Cyclic dinucleotides, cyclic di-adenosine monophosphate (c-di-AMP) and cyclic di-guanosine monophosphate (c-di-GMP) are bacterial signaling molecules essential for regulating various bacterial cellular processes. Host cells can recognize cytosolic nucleic acids and cyclic dinucleotides through a stimulator of interferon genes (STING). Such recognition can stimulate the immune response by activating the production of interferons and other pro-inflammatory cytokines. Nevertheless, the impact of bacterial cyclic dinucleotides on the cellular response of gingival cells remains unknown.

The objectives of this PhD study series were to 1) investigate the regulatory roles of bacterial cyclic dinucleotides and Porphyromonas gingivalis (Pg) lipopolysaccharides (LPS) on the cellular response of human gingival keratinocytes (HMK) and human gingival fibroblasts (HGFs), 2) evaluate cyclic dinucleotide-mediated activation of STING/TANK-binding kinase1 (TBK1)/interferon regulatory factor 3 (IRF3) pathway in HMK cells, and 3) examine the effects of cyclic dinucleotides solely or combined with Pg LPS on gingival fibroblasts' proteome response by using global proteomics analysis.

In the experiments, HMK and HGF cells were stimulated with (100, 10, 1 μM) of c-di-AMP and c-di-GMP. Cytokine expressions of HMK cells and cytokine and matrix metalloproteinase expressions of HGF cells were evaluated by the Luminex technique. Expressions of mitogen-activated protein kinases and STING/TBK1/IRF3 pathway activation in HMK cells and HGFs were measured with western blot. The gingival fibroblasts' proteome response against bacterial cyclic dinucleotides was studied using proteomics analysis.

Our results showed that cyclic dinucleotides can either enhance or inhibit LPS-regulated HMK cytokine response, and this variation depends on the type of cytokine. Moreover, STING/TBK1/IRF3 pathway operates within HMK cells and can be regulated by bacterial cyclic dinucleotides. In HGFs, cyclic dinucleotides interacted with Pg LPS to control the early cellular response, while Pg LPS predominantly influenced the late cellular response. Bacterial cyclic dinucleotides solely or with Pg LPS upregulated innate immunity-related and interferon signaling-related proteins in HGFs, in addition to controlling various other critical processes.

The results of this PhD project suggest that bacterial cyclic dinucleotides, which are previously unstudied components of periodontal bacteria, can regulate initial cellular response of gingival cells through STING/TBK1/IRF3 pathway. A thorough description of cyclic dinucleotide-regulated cellular responses in periodontium may allow us to design cyclic dinucleotide analogues that can function as STING-agonists, antagonists, and potential therapeutics