Porphyromonas gingivalis-induced reactive oxygen species activate JAK2 and regulate production of inflammatory cytokines through c-Jun.

Pathogen-induced reactive oxygen species (ROS) play a crucial role in host innate immune responses through regulating the quality and quantity of inflammatory mediators. However, the underlying molecular mechanisms of this effect have yet to be clarified. In this study, we examined the mechanism of action of ROS stimulated by Porphyromonas gingivalis in gingival epithelial cells. P. gingivalis induced the rapid production of ROS, which lead to the phosphorylation of JAK2 and increased levels of secreted proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß. Neutralization of ROS by N-acetyl-l-cysteine (NAC) abrogated the phosphorylation of JAK2 and suppressed the production of IL-6 and IL-1ß. ROS-mediated phosphorylation of JAK2 induced the phosphoactivation of c-Jun amino-terminal protein kinase (JNK) and the downstream transcriptional regulator c-Jun. Inhibition of JAK2, either pharmacologically or by small interfering RNA (siRNA), reduced both the phosphorylation of these molecules and the production of proinflammatory cytokines in response to P. gingivalis. Furthermore, pharmacological inhibition or siRNA-mediated gene silencing of JNK or c-Jun mimicked the effect of JAK2 inhibition to suppress P. gingivalis-induced IL-6 and IL-1ß levels. The results show that ROS-mediated activation of JAK2 is required for P. gingivalis-induced inflammatory cytokine production and that the JNK/c-Jun signaling axis is involved in the ROS-dependent regulation of IL-1ß and IL-6 production.

Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases.

Filifactor alocis infection and inflammatory responses in the mouse subcutaneous chamber model.

Recent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10(9) F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.

The role of JAK-3 in regulating TLR-mediated inflammatory cytokine production in innate immune cells.

The role of JAK-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific small interfering RNA or JAK3 gene knockout resulted in an increase in TLR-mediated production of proinflammatory cytokines while concurrently decreasing the production of IL-10. Inhibition of JAK3 suppressed phosphorylation of PI3K downstream effectors including Akt, mammalian target of rapamycin complex 1, glycogen synthase kinase 3ß (GSK3ß), and CREB. Constitutive activation of Akt or inhibition of GSK3ß abrogated the capability of JAK3 inhibition to enhance proinflammatory cytokines and suppress IL-10 production. In contrast, inhibition of PI3K enhanced this regulatory ability of JAK3 in LPS-stimulated monocytes. At the transcriptional level, JAK3 knockout lead to the increased phosphorylation of STATs that could be attenuated by neutralization of de novo inflammatory cytokines. JAK3 inhibition exhibited a GSK3 activity-dependent ability to enhance phosphorylation levels and DNA binding of NF-κB p65. Moreover, JAK3 inhibition correlated with an increased CD4(+) T cell response. Additionally, higher neutrophil infiltration, IL-17 expression, and intestinal epithelium erosion were observed in JAK3 knockout mice. These findings demonstrate the negative regulatory function of JAK3 and elucidate the signaling pathway by which JAK3 differentially regulates TLR-mediated inflammatory cytokine production in innate immune cells.

Expression and function of the homeostatic molecule Del-1 in endothelial cells and the periodontal tissue.

Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin). Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3) by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.

Inhibition of GSK3 abolishes bacterial-induced periodontal bone loss in mice.

The tissue destruction that characterizes periodontitis is driven by the host response to bacterial pathogens. Inhibition of glycogen synthase kinase 3ß (GSK3ß) in innate cells leads to suppression of Toll-like receptor (TLR)-initiated proinflammatory cytokines under nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 transcriptional control and promotion of cyclic adenosine monophosphate response element-binding (CREB)-dependent gene activation. Therefore, we hypothesized that the cell permeable GSK3-specific inhibitor, SB216763, would protect against alveolar bone loss induced by the key periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in a murine model. B6129SF2/J mice either were infected orally with P. gingivalis ATCC 33277; or treated with SB216763 and infected with P. gingivalis; sham infected; or exposed to vehicle only (dimethyl sulfoxide [DMSO]); or to GSK3 inhibitor only (SB216763). Alveolar bone loss and local (neutrophil infiltration and interleukin [IL]-17) and systemic (tumor necrosis factor [TNF], IL-6, Il-1ß and IL-12/IL-23 p40) inflammatory indices also were monitored. SB216763 unequivocally abrogated mean P. gingivalis-induced bone resorption, measured at 14 predetermined points on the molars of defleshed maxillae as the distance from the cementoenamel junction to the alveolar bone crest (p < 0.05). The systemic cytokine response, the local neutrophil infiltration and the IL-17 expression were suppressed (p < 0.001). These data confirm the relevance of prior in vitro phenomena and establish GSK3 as a novel, efficacious therapeutic preventing periodontal disease progression in a susceptible host. These findings also may have relevance to other chronic inflammatory diseases and the systemic sequelae associated with periodontal infections.

The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement.

Porphyromonas gingivalis is a low-abundance oral anaerobic bacterium implicated in periodontitis, a polymicrobial inflammatory disease, and the associated systemic conditions. However, the mechanism by which P. gingivalis contributes to inflammation and disease has remained elusive. Here we show that P. gingivalis, at very low colonization levels, triggers changes to the amount and composition of the oral commensal microbiota leading to inflammatory periodontal bone loss. The commensal microbiota and complement were both required for P. gingivalis-induced bone loss, as germ-free mice or conventionally raised C3a and C5a receptor-deficient mice did not develop bone loss after inoculation with P. gingivalis. These findings demonstrate that a single, low-abundance species can disrupt host-microbial homeostasis to cause inflammatory disease. The identification and targeting of similar low-abundance pathogens with community-wide impact may be important for treating inflammatory diseases of polymicrobial etiology.

Nuclear factor-kappaB p50 subunits in chronic periodontitis and Porphyromonas gingivalis lipopolysaccharide-pulsed dendritic cells.

The pathogenesis of a number of chronic inflammatory diseases has been linked to dysregulated functioning of nuclear transcription factor (NF)-kappaB. In the present study, we examine NF-kappaB activation in human oral mucosal tissues of chronic periodontitis (CP) patients. Electrophoretic mobility shift assay and DNA-binding enzyme-linked immunosorbent assays indicate elevated levels of transcriptionally repressive p50 subunits and an increased p50/p65 ratio in CP tissues compared to healthy controls. Because Porphyromonas gingivalis has been recognized to be a causal factor in CP, we used P. gingivalis lipopolysaccharide (LPS) for in vitro studies with monocyte-derived dendritic cells (MoDCs). Porphyromonas gingivalis LPS, unlike Escherichia coli LPS, induced an increased p50/p65 ratio in MoDCs. Using blocking antibodies, we demonstrated that while both Toll-like receptor 2 (TLR2) and TLR4 are required for MoDC maturation by P. gingivalis LPS, only TLR4 signaling is sufficient to induce cytokine secretion. Our results suggest that increased levels of transcriptionally repressive p50 may be characteristic of CP and might be a result of suboptimal NF-kappaB activation and dendritic cell maturation by P. gingivalis, a bacterium implicated in CP.

MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis.

Matrix metalloproteinase-9 (MMP-9) cleaves collagen, allowing leukocytes to traffic toward the vasculature and the lymphatics. When MMP-9 is unregulated by tissue inhibitor of metalloproteinase-1 (TIMP-1), this can lead to tissue destruction. Dendritic cells (DCs) infiltrate the oral mucosa increasingly in chronic periodontitis, characterized by infection with several pathogens including Porphyromonas gingivalis. In this study, human monocyte-derived DCs were pulsed with different doses of lipopolysaccharide of P. gingivalis 381 and of Escherichia coli type strain 25922, as well as whole live isogenic fimbriae-deficient mutant strains of P. gingivalis 381. Levels of induction of MMP-9 and TIMP-1, as well as interleukin-10 (IL-10), which reportedly inhibits MMP-9 induction, were measured by several approaches. Our results reveal that lipopolysaccharide of P. gingivalis, compared with lipopolysaccharide from E. coli type strain 25922, is a relatively potent inducer of MMP-9, but a weak inducer of TIMP-1, contributing to a high MMP-9/TIMP-1 ratio.Whole live P. gingivalis strain 381, major fimbriae mutant DPG-3 and double mutant MFB were potent inducers of MMP-9, but minor fimbriae mutant MFI was not. MMP-9 induction was inversely proportional to IL-10 induction. These results suggest that lipopolysaccharide and the minor and the major fimbriae of P. gingivalis may play distinct roles in induction by DCs of MMP-9, a potent mediator of local tissue destruction and leukocyte trafficking.

Targeting of DC-SIGN on human dendritic cells by minor fimbriated Porphyromonas gingivalis strains elicits a distinct effector T cell response.

The oral mucosal pathogen Porphyromonas gingivalis expresses at least two adhesins: the 67-kDa mfa-1 (minor) fimbriae and the 41-kDa fimA (major) fimbriae. In periodontal disease, P. gingivalis associates in situ with dermal dendritic cells (DCs), many of which express DC-SIGN (DC-specific ICAM-3 grabbing nonintegrin; CD209). The cellular receptors present on DCs that are involved in the uptake of minor/major fimbriated P. gingivalis, along with the effector immune response induced, are presently unclear. In this study, stably transfected human DC-SIGN(+/-) Raji cell lines and monocyte-derived DCs (MoDCs) were pulsed with whole, live, wild-type Pg381 or isogenic major (DPG-3)-, minor (MFI)-, or double fimbriae (MFB)-deficient mutant P. gingivalis strains. The influence of blocking Abs, carbohydrates, full-length glycosylated HIV-1 gp120 envelope protein, and cytochalasin D on the uptake of strains and on the immune responses was determined in vitro. We show that the binding of minor fimbriated P. gingivalis strains to Raji cells and MoDCs is dependent on DC-SIGN, whereas the double fimbriae mutant strain does not bind. Binding to DC-SIGN on MoDCs is followed by the internalization of P. gingivalis into DC-SIGN-rich intracellular compartments, and MoDCs secrete low levels of inflammatory cytokines and remain relatively immature. Blocking DC-SIGN with HIV-1 gp120 prevents the uptake of minor fimbriated strains and deregulates the expression of inflammatory cytokines. Moreover, MoDCs promote a Th2 or Th1 effector response, depending on whether they are pulsed with minor or major fimbriated P. gingivalis strains, respectively, suggesting distinct immunomodulatory roles for the two adhesins of P. gingivalis.

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.

Fimbriated Porphyromonas gingivalis is more efficient than fimbria-deficient P. gingivalis in entering human dendritic cells in vitro and induces an inflammatory Th1 effector response.

Porphyromonas gingivalis is a fimbriated mucosal pathogen implicated in chronic periodontitis (CP). The fimbriae are required for invasion of the gingival mucosa and for induction of CP in animal models of periodontitis. CP is associated with infection of immature dendritic cells (DCs) by P. gingivalis in situ and with increased numbers of dermal DCs (DDCs) and mature DCs in the lamina propria. The role of fimbriae in gaining entry into human DCs and how this modulates the inflammatory and effector immune responses, however, have not been explored. To address this, we generated monocyte-derived DCs (MDDCs) in vitro which phenotypically and functionally resemble DDCs. We show here that virulent fimbriated P. gingivalis 381, in contrast to its fimbria-deficient mutant, P. gingivalis DPG3, efficiently gains entry to MDDCs in a manner dependent on active cell metabolism and cytoskeletal rearrangement. In addition, uptake of 381, unlike DPG3, induces DCs to undergo maturation, upregulate costimulatory molecules, and secrete inflammation cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, IL-10, and IL-12. Moreover, MDDCs pulsed with 381 also stimulated a higher autologous mixed lymphocyte reaction and induced a Th1-type response, with gamma interferon (IFN-gamma) being the main cytokine. Monocytes used as controls demonstrated fimbria-dependent uptake of 381 as well but produced low levels of inflammatory cytokines compared to MDDCs. When MDDCs were pulsed with recombinant fimbrillin of P. gingivalis (10 micro g/ml), maturation of MDDCs was also induced; moreover, matured MDDCs induced proliferation of autologous CD4(+) T cells and release of IFN-gamma. Thus, these results establish the significance of P. gingivalis fimbriae in the uptake of P. gingivalis by MDDCs and in induction of immunostimulatory Th1 responses.

Human dendritic cells respond to Porphyromonas gingivalis LPS by promoting a Th2 effector response in vitro.

Understanding how mucosal pathogens modulate the immune response may facilitate the development of vaccines for disparate human diseases. In the present study, human monocyte-derived DC (MDDC)were pulsed with LPS of the oral pathogen Porphyromonas gingivalis and Escherichia coli 25922 and analyzed for: (i) production of Th-biasing/inflammatory cytokines; (ii) maturation/costimulatory molecules; and (iii) induction of allogeneic CD4+ and naive CD45RA+ T cell proliferation and release of Th1 or Th2 cytokines. We show that E. coli LPS-pulsed MDDC released Th1-biasing cytokines - consisting of high levels of IL-12 p70, IFN-gamma-inducible protein 10 (IP-10) - but also TNF-alpha, IL-10, IL-6 and IL-1beta. In contrast, no IL-12 p70 or IP-10, and lower levels of TNF-alpha and IL-10 were induced by P. gingivalis LPS. These differences were sustained at LPS doses that yielded nearly equivalent maturation of MDDC; moreover the T cell response was consistent: E. coli LPS-pulsed MDDC induced higher T cell proliferation, and T cells released more IFN-gamma and IL-2, but less IL-5 than T cells co-cultured with P. gingivalis LPS pulsed-MDDC. IL-13 was secreted by naive CD45RA+CD45RO-CD4+ T cells in response to P. gingivalisLPS-pulsed MDDC. These results suggest that human MDDC can be polarized by LPS from the mucosal pathogen P. gingivalis to induce a Th2 effector response in vitro.

Effects of enamel matrix derivative on Porphyromonas gingivalis.

BACKGROUND: Enamel matrix derivative (EMD) is used during periodontal surgery for the regeneration of periodontal tissue. It consists of the amelogenin fraction of porcine enamel matrix (AMEL) suspended in a vehicle of propylene glycol alginate (PGA). EMD-treated sites appear to heal with less inflammation. It has been suggested that antimicrobial properties of EMD might account for improved healing in vivo. The objectives of this study were: 1) to determine the antibacterial effects of EMD on the periodontal pathogen Porphyromonas gingivalis and 2) to establish the component(s) of EMD that are responsible for this effect. METHODS: The antimicrobial effects were determined in vitro using the broth dilution assay. P. gingivalis at a starting inoculum of 10(9) colony forming units/ml was treated with EMD, AMEL, and PGA in Hank's balanced salt solution (HBSS) for 1, 3, and 24 hours. The CFU/ml of P. gingivalis recovered on enriched Brucella blood agar was determined at 6 and 10 days. RESULTS: EMD (containing AMEL and PGA) or PGA alone eliminated recoverable CFUs of P. gingivalis. Interestingly, AMEL in HBSS increased recoverable CFUs from 8.62 log CFU/ml to 8.93 log CFU/ml. Further analysis of the dose response at concentrations of 0.3, 3, and 30 mg/ml of AMEL in HBSS revealed that only 30 mg/ml (clinical concentration) increased CFUs of P. gingivalis relative to baseline (from 8.8 log CFU/ml to 9.2 log CFU/ml in 3 hours). Additionally, AMEL was compared to a protein control bovine serum albumin (BSA) to determine whether this effect was unique to AMEL. A marked increase in recoverable CFUs occurred with the AMEL (increasing from 8.8 log CFU/ml to 9.47 log CFU/ml), but not with BSA. CONCLUSIONS: EMD possesses antimicrobial properties that can be attributed to the propylene glycol alginate vehicle. The amelogenin fraction of porcine enamel matrix in enamel matrix derivative (i.e., AMEL) is not antibacterial for P. gingivalis and was shown to increase recoverable CFUs.