Characterisation of Regulatory T-Cells and Th17 Cells in Chronic Inflammatory Periodontal Disease

Periodontal diseases (gingivitis/periodontitis) are common chronic inflammatory diseases that are initiated by specific bacterial complexes present in dental plaque. In health/gingivitis, there is a state of balance between the antigenic challenge in the dental plaque and the host response. However in chronic periodontitis, plaque bacteria induce an immune response, which leads to destruction of supporting structures and ultimately loss of the tooth itself. The role of T-cells in mediating the immune response in gingivitis (stable lesion) and periodontitis (progressive lesion) is well recognized. Analysis of the cytokine profile of T-cells extracted from periodontal disease tissues demonstrated that Th1 cytokines predominate in stable lesions compared with Th2 cytokines in progressive lesions. The balance between Th1 and Th2 cells has been shown to be fundamental in the immunoregulatory control of periodontal disease. Further, regulatory T-cells (Tregs) with suppressive functions have been shown to play a crucial role in the regulatory networks that control this balance. These cells express a specific transcription factor, forkhead boxp3 (FoxP3), which acts as a master regulator in their development and function and provides a means for their identification. At the same time IL17 producing CD4+ T-cells (Th17 cells) have also been suggested to play a role in the development of inflammation such that the inter-relationship between Tregs and IL17+ T-cells seems to be important in the control of chronic inflammation. Hence I hypothesize that both Tregs and IL17+ Th17 cells may have a role in the pathogenesis of periodontal disease in a positive and negative fashion. In order to test the above hypothesis the aims of the present study were; firstly, ‘to determine the presence of FoxP3+ Tregs and IL17+ Th17 cells’, secondly, ‘to identify the cell types expressing FoxP3 and IL17’, and finally, ‘to determine their functional activity in terms of gene expression and protein expression’, in diseased periodontal tissues. Chronic inflammatory periodontal disease tissues were retrieved from 29 archival FFPE specimens. Immunohistochemistry was used to identify T-cells (CD3+), B-cells (CD19+), Tregs (FoxP3+) and IL17+ cells. Plasma cells were identified based on their morphology characteristics in H&E sections. NIH Image J software was used to quantify the percentage of immunohistochemically positive cells and plasma cells per 0.12mm2 of a specimen. Initially, archival FFPE specimens were grouped based on the intensity (minimally and intensely inflamed) and nature of inflammatory infiltrate (T-cell predominant/B and plasma cell predominant). The proportion of FoxP3+ and IL17+ cells were statistically compared between these groups. The results showed more FoxP3+ cells in the B and plasma cell predominant and intensely inflamed gingival specimens when compared with T-cell predominant and minimally inflamed gingival specimens respectively. However the difference was only statistically significant at the 10% level. On the other hand very small numbers of large ovoid/plasmacytoid shaped IL17+ cells was identified. The comparison of the proportion of IL17+ cells between the groups didn’t show statistical significance. Interestingly, the numbers of FoxP3+ cells were positively correlated with the B&P/T cell ratio only within the B and plasma cell predominant gingival specimens. Fresh tissue was obtained from a further 20 patients, 10 with healthy/gingivitis and 10 with chronic periodontitis. Each gingival specimen was cut into two parts; one half was used for grouping the specimens and also for identifying the cell types expressing FoxP3 and IL17. The other half was used for the gene and protein expression experiments. Gingival specimens were grouped according to clinical expression (healthy/gingivitis and periodontitis) and the nature of inflammatory infiltrate. There was no significant difference between the groups in terms of either FoxP3+ or IL17+ cells. However there were significantly more FoxP3+ cells compared with IL17+ cells in all the specimens. Overall there were very few IL17+ cells. Double immunofluoresence revealed that all FoxP3+ cells were also CD4+, while IL17+ cells were neither CD4+ nor CD8+ suggesting that they are not T-cells and therefore not Th17 cells. Further identification of the IL17+ cells showed that they were both toluidine blue granule positive and tryptase positive suggestive of mast cells. Quantitative reverse transcriptase PCR (qRT-PCR) was used in examining the Treg and Th17 cell related gene expression profiles. The RNA was extracted and purified (using TRIzol reagent), then reverse transcribed to cDNA. For gene expression profiling a 96-well human Th17 for autoimmunity and inflammation PCR array was used with SYBR/Rox detection system. The results showed a significant up-regulation of Treg cell related genes; STAT5A, TGFβ1, and IL10, and Th17 cell related genes; STAT3, SOCS1, and TGFβ1 in the B and plasma cell predominant tissue specimens when compared with T-cell predominant tissue specimens. Interestingly, IL17 gene was not detected in any of the gingival tissue specimens investigated. The phenol-chloroform phase, which was left at the end of RNA extraction, was investigated in protein expression experiments. ELISA was used to evaluate the concentration of TGFβ1, IL10, and IL17 cytokines. The results showed higher levels of the TGFβ1 and IL10 in periodontitis and in B and plasma cell predominant gingival tissue specimens than in healthy/gingivitis and T-cell predominant gingival tissue specimens respectively. However, the difference between the groups was statistically significant only for TGFβ1 but not IL10. In accordance with the IL17 gene expression results, IL17 protein was undetected (˂ detection limit of the assay) in any of the gingival tissue specimens examined. With the current state of knowledge, the precise role of Tregs and IL17 in the pathogenesis of periodontal disease is uncertain. However, based on the findings from this study, I suggest that Tregs may have a more prominent role than IL17 in the pathogenesis of periodontal disease. Accordingly, the results of this study have also shown that Th17 cells do not exist in periodontal disease and that the source of the relatively small amounts of IL17 may in fact be mast cells