Phenotypic identification of Toll-like receptor 2 expressing cell in refractory periapical granuloma of endodontic origin

Background: Root canal infection is a common disease in human teeth. Infection arises when oral bacteria invade and colonise the pulp space through carious lesions, cracks or fractures of coronal tooth structure or breakdown of the tooth-restoration interface. The immune system keeps the intra-radicular infection in check by creating an area of inflammation in the periapical tissue around the root preventing the bacteria from spreading to other parts of the body. However, pain, abscess and tooth loss are inevitable unless the intra-radicular infection is removed by root canal treatment. The process of how human immune cells recognise bacteria was not fully understood until the recent discovery of Toll-Like receptors (TLRs). It was found that TLRs are located on surveillance cells of the innate immune system to recognize invading bacteria and recruit more potent adaptive immune cells to fight against them. Desai et al. (2011) was the first group to demonstrate TLR2 expression in refractory periapical lesions of endodontic origin. The purpose of this research is to further phenotypically characterise TLR2+ immune cells in symptomatic refractory periapical granuloma using histologic staining techniques. The information obtained from this research will increase our knowledge on the development of periapical lesions and the interactions between bacteria and the immune system. Hypothesis and Aim: It is hypotheisied that since refractory periapical granuloma of endodontic origin is a bacterial induced disease TLR2 expressing cells in the lesion will be consistent with those identified in other infection processes. The aim of this study is to phenotypically characterize TLR2 expressing immune cells within refractory periapical granulomata using a double immunofluorescence (DIF) technique to demonstrate the co-expression of cell markers that encode specific immune cells with toll-like receptor 2. Methodology: A pilot study was performed using immunoperoxidase and immunofluorescence techniques on formalin-fixed samples to standardise and validate TLR2, CD38 (lymphocytes and plasma cells), CD68 (macrophages and monocytes) and CD83 (matured dendritic cells) antibodies. The tissues used in the pilot study were symptomatic refractory periapical granulomata (n=3), lingual tonsil (positive tissue control for CD38 and CD83), mucocele (positive tissue control for CD68) and periapical scar tissue (negative tissue control). In the main experiment, a convenience sample of eight symptomatic refractory periapical granulomata obtained by apical surgery was selected from the histopathology-archived records of the University of Otago, School of Dentistry, Medlab Dental Oral pathology Diagnostic Laboratory. Histological serial sections, 4μm thick, were prepared. The expression of TLR2, CD38, CD68 and CD83 in the lesions was first confirmed with immunoperoxidase (IP) followed by double immunoflourescence (DIF) staining of TLR2 (green fluorescence) with respect to each CD antibodies (red fluorescence). All stained slides were digitally photographed. Images were analysed with Adobe® Photoshop® CS2 software. A cell was identified to co-express two targeted protein markers (ie TLR2 and a CD marker) if the processed image showed both green and red fluorescence on the cell membrane and/or cytoplasm. Result: In the pilot study, the immunostaining of each antibody worked well in both immunoperoxidase and immunofluorescence techniques. In addition, mononuclear cells stained by the CD antibodies showed morphological characteristics that were unique to the targeted cells. On the other hand, TLR2+ mononuclear cells appeared to have several cellular morphologies that resembled lymphocytes, plasma cells and macrophages. In the main experiment, TLR2+ and CD38+ cells were identified and dominated the cell population in all samples. Five samples showed abundant CD68+ cells but only located at abscess areas. Six samples contained CD83+ cells but they were infrequently observed in the lesion. Double immunoflourescence staining showed TLR2 was consistently expressed on CD38+, CD68+ and CD83+ cells in the symptomatic refractory periapical granuloma. Conclusion: Lymphocytes, plasma cells, macrophages, monocytes and mature dendritic cells express TLR2 in the symptomatic refractory periapical granuloma with lymphocytes and plasma cells being the dominant inflammatory cells expressing TLR2. On the other hand TLR2+ mature dendritic cells were scantly identified and thus may play a minor role in antigen recognition in a well-developed granuloma. Finally, the results of this study are in agreement with current literature that periapical granulomata of endodontic origin are a bacterial induced disease process and that expression of TLR2 signifies the pathogenic role of the Gram-positive bacteria within the infected root canal system in refractory periapical granuloma