Resistance of MMP9 and TIMP1 to endotoxin tolerance.

Inflammatory cytokines activate tissue collagenases such as matrix metalloproteinases (MMPs). MMPs are antagonized by tissue inhibitors of metalloproteinases (TIMPs) that attempt to regulate excessive collagenase activity during inflammatory conditions. During chronic inflammatory conditions, induction of endotoxin tolerance negatively regulates the cytokine response in an attempt to curtail excessive host tissue damage. However, little is known about how downregulation of inflammatory cytokines during endotoxin tolerance regulates MMP activities. In this study, human monocyte-derived macrophages were either sensitized or further challenged to induce tolerance with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) or Escherichia coli (EcLPS). Inflammatory cytokines, such as TNF-α and IL-1ß, and levels of MMP9 and TIMP1 were analyzed by a combination of cytometric bead array, western blot/gelatin zymography and real-time RT-PCR. Functional blocking with anti-TLR4 but not with anti-TLR2 significantly downregulated TNF-α and IL-1ß. However, MMP9 levels were not inhibited by toll-like receptor (TLR) blocking. Interestingly, endotoxin tolerance significantly upregulated TIMP1 relative to MMP9 and downmodulated MMP9 secretion and its enzymatic activity. These results suggest that regulatory mechanisms such as induction of endotoxin tolerance could inhibit MMP activities and could facilitate restoring host tissue homeostasis.

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-1ß, 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-1ß but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1ß 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.

Doxycycline counteracts bone morphogenic protein 2-induced osteogenic mediators.

BACKGROUND: Microbial colonization during wound healing may exaggerate the inflammatory response and could adversely affect the outcome of periodontal regeneration. Bone morphogenic proteins (BMPs) directly augment bone regeneration. Interestingly, inhibitors of tissue collagenases, such as sub-antimicrobial-dose doxycycline, also indirectly promote hard-tissue regeneration. In this study, it is hypothesized that BMP2-mediated bone regeneration would be positively affected by simultaneous treatment of sub-antimicrobial-dose doxycycline. METHODS: Human periodontal ligament (PDL) cells were stimulated with: 1) 10 ng/mL BMP2; 2) 1 µg/mL doxycycline; or 3) a combination of the two. The expressions of alkaline phosphatase, osteocalcin, osteonectin, and osteopontin were analyzed along with in vitro mineralized nodule formation and calcium accumulation. RESULTS: BMP2 was a potent inducer of osteocalcin/osteopontin (statistically significant at P <0.01) and osteonectin in PDL cells relative to stimulation with doxycycline. However, doxycycline relative to BMP2 (statistically significant at P <0.001) upregulated the expression of alkaline phosphatase and in vitro mineralized nodule formation. Contrary to expected results, combined BMP2 and doxycycline induced a statistically significant (P <0.001) downregulation of alkaline phosphatase, osteocalcin, osteonectin/osteopontin, and in vitro mineralized nodule formation compared to stimulation with either BMP2 or doxycycline alone. CONCLUSIONS: Combined treatment of BMP2 and doxycycline in PDL cells counteracts the osteogenic mediators. Molecular interaction of growth factors should be explored before using a combination of these biologic molecules. It is important and clinically relevant to determine whether tetracycline and its other derivatives also counteract BMP functions. Animal models should be used to confirm these in vitro results.

Bone morphogenic protein-2 induces apoptosis and cytotoxicity in periodontal ligament cells.

BACKGROUND: Periodontal ligament (PDL) expresses endogenous growth factors, such as bone morphogenic proteins (BMPs), which facilitate maintenance of tissue homeostasis. Inflammatory conditions, such as chronic periodontitis, could disrupt this homeostasis, and physiologic levels of growth factors may be insufficient to maintain tissue homeostasis. BMPs facilitate periodontal bone regeneration but also are implicated in causing tooth ankylosis and root resorption. The underlying mechanism of tooth ankylosis is unclear. However, there is evidence that BMPs induce apoptosis in progenitor cells. Little is known about BMP-induced cytotoxicity in PDL cells, which contain a population of progenitor cells. The aim of this study is to determine BMP2-induced osteogenic mediators and cytotoxic effects in PDL cells and compare these cells to osteoblasts. METHODS: Human PDL cells and primary osteoblasts were stimulated with doses of 1 to 200 ng/mL BMP2. Expression of alkaline phosphatase (ALP), in vitro mineralization along with osteonectin expression, induction of apoptosis, and cytotoxicity assays were performed. RESULTS: PDL cells and osteoblasts upregulated ALP and in vitro mineralization in a dose-dependent manner with BMP2 stimulation. However, at BMP2 concentrations >10 ng/mL, ALP, in vitro mineralization, and osteonectin were downregulated in PDL cells. Relative to osteoblasts, PDL cells were susceptible to apoptosis and cytotoxicity with 10 times lower concentration of BMP2. CONCLUSIONS: Relative to osteoblasts, PDL cells are susceptible to BMP2-induced cytotoxicity. BMP-induced tooth ankylosis is controversial and is poorly understood. Disruption of PDL homeostasis by BMP-induced apoptosis could play a role in tooth ankylosis.

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

UNLABELLED: 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.

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

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.

Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells.

Squamous cell carcinomas (SCCs) of the skin are sun-induced skin cancers that are particularly numerous in patients on T cell immunosuppression. We found that blood vessels in SCCs did not express E-selectin, and tumors contained few cutaneous lymphocyte antigen (CLA)(+) T cells, the cell type thought to provide cutaneous immunosurveillance. Tumors treated with the Toll-like receptor (TLR)7 agonist imiquimod before excision showed induction of E-selectin on tumor vessels, recruitment of CLA(+) CD8(+) T cells, and histological evidence of tumor regression. SCCs treated in vitro with imiquimod also expressed vascular E-selectin. Approximately 50% of the T cells infiltrating untreated SCCs were FOXP3(+) regulatory T (T reg) cells. Imiquimod-treated tumors contained a decreased percentage of T reg cells, and these cells produced less FOXP3, interleukin (IL)-10, and transforming growth factor (TGF)-beta. Treatment of T reg cells in vitro with imiquimod inhibited their suppressive activity and reduced FOXP3, CD39, CD73, IL-10, and TGF-beta by indirect mechanisms. In vivo and in vitro treatment with imiquimod also induced IL-6 production by effector T cells. In summary, we find that SCCs evade the immune response at least in part by down-regulating vascular E-selectin and recruiting T reg cells. TLR7 agonists neutralized both of these strategies, supporting their use in SCCs and other tumors with similar immune defects.

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-kappaB 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.

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-kappaB 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-1beta in response to endotoxin. Thus, we provide more definitive evidence for a basic mechanism of immunoregulation in the oral mucosa.

Oral mucosal endotoxin tolerance induction in chronic periodontitis.

The oral mucosa is exposed to a high density and diversity of gram-positive and gram-negative bacteria, but very little is known about how immune homeostasis is maintained in this environment, particularly in the inflammatory disease chronic periodontitis (CP). The cells of the innate immune response recognize bacterial structures via the Toll-like receptors (TLR). This activates intracellular signaling and transcription of proteins essential for the induction of an adaptive immune response; however, if unregulated, it can lead to destructive inflammatory responses. Using single-immunoenzyme labeling, we show that the human oral mucosa (gingiva) is infiltrated by large numbers of TLR2(+) and TLR4(+) cells and that their numbers increase significantly in CP, relative to health (P < 0.05, Student's t test). We also show that the numbers of TLR2(+) but not TLR4(+) cells increase linearly with inflammation (r(2) = 0.33, P < 0.05). Double-immunofluorescence analysis confirms that TLR2 is coexpressed by monocytes (MC)/macrophages (mphi) in situ. Further analysis of gingival tissues by quantitative real-time PCR, however, indicates that despite a threefold increase in the expression of interleukin-1beta (IL-1beta) mRNA during CP, there is significant (30-fold) downregulation of TLR2 mRNA (P < 0.05, Student's t test). Also showing similar trends are the levels of TLR4 (ninefold reduction), TLR5 (twofold reduction), and MD-2 (sevenfold reduction) mRNA in CP patients compared to healthy persons, while the level of CD14 was unchanged. In vitro studies with human MC indicate that MC respond to an initial stimulus of lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) or Escherichia coli (EcLPS) by upregulation of TLR2 and TLR4 mRNA and protein; moreover, IL-1beta mRNA is induced and tumor necrosis factor alpha (TNF-alpha), IL-10, IL-6, and IL-8 proteins are secreted. However, restimulation of MC with either PgLPS or EcLPS downregulates TLR2 and TLR4 mRNA and protein and IL-1beta mRNA and induces a ca. 10-fold reduction in TNF-alpha secretion, suggesting the induction of endotoxin tolerance by either LPS. Less susceptible to tolerance than TNF-alpha were IL-6, IL-10, and IL-8. These studies suggest that certain components of the innate oral mucosal immune response, most notably TLRs and inflammatory cytokines, may become tolerized during sustained exposure to bacterial structures such as LPS and that this may be one mechanism used in the oral mucosa to attempt to regulate local immune responses.