The role of TLR2 and TLR4 in the production of cytokines by human periodontal fibroblasts deficient of these receptors.

• Grant number: 09/07376-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2009
• End date: October 31, 2012
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Carlos Ferreira dos Santos
• Grantee: Ana Carolina Morandini Ramos
• Host Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil

Abstract

When challenged by different agents, periodontal fibroblasts are able to respond through the release of substances, such as cytokines and chemokines that participate in an active way in the inflammatory process. It has been recognized that Toll like receptors (TLR) are stimulated by these molecules and in particular TLR2 and TLR4 has been strongly associated with the host immune response in periodontal ligament and gingival tissues. However, the expression of TLR2 and TLR4 in periodontal fibroblasts remains controversial. Moreover, there are no evidences of periodontal ligament and gingival fibroblasts deficient of these receptors regarding the release of cytokines like MIP-1±, SDF-1, IL-6 e IL-10. The objectives of this study are: to evaluate the expression of TLR2 and TLR4 by periodontal ligament and gingival fibroblasts as well as to investigate the production of cytokines by fibroblasts deficient of these receptors. Human periodontal ligament and gingival fibroblasts will be cultured from biopsies of the same donors and will be used between the 4th and 8th passage. After confluence in 24-well plates, the culture medium alone (control) or with 0,1 - 10 ug/mL of LPS from P. gingivalis will be added in duplicate. Flow cytometry analysis will be performed in stimulated and non-stimulated cells to evaluate and to compare the expression of TLR2 and TLR4. In addition, gene silencing will be done by the transfection of synthetic RNA interference (RNAi), followed by molecular assays as research tools to check the transfection efficacy of the cell cultures like RealTimePCR and Western Blot. Subsequently, for detection of the protein production of cytokines by the fibroblasts, ELISA will be performed.