1Neurosciences Research Center, Tabriz University of Medical Science, Tabriz, Iran
2Immunology Research Center, Tabriz University of Medical Science, Tabriz, Iran
3Department of Immunology, Tabriz Branch, Islamic Azad University, Tabriz, Iran
Objective(s): High Mobility Group Box1 (HMGB1) is a nonhistone, DNA-binding protein that serves a crucial role in regulating gene transcription and is involved in a variety of proinflammatory, extracellular activities. The aim of this study was to explore whether HMGB1 stimulation can up-regulate the expression of Toll-like Receptor 2 (TLR2) and Toll-like Receptor 4 (TLR4) on macrophages from pulpitis and to clarify the subsequent events involving Th17 cells and Th17 cell-associated cytokine changes. Materials and Methods: Having prepared dental pulp tissues of pulpitis and healthy controls, macrophage were isolated and cultured. Macrophages were thereafter stimulated by HMGB1 time course. RT-QPCR, flowcytometer, immunofluorescence, Western blotting, and ELISA techniques were used in the present research. Results: Our results showed that the expression of TLR2 and TLR4 on macrophages stimulated with HMGB1 increased in pulpitis compared with controls (macrophages without HMGB1 stimulation) with a statistical signiﬁcance (P<0.001). In addition, the levels of IL-17, IL-23, and IL-6 in supernatants from cultured macrophages stimulated with HMGB1 from pulpitis increased, and NF-kB, the downstream target of TLR2 and TLR4, also showed a marked elevation after macrophages’ stimulation by HMGB1. Conclusion: The evidence from the present study suggests that the enhanced TLR2 and TLR4 pathways and Th17 cell polarization may be due to HMGB1 stimulation in pulpitis.
1. Hahn CL, Liewehr FR. Update on the adaptive immune responses of the dental pulp. J Endod 2007; 33:773-781.
2. Desai S, Love R, Rich A, Seymour G. Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells? Int Endod J 2011; 44:87-99.
3. Yonehiro J, Yamashita A, Yoshida Y, Yoshizawa S, Ohta K, Kamata N, et al Establishment of an ex vivo pulpitis model by co‐culturing immortalized dental pulp cells and macrophages. Int Endod J 2012; 45:1103-1108.
4. Lin BC, Zhao YM, Yang J, Ge LH. Root resorption of primary molars without successor teeth. An experimental study in the beagle dog. Eur J Oral Sci 2012; 120:147-152.
5. Hamm J, Pulito R, Benedetto S, Barberis L, Hirsch E, Poli V, et al. Magnetically enriched bone marrow‐derived macrophages loaded in vitro with iron oxide can migrate to inflammation sites in mice. NMR Biomed 2008; 21:120-128.
6. Chokechanachaisakul U, Kaneko T, Yamanaka Y, Kaneko R, Katsube K-i, Kobayashi H, et al. Gene expression analysis of resident macrophages in lipopolysaccharide-stimulated rat molar pulps. J Endod 2011; 37:1258-1263.
7. Bruno K, Silva J, Silva T, Batista A, Alencar A, Estrela C. Characterization of inflammatory cell infiltrate in human dental pulpitis. Int Endod J 2010; 43:1013-1021.
8. Caviedes-Bucheli J, Moreno GC, López MP, Bermeo-Noguera AM, Pacheco-Rodríguez G, Cuellar A, et al. Calcitonin gene-related peptide receptor expression in alternatively activated macrophages/macrophages during irreversible pulpitis. J Endod 2008; 34:945-949.
9. Izumi T, Kobayashi I, Okamura K, Matsuo K, Kiyoshima T, Ishibashi Y, et al. An immunohistochemical study of HLA-DR and alpha 1-antichymotrypsin-positive cells in the pulp of human non-carious and carious teeth. Arch Oral Biol 1996; 41:627-630.
10. Schmalz G, Krifka S, Schweikl H. Toll-like Receptors, LPS, and dental monomers. Adv Dent Res 2011; 23:302-306.
11. Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol 2005; 5:331-342.
12. Yang D, Chen Q, Yang H, Tracey KJ, Bustin M, Oppenheim JJ. High mobility group box-1 protein induces the migration and activation of human dendritic cells and acts as an alarmin. J Leukoc Biol 2007; 81:59-66.
13. Sugars R, Karlström E, Christersson C, Olsson M-L, Wendel M, Fried K. Expression of HMGB1 during tooth development. Cell Tissue Res 2007; 327:511-519.
14. Kim YS, Jung HK, Kwon TK, Kim CS, Cho JH, Ahn DK, et al. Expression of Transient Receptor Potential Ankyrin 1 in Human Dental Pulp. J Endod 2012; 38:1087-1092.
15. Michaelson P, Holland G. Is pulpitis painful? Int Endod J 2002; 35:829-832.
16. Kakehashi S, Stanley H, Fitzgerald R. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965; 20:340-349.
17. Närhi M, Jyväsjärvi E, Virtanen A, Huopaniemi T, Ngassapa D, Hirvonen T. Role of intradental A-and C-type nerve fibres in dental pain mechanisms. Proc Finn Dent Soc 1991; 88:507-516.
18. Hahn CL, Liewehr FR. Relationships between caries bacteria, host responses, and clinical signs and symptoms of pulpitis. J Endod 2007; 33:213-219.
19. Chen E, Abbott PV. Dental pulp testing: a review. Int J Dent 2009; 2009:
20. Randolph GJ, Inaba K, Robbiani DF, Steinman RM, Muller WA. Differentiation of phagocytic macrophages into lymph node dendritic cells in vivo. Immunity 1999; 11:753-761.
21. Barreiro LB, Ben-Ali M, Quach H, Laval G, Patin E, Pickrell JK, et al. Evolutionary dynamics of human Toll-like receptors and their different contributions to host defense. PLoS Genet 2009; 5:e1000562.
22. Hirao K, Yumoto H, Takahashi K, Mukai K, Nakanishi T, Matsuo T. Roles of TLR2, TLR4, NOD2, and NOD1 in pulp fibroblasts. J Dent Res 2009; 88:762-767.
23. Mutoh N, Watabe H, Chieda K, Tani-Ishii N. Expression of Toll-like receptor 2 and 4 in inflamed pulp in severe combined immunodeficiency mice. J Endod 2009; 35:975-980.
24. Mutoh N, Tani-Ishii N, Tsukinoki K, Chieda K, Watanabe K. Expression of toll-like receptor 2 and 4 in dental pulp. J Endod 2007; 33:1183-1186.
25. Yang H, Hreggvidsdottir HS, Palmblad K, Wang H, Ochani M, Li J, et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc Natl Acad Sci U S A 2010; 107:11942-11947.
26. Curtin JF, Liu N, Candolfi M, Xiong W, Assi H, Yagiz K, et al. HMGB1 mediates endogenous TLR2 activation and brain tumor regression. PLoS Med 2009; 6:e1000010.
27. Akirav EM, Preston-Hurlburt P, Garyu J, Henegariu O, Clynes R, Schmidt AM, et al. RAGE expression in human T cells: a link between environmental factors and adaptive immune responses. PloS One 2012; 7:e34698.
28. Park JS, Arcaroli J, Yum H-K, Yang H, Wang H, Yang KY, et al. Activation of gene expression in human neutrophils by high mobility group box 1 protein. Am J Physiol Cell Physiol 2003; 284:C870-C879.
29. Shi Y, Sandoghchian Shotorbani S, Su Z, Liu Y, Tong J, Zheng D, et al. Enhanced HMGB1 expression may contribute to Th17 cells activation in rheumatoid arthritis. Clin Dev Immunol 2011; 2012:
30. Su Z, Sun C, Zhou C, Liu Y, Zhu H, Sandoghchian S, et al. HMGB1 blockade attenuates experimental autoimmune myocarditis and suppresses Th17‐cell expansion. Eur J Immunol 2011; 41:3586-3595.
31. He Z, Shotorbani S, Jiao Z, Su Z, Tong J, Liu Y, et al. HMGB1 Promotes the Differentiation of Th17 via Up‐Regulating TLR2 and IL‐23 of CD14+ Macrophages from Patients with Rheumatoid Arthritis. Scand J Immunol 2012; 76:483-490.