Immunomodulatory Effects of N-Acetyl Cysteine Treated SCAP.

INTRODUCTION: Stem cells of the apical papilla (SCAP) play an important role in regenerative endodontic procedures (REPs). Previous studies have shown that during REPs, bacteria can activate the innate immune system and cause indirect stem cell toxicity, leading to the lysis of SCAP. N-acetylcysteine (NAC)-treated cells are resistant to apoptosis and have increased differentiation capabilities. The immunomodulatory properties of NAC-treated SCAP are still unknown. Hence, the aim of this study is to evaluate the interactions of SCAP pretreated with and without NAC with the immune system. METHODS: Flow cytometric analysis was performed to assess the effects of NAC on SCAP viability. Human SCAP were then cultured and were either pretreated with NAC or non-treated and co-cultured with human peripheral blood mononuclear cells. A lactate dehydrogenase assay was performed to evaluate the levels of immune cell mediated apoptosis, followed by an enzyme-linked immunosorbent assay (ELISA) to measure levels of proinflammatory cytokines for these co-cultures. Data were analyzed using analysis of variance with post hoc Tukey test. RESULTS: Cells treated with NAC had similar levels of viability as the controls. SCAP pretreated with NAC had significantly lower immune cell-mediated cytotoxicity to nonactivated and activated peripheral blood mononuclear cells. The ELISA results showed that SCAP pretreated with NAC induced lower levels of proinflammatory cytokines. CONCLUSIONS: SCAP pretreated with NAC have a higher chance of surviving the activated immune system. This information may provide a better insight into the properties of these stem cells and may be the key to making REPs more predictable.

Characterization of the Cellular Responses of Dental Mesenchymal Stem Cells to the Immune System.

INTRODUCTION: Dental stem cells have gained importance recently and are being used for various purposes in regenerative medicine and dentistry. Although much research has been done to show the various properties of these dental stem cells, the immunomodulatory properties of some of these stem cells are still unknown. This is important considering these cells are being used routinely. Therefore, the aim of this study was to investigate the interactions between the activated immune cells and 3 types of dental-derived mesenchymal stem cells: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells of the apical papilla (SCAP). METHODS: SCAP, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and periodontal ligament fibroblasts were cultured, and various assays were performed including a proliferation assay, flow cytometric analysis, lactate dehydrogenase and chromium-51 cytotoxicity assays, and an enzyme-linked immunosorbent assay to evaluate the interactions of these dental stem cells when cocultured with either peripheral blood mononuclear cells or natural killer cells. RESULTS: SCAP were less resistant to immune cell-mediated cytotoxicity as seen from the results obtained from the LDH and chromium-51 cytotoxicity assays. The flow cytometric analysis showed a lower resilience of SCAP to cytotoxic compounds. The enzyme-linked immunosorbent assay results demonstrated that the SCAP induced high levels of proinflammatory cytokine secretion compared with the other dental stem cells. CONCLUSIONS: SCAP did not perform as well as the other dental stem cells. This could in turn affect their survival and differentiation abilities as well as their functionality. This may be an important aspect to consider when selecting dental stem cells for various regenerative procedures.

Genotype-specific regulation of oral innate immunity by T2R38 taste receptor.

The bitter taste receptor T2R38 has been shown to regulate mucosal innate immune responses in the upper airway epithelium. Furthermore, SNPs in T2R38 influence the sensitivity to 6-n-propylthiouracil (PROP) and are associated with caries risk/protection. However, no study has been reported on the role of T2R38 in the innate immune responses to oral bacteria. We hypothesize that T2R38 regulates oral innate immunity and that this regulation is genotype-specific. Primary gingival epithelial cells carrying three common genotypes, PAV/PAV (PROP super-taster), AVI/PAV (intermediate) and AVI/AVI (non-taster) were stimulated with cariogenic bacteria Streptococcus mutans, periodontal pathogen Porphyromonas gingivalis or non-pathogen Fusobacterium nucleatum. QRT-PCR analyzed T2R38 mRNA, and T2R38-specific siRNA and ELISA were utilized to evaluate induction of hBD-2 (antimicrobial peptide), IL-1α and IL-8 in various donor-lines. Experiments were set up in duplicate and repeated three times. T2R38 mRNA induction in response to S. mutans was highest in PAV/PAV (4.3-fold above the unstimulated controls; p<0.05), while lowest in AVI/AVI (1.2-fold). In PAV/PAV, hBD-2 secretion in response to S. mutans was decreased by 77% when T2R38 was silenced. IL-1α secretion was higher in PAV/PAV compared to AVI/PAV or AVI/AVI with S. mutans stimulation, but it was reduced by half when T2R38 was silenced (p<0.05). In response to P. gingivalis, AVI/AVI showed 4.4-fold increase (p<0.05) in T2R38 expression, whereas the levels in PAV/PAV and AVI/PAV remained close to that of the controls. Secretion levels of IL-1α and IL-8 decreased in AVI/AVI in response to P. gingivalis when T2R38 was silenced (p<0.05), while the changes were not significant in PAV/PAV. Our data suggest that the regulation of gingival innate immunity by T2R38 is genotype-dependent and that the ability to induce a high level of hBD-2 by PAV/PAV carriers may be a reason for protection against caries in this group.

Titanates and Titanate-Metal Compounds in Biological Contexts.

Metal ions are notorious environmental contaminants, some causing toxicity at exquisitely low (ppm-level) concentrations. Yet, the redox properties of metal ions make them attractive candidates for bio-therapeutics. Titanates are insoluble particulate compounds of titanium and oxygen with crystalline surfaces that bind metal ions; these compounds offer a means to scavenge metal ions in environmental contexts or deliver them in therapeutic contexts while limiting systemic exposure and toxicity. In either application, the toxicological properties of titanates are crucial. To date, the accurate measurement of the in vitro toxicity of titanates has been complicated by their particulate nature, which interferes with many assays that are optical density (OD)-dependent, and at present, little to no in vivo titanate toxicity data exist. Compatibility data garnered thus far for native titanates in vitro are inconsistent and lacking in mechanistic understanding. These data suggest that native titanates have little toxicity toward several oral and skin bacteria species, but do suppress mammalian cell metabolism in a cells-pecific manner. Titanate compounds bind several types of metal ions, including some common environmental toxins, and enhance delivery to bacteria or cells. Substantial work remains to address the practical applicability of titanates. Nevertheless, titanates have promise to serve as novel vehicles for metal-based therapeutics or as a new class of metal scavengers for environmental applications.

DNA methylation differentially regulates cytokine secretion in gingival epithelia in response to bacterial challenges.

Epigenetic modifications are changes in gene expression without altering DNA sequence. We previously reported that bacteria-specific innate immune responses are regulated by epigenetic modifications. Our hypothesis is that DNA methylation affects gingival cytokine secretion in response to bacterial stimulation. Gingival epithelial cells (GECs) were treated with DNMT-1 inhibitors prior to Porphyromonas gingivalis (Pg) or Fusobacterium nucleatum (Fn) exposure. Protein secretion was assessed using ELISA. Gene expression was quantified using qRT-PCR. The ability of bacteria to invade inhibitor pretreated GECs was assessed utilizing flow cytometry. Changes were compared to unstimulated GECs. GEC upregulation of IL-6 and CXCL1 by Pg or Fn stimulation was significantly diminished by inhibitor pretreatment. Pg stimulated IL-1α secretion and inhibitor pretreatment significantly enhanced this upregulation, while Fn alone or with inhibitor pretreatment had no effect on IL-1α expression. GEC upregulation of human beta-definsin-2 in response to Pg and Fn exposure was enhanced following the inhibitor pretreatment. GEC susceptibility to bacterial invasion was unaltered. These results suggest that DNA methylation differentially affects gingival cytokine secretion in response to Pg or Fn. Our data provide basis for better understanding of how epigenetic modifications, brought on by exposure to oral bacteria, will subsequently affect host susceptibility to oral diseases.

Ellagic acid modulates the expression of oral innate immune mediators: potential role in mucosal protection.

BACKGROUND: Ellagic acid (EA) found in various fruits such as pomegranates, blackberries, raspberries, strawberries, and walnuts has different pharmacological functions including antioxidant, antitumor, antiallergic, anti-inflammatory, antibacterial, and antiviral activities. It is not known, however, if EA could enhance mucosal innate immunity. Our goal was to determine the effects of EA on the expression of innate immune mediators produced by oral epithelial cells. METHODS: Culture of primary human gingival epithelial cells (HGEs) was performed in duplicate, and after the primary HGEs had been treated with EA at a concentration ranging from 12.5 to 100 µM for 18 h the cells and supernatants were harvested. The expression of innate immune mediators including human ß-defensin 2 (hBD2), secretory leukocyte protease inhibitor (SLPI), and various cytokines and chemokines was measured at both transcriptional and translational levels by using quantitative real-time PCR, ELISA, and Luminex assay. RESULTS: In the presence of EA, the expression of hBD2-and SLPI mRNA was 3.7-folds and 2.6-folds greater than untreated controls, respectively, and consistent with their secreted protein levels. For cytokines and chemokines, increased expression of RANTES, IL-2, and IL-1ß was found in response to EA. In contrast, EA decreased the expression of IL-6, IL-8, and TNF-α. CONCLUSIONS: This study demonstrated that oral innate immunity is affected by EA found in fruits. Thus, it may play some roles in mucosal innate immunity. The potential of EA for modulating the innate immune mediators may lead to developing a new topical agent to treat and/or prevent immune-mediated oral diseases.