿P < .05 and ▿▿P < .01 (n = 4) compared with wild-type mice. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 SRWe induces TSLP mRNA (A, C, and E) and protein (B, D, and F) by murine corneal and conjunctival epithelia through a TLR4- and MyD88-dependent pathway. Fig 4, A and B, BALB/c mice were topically administered SRWe at 150 μg/5 μL per eye without or with preinstilled rat anti-mouse TLR4 antibody (1 μg/5 μL per eye) or its isotype rat IgG2a. Fig 4, C and D, TSLP induction by topically challenged SRWe or LPS (5 μg/5 μL per eye) in wild-type and Tlr4-d BALB/C mice. Fig 4, E and F, TSLP induction by means of topical challenge in wild-type MyD88+/+ and MyD88−/− mice. Untreated or PBS-treated mice were used as controls. Results shown are means ± SDs (n = 4). ∗P < .05 and ∗∗P < .01 compared with PBS-treated controls. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 SRWe induces TSLP mRNA and protein by HCECs through TLR4 and NF-κB signaling pathways. A and B, Primary HCECs were treated with 0.1 to 50 μg/mL SRWe for 4 hours for TSLP mRNA or 48 hours for TSLP protein measurement in the supernatants. C and D, HCECs were preincubated with murine TLR4 antibody (10 μg/mL), isotype murine IgG2a κ, or the NF-κB activation inhibitor quinazoline (NFκB-I, 10 μmol/L) for 1 hour before adding 10 μg/mL SRWe for 4 hours for TSLP mRNA or 48 hours for TSLP protein measurement in the supernatants. Results shown are means ± SDs (n = 4). ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Serum SRW-specific IgE levels in an SRW-sensitized EAC model in different strains. Results shown are means ± SDs (n = 3). ∗P < .05 compared with untreated (UT) mice. Journal of Allergy and Clinical Immunology 2011 128, 1318-1325.e2DOI: (10.1016/j.jaci.2011.06.041) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions