Characterisation of innate T cells in response to oral bacterial infection
AuthorFong, Shao Bing
AffiliationMelbourne Dental School
Document TypePhD thesis
Access StatusOpen Access
© 2015 Dr. Shao Bing Fong
Chronic periodontitis is an inflammatory disease of the supporting tissues of teeth that is characterised by bone resorption and if left untreated can result in eventual tooth loss. The subgingival plaque bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have been closely associated with chronic periodontitis. T cell immunity during periodontal infections has been well-documented, mainly involving the adaptive immunity which includes Th1 and Th2 responses. A small number of clinical studies have reported the presence of IL-17 and Th17-related cytokines in the gingival crevicular fluid and diseased tissues of periodontitis patients. However, the roles of innate T cells and Th17-related responses in disease progression is unclear. This study shows that NKT cells play a role in pro-inflammatory cytokine production, contributing to P. gingivalis-induced bone loss in the mouse periodontitis model. Inflammation and bone resorption was reduced in the absence of NKT cells or CD1d, the corresponding activating receptor for the TCR of NKT cells. As NKT cell responses were likely to be associated with glycolipid antigens, a major glycolipid of P. gingivalis was isolated, Pg-GL1, which was found to induce NKT cell and B cell activation. Results from knock-out mice studies suggested that NKT activation by Pg-GL1 occurs through a mixed requirement for TCR/CD1d-engagement and additional cytokines. Furthermore, Pg-GL1 was shown to induce the secretion of IL-1β, IL-6, IL-12(p40), IL-17, and G-CSF, cytokines that have been associated with inducing Th17-responses and, as contributing factors during periodontal inflammation and bone resorption. Similar to P. gingivalis, three major glycolipids (termed Tf-GL1, Tf-GL2, Tf-GL3) of T. forsythia were found to highly activate NKT cells and B cells. Alkaline treatment of T. forsythia glycolipids revealed that Tf-GL2 was alkali-resistant, while Tf-GL1 and Tf-GL3 were susceptible and their deacylated forms showed the presence of complex carbohydrates. Mass spectrometry analysis revealed a wide structural variation in the carbohydrate head-group and acyl chain lengths. Base-labile Tf-GL1 and Tf-GL3 were identified to be diacylgylcerolipids while base-resistant Tf-GL2 was proposed to be a glycosphingolipid. The structure of the third glycolipid, Tf-GL3, was predicted to be highly complex and thus may have contributed to its lower antigenicity compared with Tf-GL1 and Tf-GL2. When the stimulatory ability of T. forsythia glycolipids was further investigated, only Tf-GL2 was able to induce IL-17 production when cultured with purified NKT cells and BMDCs. As only Tf-GL2 was able to induce cytokine production, it was concluded that structural characteristics play an important role in antigen potency. When mice were orally infected with P. gingivalis, the CD27- γδ T cell sub-population in the maxillary epithelium was found to be activated after 14 days post-infection. Using pHrodo™ technology, it was shown that γδ T cells were able to phagocytose P. gingivalis, and this was confirmed using super resolution imaging. γδ T cells were then classified based on their ability to phagocytose bacteria and their expression of CD27, namely, “pHrodohi CD27-”, “pHrodomed/hi CD27+” and “pHrodolo CD27+” γδ T cell sub-populations. Naïve γδ T cells from thymus and spleen expressed a number of migratory markers and antigen presentation molecules, and the expression levels of these markers between CD27- and CD27+ γδ T cells were not significantly different. However, when γδ T cells were exposed to bacteria either through priming or in culture, the sub-populations of γδ T cells displayed differences in surface marker expression and cytokine secretion. Amongst the sub-populations, it was found that splenic γδ T cells that present a strong ability to phagocytose bacteria (pHrodohi or pHrodomed/hi) expressed high concentrations of cytokines and chemokines, which was most evidently observed when primed with heat-killed P. gingivalis. As the pHrodolo CD27+ γδ T cell sub-population expressed a high level of MHC molecules and were slower in phagocytosis, it was hypothesised that this sub-population is involved in antigen presentation, similar to dendritic cells. The results from this study provide a preliminary understanding of the functional roles of NKT and γδ T cells in response to oral bacteria and their bacterial products. As these innate T cells contribute to the induction of an inflammatory or non-inflammatory response, they are crucial in the determination of early responses during infection. As such, the expansion of this investigation may aid in the development of therapeutics to control inflammation and prevent the progression of chronic periodontitis.
Keywordsimmunology; microbiology; innate immunity; NKT cells; gamma-delta T cells; periodontitis; glycolipids; porphyromonas gingivalis; Tannerella forsythia; oral bacteria; cytokines
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