Involvement of toll-like receptors in autoimmune sialoadenitis of the non-obese diabetic mouse Shigeyoshi Shimizu 1 , Yoshihito Kurashige 2 , Michiko Nishimura 1 , Mami Yamazaki 1 , Jun Sato 1 , Masato Saitoh 2 , Denis Selimovic 3 , Yoshihiro Abiko 1 1 Division of Oral Medicine and Pathology, School of Dentistry, Health Sciences University of Hokkaido, Sapporo, Japan; 2 Division of Pediatric Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Sapporo, Japan; 3 Department of Oral Medicine, Surgery and Pathology, Dental Faculty, University Hospitals of Strasbourg, Strasbourg, France The aim of this study was to characterize the expression of Toll-like receptors (TLRs) during the development of sialoadenitis in the non-obese diabetic mouse. Sub- mandibular glands were dissected from non-obese diabetic mice at 4, 8, 10, 12, and 16 weeks of age. The mRNA expression levels of TLR1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, MyD88, and TRIF was quantified using real-time reverse transcription polymerase chain reaction. The mRNA expression levels in 4-week-old non-obese diabetic mice were used as controls. The expression levels of TLR1, 2, 4, and 9 were significantly higher at 8, 10, 12, and 16 weeks than the levels in the controls. The expression level of TLR3 was significantly higher at 16 weeks than in the controls. A group of mice were given drinking water containing 4.75% chloroquine starting at 4 weeks. Chloroquine caused a significant decrease in the expression of TLR1, 2, 3, 4, and 9 at 16 weeks compared with control mice who did not re- ceive chloroquine. The areas of lymphocyte infiltration seen on serial sections of submandibular glands in the mice receiving chloroquine were significantly smaller than the areas of infiltration in control glands. Increased expression of Toll-like receptors may be involved in the development and / or progression of sialoadenitis in the non-obese dia- betic mouse. Toll-like receptors may be a therapeutic target for autoimmune sialoadenitis. J Oral Pathol Med (2012) Keywords: autoimmune sialoadenitis; salivary gland; toll-like receptors; xerostomia Introduction Sjo¨gren syndrome (SS) is an autoimmune disease characterized by chronic inflammation, primarily of the lacrimal and salivary glands, leading to hyposecre- tion of these glands and dry eyes and dry mouth. Hyposalivation causes dysphagia and increasing num- bers of species of flora, and is a risk factor for caries, periodontitis, and oral candidiasis. The pathogenesis of Sjo¨gren syndrome remains largely unknown, although environmental factors, immune disorders and auto- nomic defects have been identified (1, 2). Toll-like receptors (TLR) belong to a conserved family of type I transmembrane receptors that function as innate immune receptors. TLRs are typically expressed on macrophages, dendritic cells, epithelial cells, and endothelial cells, where they provide rapid early responses to microbial danger signals, including the induction of secretion of proinflammatory cytokines that recruit and activate additional immune responses (3–5). There are 10 human (TLR1-10) and 12 mouse (TLR 1-9, 11-13) TLRs (3–5). Each member of the family has been shown to recognize distinct elements, known as pathogen-associated molecular patterns, of bacteria, fungi, or viruses. TLR2 can form a hetereodi- mer with either TLR1 or TLR6 to recognize bacterial tri- or diacyl lipopeptides. The heterodimer TLR2 / 6 recognizes lipoteichoic acid, which is associated with the cell walls of Gram-positive bacteria. TLR2 also recog- nizes the peptidoglycan component of bacteria and fungi. TLR3, 4, 5, and 6 recognize double-stranded RNA, Gram-negative bacterial lipopolysaccharide, and bacterial flagellin. TLR7 and 8 recognize single-stranded RNAs found in certain viruses. TLR9 recognizes hypomethylated CpG motifs of bacterial double- stranded DNA. The pathogen-associated molecular pattern recognized by TLR10 is unknown. TLR 11, 12, and 13 are detected in the mouse but not in the human. TLR11 recognizes profilin-like protein, and the pathogen-associated molecular patterns recognized by TLR12 and 13 are unknown. Inappropriate expression of TLRs has been demon- strated in autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel dis- ease, systemic lupus erythematosus, and psoriasis (3–5). Correspondence: Yoshihiro Abiko, Division of Oral Medicine and Pathology, School of Dentistry, Health Sciences University of Hok- kaido, Sapporo, Hokkaido 002-8072, Japan. Tel: +81 11 778 7558, Fax: +81 11 770 5035, E-mail: yoshi-ab@hoku-iryo-u.ac.jp Accepted for publication February 2, 2012 doi: 10.1111/j.1600-0714.2012.01136.x J Oral Pathol Med ª 2012 John Wiley & Sons A/S Æ All rights reserved wileyonlinelibrary.com/journal/jop