Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis

Background: Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). Objective: We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. Methods: We performed an in vitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. A human nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption in vitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier in vivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. Results: rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption in vitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption in vivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. Conclusion: Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen–induced AR

Activation of group 2 innate lymphoid cells exacerbates and confers corticosteroid resistance to mouse nasal type 2 inflammation

Both Th2 cells and group 2 innate lymphoid cells (ILC2s) contribute to allergic diseases. However, their exact role and relationship in nasal allergic disorders are unclear. In this study, we investigated the cooperation of Th2 cells and ILC2s in a mouse model of nasal allergic disorder. To differentially activate Th2 cells and/or ILC2s in nasal mucosa, mice were intra-nasally administered ovalbumin (OVA) antigen, papain, an ILC2-activator, or both for 2 weeks. Epithelial thickness and number of eosinophils in the nasal mucosa were evaluated at 24 h after the final challenge. Intra-nasal administration of OVA and papain preferentially activated Th2 cells and ILC2s, respectively, in the nose. Both OVA and papain increased the nasal epithelial thickness and number of eosinophils, and their coadministration significantly enhanced the symptoms. Although T-/B-cell-deficient mice showed severely decreased nasal symptoms induced by OVA or OVA-plus-papain, the mice still showed slight papain-induced nasal symptoms. In ILC2-deficient mice, OVA-plus-papain-induced nasal symptoms were suppressed to the same level as OVA-alone. Similarly, IL-33- and ST2-deficient mice showed decreased OVA-plus-papain-induced nasal symptoms. IL-5 induced eosinophilia only, but IL-13 contributed to both nasal epithelial thickening and eosinophilia induced by OVA-plus-papain. Dexamethasone ameliorated OVA-alone-induced nasal epithelial thickening. However, OVA-plus-papain-induced nasal epithelial thickening was only partially controlled by dexamethasone. These results demonstrate that IL-33/ST2-pathway-mediated ILC2 activation exacerbated Th2-cell-induced nasal inflammation by producing IL-13. Although Th2-cell-alone-induced nasal inflammation was controlled by corticosteroid treatment, the activation of ILC2s conferred treatment resistance. Therefore, ILC2s and their activators could be therapeutic targets for treatment-refractory nasal allergic disorders

Murine allergic rhinitis and nasal th2 activation are mediated via tslp- and il-33-signaling pathways

Thymic stromal lymphopoietin (TSLP) and IL-33 are epithelium-derived proallergic cytokines that contribute to allergic diseases. Although the involvement of TSLP in allergic rhinitis (AR) is suggested, the exact role of TSLP in AR is poorly understood. Furthermore, the relative contribution of TSLP and IL-33 in nasal allergic responses has not been described. In this study, we examined the roles of TSLP and IL-33 in AR by analyzing acute and chronic AR models. Acute AR mice were intraperitoneally immunized with ragweed, then intranasally challenged with ragweed pollen for four consecutive days. Chronic AR mice were nasally administrated ragweed pollen on consecutive days for 3 weeks. In both models, TSLP receptor (TSLPR)-deficient mice showed defective sneezing responses and reduced serum ragweed-specific IgE levels compared with wild-type (WT) mice. Analyses of bone-marrow chimeric mice demonstrated that hematopoietic cells were responsible for defective sneezing in TSLPR-deficient mice. In addition, FcεRI + -cell-specific TSLPR-deficient mice showed partial but significant reduction in sneezing responses. Of note, T h2 activation and nasal eosinophilia were comparable between WT and TSLPR-deficient mice. ST2- and IL-33-deficient mice showed defective T h2 activation and nasal eosinophilia to acute, but not chronic, ragweed exposure. TSLPR and ST2 double-deficient mice showed defective T h2 activation and nasal eosinophilia even after chronic ragweed exposure. These results demonstrate that TSLPR signaling is critical for the early phase response of AR by controlling the IgE-mast-cell/basophil pathway. The IL-33/ST2 pathway is central to nasal T h2 activation during acute allergen exposure, but both TSLPR and ST2 contribute to T h2 responses in chronically allergen-exposed mice

Nasal Sensitization with Ragweed Pollen Induces Local-Allergic-Rhinitis-Like Symptoms in Mice

<div><p>Recently, the concept of local allergic rhinitis (LAR) was established, namely rhinitis symptoms with local IgE production and negative serum antigen-specific IgE. However, the natural course of LAR development and the disease pathogenesis is poorly understood. This study investigated the pathophysiology of mice with allergic rhinitis that initially sensitized with ragweed pollen through the nasal route. Mice were nasally administrated ragweed pollen over consecutive days without prior systemic immunization of the allergen. Serial nasal sensitization of ragweed pollen induced an allergen-specific increase in sneezing, eosinophilic infiltration, and the production of local IgE by day 7, but serum antigen-specific IgE was not detected. Th2 cells accumulated in nose and cervical lymph nodes as early as day 3. These symptoms are characteristic of human LAR. Continual nasal exposure of ragweed pollen for 3 weeks resulted in the onset of classical AR with systemic atopy and adversely affected lung inflammation when the allergen was instilled into the lung. <i>Fcer1a</i>^−/− mice were defective in sneezing but developed normal eosinophilic infiltration. Contrary, <i>Rag2</i>^−/− mice were defective in both sneezing and eosinophilic infiltration, suggesting that T cells play a central role in the pathogenesis of the disease. These observations demonstrate nasal allergen sensitization to non-atopic individuals can induce LAR. Because local Th2 cell accumulation is the first sign and Th2 cells have a central role in the disease, a T-cell-based approach may aid the diagnosis and treatment of LAR.</p></div

Repeated nasal ragweed sensitization induces systemic atopy.

<p>Nasal, ragweed (RW)-sensitized mice were analyzed 1 day after final sensitization. (A) Experimental schema. (B) Serum immunoglobulin levels. (C) Production of cytokines in cLN cells. (D–F) Histological examinations. (D) Hematoxylin and eosin (HE) staining of coronal section of nasal cavity. (E) HE staining of nasal lateral mucosa; black square in (D). (F) Periodic acid-Schiff staining of nasal septum; red square in (D). (G) Presence of eosinophils in nasal mucosa. Data representative of three independent experiments (means, SEMs, n = 5). *P<0.05, ***P<0.001. N.D. not detected.</p

Nasal sensitization of ragweed adversely affects allergic inflammation in lungs.

<p>Nasal, ragweed (RW)-sensitized mice were intratracheally challenged with ragweed 3 days after final sensitization. (A) Experimental schema. (B) BALF CD45⁺ cells and eosinophils. Hematoxylin and eosin (C) and Periodic acid-Schiff (D) staining of lungs. Data representative of three independent experiments (means and SEMs, n = 4). *P<0.05, **P<0.01.</p

Local IgE production by nasal ragweed sensitization.

<p>Mice were nasally administered ragweed (RW) pollen on indicated consecutive days. GLTs and PSTs expression in cLN (A) and nasal (B) B cells. (C) qPCR analysis of <i>Aicda</i> expression in cLN and nasal B cells. Data representative of three independent experiments (n = 2). Nasal B cells were pooled from five mice.</p