Yukinori Kato

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, Th2 activation and nasal eosinophilia were comparable between WT and TSLPR-deficient mice. ST2- and IL-33-deficient mice showed defective Th2 activation and nasal eosinophilia to acute, but not chronic, ragweed exposure. TSLPR and ST2 double-deficient mice showed defective Th2 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 Th2 activation during acute allergen exposure, but both TSLPR and ST2 contribute to Th2 responses in chronically allergen-exposed mice.

Nasal sensitization with ragweed pollen induces local-allergic-rhinitis-like symptoms in mice.

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. Fcer1a −/− mice were defective in sneezing but developed normal eosinophilic infiltration. Contrary, Rag2 −/− 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.

Proallergic cytokines and group 2 innate lymphoid cells in allergic nasal diseases

Recent advances in our understanding of proallergic cytokines and group 2 innate lymphoid cells (ILC2s) indicate their critical roles in type 2 immunity-mediated disorders. Proallergic cytokines, interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin, are released from epithelial cells in inflamed tissues and drive type 2 inflammation by acting on innate and acquired immune systems. ILC2s are an innate immune population that responds to proallergic cytokines by producing type 2 cytokines. In line with allergic disorders in the lung, skin, and intestine, emerging evidence suggests the involvement of proallergic cytokines and ILC2s in allergic nasal diseases such as chronic rhinosinusitis with polyps (CRSwNP), allergic fungal rhinosinusitis, and allergic rhinitis (AR). In CRSwNP patients, both proallergic cytokine levels and ILC2s frequency are increased in the nasal mucosa. Increased proallergic cytokine levels correlate with poorer disease outcomes in CRSwNP. Levels of nasal proallergic cytokines are also elevated in AR patients. In addition, animal studies demonstrate that cytokines are essential for the development of AR. It is becoming clear that the proallergic cytokine/ILC2s axis participates in allergic diseases by multiple mechanisms dependent upon the inflammatory context. Thus, a thorough understanding of these cytokines and ILC2s including their tissue- and disease-specific roles is essential for targeting the pathways to achieve therapeutic applications.

Nasal delivery of Japanese cedar pollen Cryj1 by using self-gelling immunostimulatory DNA for effective induction of immune responses in mice.

To develop an immunotherapeutic vaccine for treatment of allergic rhinitis, we developed a controlled release formulation of Cryj1, a major Japanese cedar pollen allergen, with immunostimulatory potency. Two sets of hexapod-like structured DNA (hexapodna) were prepared using six oligodeoxynucleotides (ODNs) each, including ODNs with an unmethylated cytosine-phosphate-guanine (CpG) sequence (CpG motif), to obtain an immunostimulatory DNA hydrogel (sDNA hydrogel). A non-immunostimulatory DNA hydrogel (nsDNA hydrogel) was also prepared using ODNs with no CpG motifs. The sDNA hydrogel was more effective than its components or the nsDNA hydrogel for production of interleukin (IL)-12 after addition to murine macrophage-like RAW264.7 cells or after intranasal administration to mice. Then, a Cryj1-loaded sDNA hydrogel (Cryj1/sDNA hydrogel) formulation was prepared by mixing solutions containing both Cryj1 and hexapodna. Cryj1 was slowly released from the sDNA hydrogel in phosphate-buffed saline. After intranasal administration of the fluorescein isothiocyanate (FITC)-labeled Cryj1/sDNA hydrogel in mice, FITC-Cryj1 was retained in the nasal cavity for a longer period than FITC-Cryj1 mixed with hexapodna in solution. Intranasal immunization of mice with the Cryj1/sDNA hydrogel resulted in high levels of Cryj1-specific IgG in nasal lavage fluid (NFL), IL-12 and interferon-γ release from spleen cells after re-stimulation with Cryj1 when compared with intranasal immunization with the other formulations examined. These results indicate that the self-gelling immunostimulatory DNA hydrogel is an effective formulation for controlled induction of allergen-specific immune responses.

The significant expression of TRPV3 in nasal polyps of eosinophilic chronic rhinosinusitis

BACKGROUND The number of patients with eosinophilic chronic rhinosinusitis (ECRS) has been increasing in recent years in Japan. In ECRS, nasal polyps recur immediately after endoscopic sinus surgery. The molecular biological mechanism underlying the refractoriness of ECRS is unclear. METHODS Whole-transcriptome analysis with next-generation sequencing (RNA-seq) was conducted to investigate the molecular biological mechanism of ECRS. Real-time PCR, immunohistochemical staining, and immunofluorescence staining were performed to validate the results of RNA-seq. RESULTS RNA-seq analysis revealed that in the nasal polyps of ECRS, the levels of 3 transcripts were elevated significantly and those of 7 transcripts were diminished significantly. Among the genes encoding these transcripts, TRPV3 (transient receptor potential cation channel, subfamily V, member 3) was identified as the only gene that is highly expressed in ECRS nasal polyps but this genes expression was not previously detected using DNA microarray analysis in peripheral blood eosinophils. TRPV3 is newly identified here as a gene transcribed in ECRS. Our analysis also revealed that TRPV3 was highly expressed in the infiltrating eosinophils and mucosal epithelium of the nasal polyps of ECRS, and further that the more severe the refractoriness was after surgery, the higher the TRPV3 expression was in nasal polyps. CONCLUSIONS TRPV3 might play a role in the refractoriness of ECRS. Additional studies are required to evaluate the function of TRPV3 in ECRS.

DNA Methylation of Proximal PLAT Promoter in Chronic Rhinosinusitis With Nasal Polyps

Background Nasal polyps (NP) are characterized by pseudocysts derived from stromal tissue edema and cause persistent infections in patients with chronic rhinosinusitis (CRS). A low level of tissue-type plasminogen activator (gene name PLAT) is considered a cause of stromal tissue edema because of insufficient plasmin activation in NP; however, the mechanism regulating PLAT gene expression levels is still unclear. The epigenetic mechanism regulating the PLAT gene expression has been studied in other tissues. Objective We aimed to investigate the methylation levels in the proximal PLAT promoter and their effects on gene expression in NP tissue. Methods We investigated the methylation levels at 3 CpG sites in the proximal PLAT promoter regions (−618, −121, and −105 with respect to the transcription initiation site) by bisulfite pyrosequencing and their effects on the gene expression by quantitative real-time polymerase chain reaction (qPCR) in 20 paired samples of NP and inferior turbinate tissue (IT) from patients with CRS. Results The DNA methylation levels at all CpG sites were higher (P < .01), and the PLAT expression was lower (P < .001) in NP compared with IT. The methylation changes at the −618 site showed a negative correlation with the gene expression changes between NP and IT (r = −.65, P < .01). Conclusions Hypermethylation of PLAT promoter may downregulate the gene expression in NP, leading to excessive fibrin deposition by aberrant coagulation cascade. DNA methylation of proximal PLAT promoter may contribute to NP growth and have a potential as a new therapeutic target.

Expression and Functional Analysis of CST1 in Intractable Nasal Polyps

Abstract In this study, we found Cystatin SN (CST1), a type 2 cystatin subfamily member, to be highly expressed in nasal polyps from patients with intractable chronic rhinosinusitis (CRS) with nasal polyps, using a whole‐transcript analysis with next‐generation sequencing. Eosinophilic CRS (ECRS) involves nasal polyps that are refractory and recur immediately after endoscopic sinus surgery. We hypothesized that CST1 may contribute to the pathogenesis of ECRS. We examined the expression of CST1 in nasal polyps from patients with ECRS by assessing mRNA expression levels using real‐time PCR and immunohistochemistry. CST1 showed significantly greater expression in the epithelial cells of nasal polyps from patients with ECRS than in those from patients who did not have ECRS (non‐ECRS). In particular, CST1 showed very strong expression in patients with severe ECRS. The expression of CST1 may be correlated with the recurrent and refractory nature of ECRS. We examined the function of CST1 using nasal epithelial cells and nasal fibroblasts. Stimulation by a combination of IL‐4 plus double‐stranded RNA plus CST1 significantly elevated mRNA expression levels and protein levels of TSLP in nasal epithelial cells. Stimulation by TSLP or IL‐33 significantly elevated mRNA expression levels of CST1 in nasal epithelial cells. Stimulation of CST1 significantly elevated mRNA expression levels of CCL11 and POSTN in nasal fibroblasts. CST1 could amplify eosinophilic infiltration and T‐helper cell type 2 inflammation by interacting with epithelial‐derived cytokines and fibroblasts on nasal polyps. CST1 may be involved in the pathogenesis of ECRS, and may contribute to the severity and recurrence of CRS with nasal polyps after endoscopic sinus surgery.