Dissertation committee:
1. Γκαγκάρη Ελένη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
2. Θεοχαρίδης Θεοχάρης, Καθηγητής TUFTS, USA
3. Αντωνίου Χριστίνα, Ομότιμη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
4. Στρατηγός Αλέξανδρος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
5. Νικολαίδου Ηλέκτρα, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
6. Γρηγορίου Σταμάτης, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
7. Βούρος Ιωάννης, Καθηγητής, Οδοντιατρική Σχολή, ΑΠΘ
Summary:
Background: Periodontal diseases are a group of common chronic infectious diseases associated with pathogenic microorganisms forming the dental biofilm, affecting the supporting structures of teeth, and leading to their progressive destruction and tooth loss. Periodontitis is the 6th most prevalent disease in the world and the primary cause for tooth loss in adults. Recently, the role of the immune system in the progression of periodontitis was highlighted, indicating that bacterial antigens can trigger an immunopathologic reaction and that the host response is an important factor in determining the extent and severity of the disease. Periodontal tissue breakdown is a result of the complex interplay between the pathogenic bacteria forming the biofilm and the host's immune responses. Porphyromonas gingivalis (P. gingivalis) is the species most highly associated with the chronic form of periodontitis and can be detected in up to 85% of the diseased sites. The major virulence factors produced by P. gingivalis are its lipopolysaccharides (LPS). LPS is an outer membrane component recognized by pattern recognition receptors on immune cells and specifically by the toll like receptors (TLR). TLR4 have been identified and characterized as the main pathogen sensor against LPS of most Gram-negative bacteria. During the past decade, the importance of mast cells (MCs) in the defense mechanism against bacterial infections has been increasingly recognized. MCs are multifunctional, immune system secretory cells, well known for their involvement in a wide variety of physiological and pathological processes, including mainly allergic and anaphylactic reactions, through the release of inflammatory mediators. Increasing evidence indicates that MCs are also critical for the pathogenesis of inflammatory diseases such as arthritis, atopic dermatitis, psoriasis, multiple sclerosis, and oral inflammation including periodontal diseases. The increase of MCs numbers, in vivo, in periodontally affected tissues, has drawn attention with respect to the possible participation of MCs in the defense mechanism and destructive events in periodontal disease.
Objectives: This in vitro study was designed to a) examine the effects of P. gingivalis LPS on the expression and release of inflammatory mediators from the LAD2 human MC line b) to compare the differential effects of P. gingivalis and E. coli LPS on the expression and release of these mediators from human MCs and c) to assess the functional role of TLR2 and TLR4 in mediator release from MCs stimulated with either LPS. . The mediators selected were the tumor necrosis factor (TNF-α), the vascular endothelial growth factor (VEGF) and the monocyte chemoattractant protein (MCP-1) as they have been documented to be significantly implicated in the initiation and progression of periodontal disease,
Materials and Methods: LAD2 MCs (kindly supplied by Dr. Kirshenbaum, National Institute of Health, Bethesda, Maryland) derived from a human MC leukemic patient were cultured in StemPro-34 medium supplemented with 100 U/mL of penicillin–streptomycin and 100 ng/mL of Recombinant human stem cell factor (rhSCF). All cells were used during their logarithmic growth period. Substance P (SP) was diluted in Milli-Q water, and a stock solution (10mM) was prepared. Commercially available preparation of P. gingivalis LPS (Invivogen, CA) and E. coli 0111: B4 LPS (Sigma-Aldrich) was used. Bacterial LPS was purified by the supplier to be free from contaminating lipoproteins. SP and LPS were dissolved in sterile distilled water. ELISA kits for TNF-α, VEGF and MCP-1 were obtained from R&D Systems. Purified monoclonal antibodies to human TLR4 and TLR2 (100μg Mab-hTRL4, Mab-hTRL2) were obtained from Invivogen. TNF-α, VEGF, and MCP-1 Taqman probes and Taqman Master Mix were purchased from Applied Biosystems. Beta-hexosaminidase (β-hex) release was assayed as an index of MC degranulation. LAD2 cells (0.5 × 105) were stimulated with LPS (1 μg/ml) and SP (2 μmol/L) for 30 min. SP was used as the positive control and medium alone as the negative control. Results were expressed as the percentage of β-hex released over the total amount present in LAD2 cells. In order to examine the mediator expression on mRNA level, LAD2 cells were stimulated with SP (2 μM), P. gingivalis and E. coli LPS (1 ng/mL) for 6 h. Total mRNA was extracted with a RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Relative mRNA abundance was determined from standard curves run within each experiment. An iScript cDNA synthesis kit (Bio-Rad Laboratories) was used for reverse transcription of each sample. Quantitative real-time PCR was performed with TaqMan gene expression assays (Applied Biosystem) for TNF (Hs99999043_m1), MCP1 (Hs00234140_m1) and VEGF (Hs00900055_m1). Samples were run at 45 cycles by using a real-time PCR system (7300, Applied Biosystems). The mRNA gene expression was normalized to human GAPDH endogenous control (Applied Biosystems). Τo measure de novo–synthesized cytokine/chemokine release, LAD2 cells (1 × 105) were stimulated for 24 h, with a minimum and a maximum concentration of LPS (1 ng/ml and 1 μg/ml respectively). The cells were also stimulated with either SP (2 μM) serving as the positive control or media alone serving as the negative control for 24h. The supernatant fluids were collected by centrifugation (5 min, 150 x g), stored at –20° and assayed for TNF-α, VEGF and MCP-1 release using enzyme-linked immunosorbent assay (ELISA) kits according to the protocol suggested by the manufacturer (R&D Systems). To assess the functional role of TLR2 and TLR4, MCs were incubated with either anti-TLR2 or anti-TLR4 polyclonal antibody (2 μg/ml) for 1 h before stimulation with P. gingivalis or E. coli LPS (1 μg/ml). Supernatants fluids were collected and assayed for mediators by Elisa after 24 h. The positive controls were MCs incubated with LPS, but without anti-TLRs, whereas the negative controls were unstimulated MCs. All experiments were performed in triplicates and repeated at least 3 times (n=3). The results are presented as means ± Standard Deviation. Data between different treatment groups were analyzed by using the unpaired 2-tailed Student’s t test (GraphPad Prism 6). Mean values of the parameters were tested by means of the least significant difference test at significance level p < 0.05.
Results: Both LPSs used did not induce MCs degranulation in contrast to SP (P<0.05). The stimulation of LAD2 cells with P. gingivalis LPS resulted in a small, but statistically significant increase, in gene expression for all the three mediators (p<0.05). The same results were observed for E. coli LPS regarding the TNF-α and VEGF gene expression (p<0.05). On the contrary, for MCP-1 mRNA expression, even if there was a small increase, it was not statistically significant from the control group (p>0.05). Regarding the two different LPSs used, no statistically significant difference in gene expression was observed. The stimulation of LAD2 cells with SP induced the most potent increase in gene expression for all mediators that was statistically significant from both the Control and the LPSs groups. Regarding the release on a protein level, both LPS concentrations tested (1 ng/ml, 1 μg/ml) led to de novo release of all the three mediators studied, compared to unstimulated cells (p<0.05). P. gingivalis LPS and E. coli LPS, at the same concentration, triggered approximately, the same amount of de novo release of TNF-α, VEGF and MCP-1, as there was no statistically significant difference (p>0.05). However, there was a tendency of higher levels of mediator release for P. gingivalis LPS in both tested concentrations compared to E. coli LPS. A dose response relationship could not be documented for both LPSs in our study. The release of TNF-α, VEGF and MCP-1 reached the highest concentrations, which were statistically significant in relation to the control and the LPSs Groups (p<0.001) when stimulated with SP (p<0.05). The levels of the three mediators studied, were reduced after pre-treatment with anti-TLR4 and anti-TLR2 antibodies in both LPSs Groups. TNF-α levels were significantly reduced in the P. gingivalis Group, after incubation with anti-TLR2 antibody whereas in the E. coli Group, the reduction was more prominent after incubation with the anti-TLR4 antibody. Both anti-TLR4 and anti-TLR2 antibodies significantly reduced VEGF levels after stimulation with either P. gingivalis or E. coli LPS (p<0.05). Interestingly, pre-incubation of LAD2 cells with either antiTLR4 or anti-TLR2 antibodies did not statistically significantly reduce the levels of MCP-1 in either of the LPSs Groups (p>0.05).
Conclusions: The increase of MCs in periodontally affected tissues, has called attention with respect to the possible participation of MCs in the defense mechanism and destructive events in periodontal inflammation. Overall, we reported for the first time, that LPS from P. gingivalis selectively, i.e. without degranulation, stimulated MCs to generate and release mediators that have been documented to play an important role in the initiation and progression of periodontal disease, with the same potency as E. coli LPS, TNF-α, VEGF and MCP-1. In addition, we documented that P. gingivalis LPS could function through both TLR2 and TLR4 in MCs. It is undisputable that MCs play a crucial role in the development of inflammation during many pathological processes including allergic reactions as well as during bacterial infection. Therefore, our findings could indicate that MCs might be involved in the emergence of inflammatory processes evolved in response to P. gingivalis infection such as periodontal disease. In addition, the recent identification of a neurogenic component in periodontal disease and the existence of spatial interactions between nerves and MCs in a neural-immune network with the significant effect of SP on MCs, that was documented in our study as well, opens new possibilities for altering the function of these critical immune cells. In the future, it may be possible to develop novel approaches that influence the release of inflammatory molecules and neuropeptides to ameliorate MCs driven inflammation including periodontal disease.
