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Functional Analysis of the Thymic Stromal Lymphopoietin Variants in Human Bronchial Epithelial Cells

¹ Laboratory for Respiratory Diseases, Center for Genomic Medicine, The Institute of Physical and Chemical Research (RIKEN), Kanagawa, Japan; ² Department of Pediatric Allergy, Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, Habikino, Osaka, Japan; ³ School of Human Nursing, The University of Shiga Prefecture, Shiga, Japan; ⁴ Miyatake Asthma Clinic, Osaka, Japan; ⁵ Department of Otolaryngology, Japanese Red Cross Society, Wakayama Medical Center, Wakayama, Japan; ⁶ Department of Medical Genetics, Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, Japan; ⁷ Department of Pediatrics, Dokkyo University School of Medicine, Tochigi, Japan; ⁸ Department of Pediatric Allergy, National Sagamihara Hospital, Clinical Research Center for Allergy and Rheumatology, Kanagawa, Japan; ⁹ Department of Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; ¹⁰ Laboratory of Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; and ¹¹ Immunology Program, Benaroya Research Institute, Seattle, Washington

Correspondence and requests for reprints should be addressed to Mayumi Tamari, M.D., Ph.D., Laboratory for Respiratory Diseases, Center for Genomic Medicine, Institute of Physical and Chemical Research (RIKEN), 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail: tamari{at}src.riken.jp.

Thymic stromal lymphopoietin (TSLP) is an IL-7–like cytokine that triggers dendritic cell–mediated T helper (Th)2 inflammatory responses, and is implicated in the pathogenesis of allergic diseases in humans. Two TSLP splice variants have been reported. To find functional genetic variants that might contribute to disease, we conducted analyses of single nucleotide polymorphisms (SNPs) of the TSLP gene in human bronchial epithelial cells. We surveyed SNPs on the TSLP gene by sequencing genomic DNA from 36 subjects, and characterized the linkage disequilibrium of the gene. We examined whether the SNPs have functional effects on mRNA expression or protein production using real-time PCR, reporter gene analysis, and enzyme-linked immunosorbent assay. We identified a total of 23 polymorphisms in the TSLP gene. The long form of TSLP, which is associated with allergic inflammation, was highly induced by poly(I:C) (double-stranded RNA) stimulation in normal human bronchial epithelial cells (NHBE) (P = 0.0060). The SNP rs3806933 (–847C > T) in the promoter region of long-form TSLP was found to create a binding site for the transcription factor activating protein (AP)-1, and in vitro functional analyses demonstrated that the SNP enhanced AP-1 binding to the regulatory element. The functional variant increased promoter-reporter activity of long-form TSLP in response to poly(I:C) stimulation in NHBE. Functional genetic polymorphism of the TSLP gene appears to contribute to Th2-polarized immunity through higher TSLP production by bronchial epithelial cells in response to viral respiratory infections.

Key Words: bronchial epithelial cells • dsRNA • polymorphisms • splicing variants • TSLP

CLINICAL RELEVANCE We identified a single nucleotide polymorphism that creates a binding site for activating protein-1 and affects the transcriptional efficiency of the long-form TSLP induced by poly(I:C) in bronchial epithelial cells. The variant may be involved in the pathogenesis of T helper (Th)2-mediated diseases.