Technical Advance: Decreased helper T cells and increased natural killer cells in chronic periodontitis analyzed by a novel method for isolating resident lymphocytes

Abstract A novel oral mucosal gingival explant culture facilitates isolation of tissue resident mononuclear cells that represent tissue resident population, and are functional. During CP, the gingival environment is primed to recruit and activate homing lymphocytes. However, detailed phenotypic and functional characterization of gingival tissue resident lymphocytes has been challenging as a result of limitations associated with available isolation methods and limited availability of human samples. This study aimed to develop a novel explant culture technique for effectively isolating human gingival lymphocytes. This technique takes advantage of the natural tendency of MNCs to migrate toward a chemokine gradient generated by the gingival fibroblasts. The explant system allowed isolation of MNCs with ∼95× higher yield relative to conventional approaches. The MNC yield correlates directly with wet weights of the tissues, and maximal MNCs are isolated during the 2nd day of the culture. The explant culture method and conventional approach produce similar MNC subpopulations such as Th, Tc, and B cells. Chemokines associated with MNC migration but not cytokines associated with MNC proliferation and differentiation were produced in the explant culture. Moreover, MNC migration in response to the secreted chemokines was inhibited by PTX. T cells did not undergo proliferation during the culture. However, the isolated T cells responded to mitogenic stimulation ex vivo. A statistically, significantly decreased Th cell with reduced CD25 expression along with increased NK and NKT cells in CP are shown. However, the number of naïve NK cells was decreased significantly in MNCs, suggesting activation of NK cells in CP.</jats:p

TLR signaling that induces weak inflammatory response and SHIP1 enhances osteogenic functions

Toll-like receptor (TLR)-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 (P3C) and Escherichia coli lipopolysaccharide (EcLPS) were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism (Pg1690) or TLR4 antagonism (Pg1449) and mutant E. coli LPS (LPxE/LPxF/WSK) were used. IL-1β, SH2-containing inositol phosphatase-1 (SHIP1) that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane (3AC) was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis

Upregulation of Immunoregulatory Src Homology 2 Molecule Containing Inositol Phosphatase and Mononuclear Cell Hyporesponsiveness in Oral Mucosa during Chronic Periodontitis

Our group and others have shown in vitro that repeated exposure of human mononuclear cells (MNC) to lipopolysaccharide can induce endotoxin tolerance, evidenced by downregulation of TLR2 and TLR4 mRNA and surface protein; moreover, the ability of the MNC to secrete inflammatory cytokines is reduced. In situ studies performed on diseased and healthy gingiva suggest that a similar pattern of endotoxin tolerance occurs in human oral mucosa with chronic periodontitis (CP). We hypothesized that this represents a fundamental immunoregulatory mechanism to restore immune homeostasis and protect the host from further tissue damage. In the current study, we extend these published studies by providing evidence that Src homology 2 containing inositol phosphatase, an inhibitor of NF-κB activation and a negative regulator of the immune response, is upregulated in the oral mucosa during CP compared to its level during gingival health. We have also isolated MNC from patients with CP and those with healthy gingiva and show that MNC from CP subjects have a reduced capacity to upregulate TLR2, TLR4, and interleukin-1β in response to endotoxin. Thus, we provide more definitive evidence for a basic mechanism of immunoregulation in the oral mucosa

Antigen Capture of Porphyromonas gingivalis by Human Macrophages Is Enhanced but Killing and Antigen Presentation Are Reduced by Endotoxin Tolerance▿

The innate and the adaptive arms of the mucosal immune system must be coordinated to facilitate the control of pathogenic invasion while maintaining immune homeostasis. Toll-like receptors, able to activate the cell to produce bactericidal and inflammatory cytokines but also able to upregulate antigen (Ag)-presenting and costimulatory molecules, are particularly important in this regard. We have previously shown that the chronically infected oral mucosa is in a state of endotoxin tolerance, as evidenced by the downregulation of Toll-like receptors 2 and 4 and of inflammatory cytokines and the upregulation of SH2-containing inositol phosphatase, an inhibitor of NF-κB signaling. In the present study, we hypothesized that endotoxin tolerance would influence the ability of human macrophages to engage in Ag capture and killing of the oral pathogen Porphyromonas gingivalis and to upregulate costimulatory molecules and stimulate autologous T-cell proliferation. We show that uptake, but not killing, of P. gingivalis 381 is enhanced by endotoxin tolerance. Reduced killing is possibly due to a reduction of the intracellular lysosomes. We further show that the expression of the Ag-presenting molecule HLA-DR and costimulatory molecules CD40 and CD86 is dampened by endotoxin tolerance to the constitutive level. This, along with our previous evidence for reduction in immunostimulatory cytokines, is consistent with the observed decrease in the induction of autologous CD4+ T-cell proliferation by endotoxin-tolerized macrophages. Overall, these studies suggest that endotoxin tolerance, as observed in the inflamed oral mucosa, potentiates the innate Ag capture activity of macrophages but diminishes the potential of human macrophages to initiate the adaptive immune response. In conclusion, endotoxin tolerance, while helpful in bacterial clearance and in surmounting excessive inflammatory tissue damage, could potentially reduce the (protective) adaptive immune response during chronic infections such as periodontitis

Non-surgical therapy for the management of peri-implantitis: A systematic review

Peri-implantitis is characterized by mucosal inflammation and loss of supporting peri-implant bone. Objective: The objective of this systematic review was to evaluate the efficacy and safety of non-surgical treatment of peri-implantitis. Materials and methods: MEDLINE, Embase, and Web of Science were searched to identify randomized clinical trial studies that assessed non-surgical treatment of peri-implantitis with a minimum follow-up period of 3 months. Results: From a total of 29 abstracts, nine trials were included in this systematic review. Adjunctive local delivery of antibiotics, submucosal glycine powder air polishing, or Er:YAG laser treatment resulted in greater reduction in bleeding on probing compared with submucosal debridement using curettes with adjunctive irrigation with chlorhexidine. In addition, greater reductions in probing depths were found following adjunctive local delivery of antibiotics. The evidence neither supported nor refuted the clinical efficacy of submucosal debridement using curettes or ultrasonic scalers alone. No progressive bone loss was found following any of the assessed treatments over a maximum observation period of 12 months. Only two studies reported implant survival rates, which were 100% over 6 months. Conclusions: The available evidence suggested that submucosal debridement with adjunctive local delivery of antibiotics, submucosal glycine powder air polishing, or Er:YAG laser treatment may reduce clinical signs of peri-implant mucosal inflammation to a greater extent relative to submucosal debridement using curettes with adjunctive irrigation with chlorhexidine. Long-term randomized controlled trials are needed to assess the efficacy of non-surgical therapy on progressing bone loss, implant survival rates, and measures of oral health-related quality of life. © 2012 John Wiley & Sons A/S.link_to_subscribed_fulltex