Linagliptin Inhibits Interleukin-6 Production Through Toll-Like Receptor 4 Complex and Lipopolysaccharide-Binding Protein Independent Pathway in vitro Model

Abstract

Hiroshi Saito,1 Yuya Nakamura,2,3 Masahiro Inagaki,2,4 Shiho Yamadera,5 Hideo Misawa,2,3 Naoki Sato,2,6 Tatsunori Oguchi,2 Takae Inagaki,7 Yuya Tsuji,2 Mayumi Tsuji,2 Isao Ohsawa,3 Hiromichi Gotoh,3 Yuji Kiuchi2 1JA Hiroshima Genneral Hospital, Hatsukaichi City, Hiroshima, Japan; 2Department of Pharmacology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan; 3Saiyu Soka Hospital, Soka City, Saitama, Japan; 4Faculty of Arts and Sciences at Fujiyoshida, Showa University, Fujiyoshida City, Yamanashi, Japan; 5Department of Hospital Pharmaceutics, Showa University School of Pharmacy, Shinagawa-ku, Tokyo, Japan; 6Department of Research Center, Tanabe Pharmacy Inc., Chuo-ku, Tokyo, Japan; 7Graduate School of Nursing and Rehabilitation Science, Yokohama City, Kanagawa, JapanCorrespondence: Yuya Nakamura Email [email protected]: Lipopolysaccharides (LPS) induce inflammation by binding to the Toll-like receptor (TLR) 4 complex, including LPS-binding protein (LBP). The anti-inflammatory effects of linagliptin in LPS-induced inflammation in the TLR4-independent pathway have not been examined before. We examined the anti-inflammatory effects of linagliptin in the TLR4- and the LBP-independent pathway.Methods: U937 cells were cultured in the medium supplemented with 10% fetal bovine serum (FBS) and treated with 100 nM phorbol myristate acetate for 48 h. Cells were then left untreated or were treated with 10 μg/mL anti-TLR4 antibodies alone or in combination with linagliptin for 1 h in media supplemented with or without 10% FBS. The cells were divided into 5 groups: a) control cells (untreated) b) cells treated with LPS c) cells treated with 10 μg/mL anti-TLR4 antibodies d) cells treated with LPS and 10 μg/mL anti-TLR4 antibodies and e) cells treated with LPS, 10 μg/mL anti-TLR4 antibodies, and linagliptin. The LPS concentrations used were 50 pg/mL or 100 pg/mL for cells treated in the presence of 10% FBS and 100 pg/mL or 1 μg/mL for cells treated in the absence of FBS. Linagliptin concentrations of 1 nM, 10 nM, and 100 nM were used for treatment. The supernatants were analyzed for interleukin (IL)-6 production after 24 h of various treatments.Results: LPS increased IL-6 production compared to the untreated control cells, and anti-TLR4 antibody suppressed LPS-induced increased IL-6 levels. Linagliptin suppressed LPS-induced IL-6 production in a concentration-dependent manner in the presence of FBS. However, only 100 nM linagliptin could suppress LPS-induced IL-6 production in the absence of FBS.Conclusion: Concentration-dependent and -independent inflammatory suppression was observed following linagliptin treatment after LPS induction in an experimental model of TLR4 inhibition by anti-TLR4 antibodies. Our results showed that linagliptin may inhibit inflammation through multiple mechanisms centered around the TLR-4-mediated pathway.Keywords: lipopolysaccharide, U937 cells, fetal bovine serum, anti- toll-like receptor 4 antibody, interleukin-6

Keywords

• lipopolysaccharide
• u937 cells
• fetal bovine serum
• anti- toll-like receptor 4 antibody
• interleukin-6