Oral immunization with Porphyromonas gingivalis outer membrane protein and CpG oligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity

The aim of this study was to evaluate the efficacy of an oral vaccine containing the 40‐kDa outer membrane protein of Porphyromonas gingivalis (40K‐OMP) and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) to control oral infection by P. gingivalis. Oral immunization with 40K‐OMP plus CpG ODN induced significant 40K‐OMP‐specific serum immunoglobulin G (IgG), IgA, and saliva IgA antibody responses. The 40K‐OMP‐specific CD4⁺ T cells induced by oral 40K‐OMP plus CpG ODN produced both T helper type 1 (Th1; interferon‐γ) and Th2 (interleukin‐4) cytokines. Furthermore, increased frequencies of CD11c⁺ B220⁺ dendritic cells (DCs) and CD11c⁺ CD11b⁺ DCs with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II molecules were noted in spleen, Peyer’s patches, and cervical lymph nodes. Immunized mice were then infected orally with P. gingivalis to determine whether the immune responses induced by oral 40K‐OMP plus CpG ODN were capable of suppressing the bone resorption caused by P. gingivalis infection. Mice given 40K‐OMP plus CpG ODN showed significantly reduced bone loss associated with oral infection by P. gingivalis. Oral administration of 40K‐OMP together with CpG ODN induces Th1‐type and Th2‐type cells, which provide help for protective immunity against P. gingivalis infection. This may be an important tool for the prevention of chronic periodontitis.     

Gingipain‐dependent augmentation by Porphyromonas gingivalis of phagocytosis of Tannerella forsythia

In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that manipulates host immune responses leading to dysbiotic oral microbial communities. Arg‐gingipains (RgpA and RgpB) and Lys‐gingipain (Kgp) are responsible for the majority of bacterial proteolytic activity and play essential roles in bacterial virulence. Therefore, gingipains are often considered as therapeutic targets. This study investigated the role of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in a multiplicity of infection‐dependent and gingipain‐dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in mixed infection, and this coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, the direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected macrophages, more T. forsythia remained in cells coinfected with gingipain‐expressing P. gingivalis than in cells coinfected with the gingipain‐null mutant or infected only with T. forsythia at 24 and 48 h post‐infection. Collectively, these results suggest that P. gingivalis, mainly via its gingipains, alters the clearance of T. forsythia, and provide some insights into the role of P. gingivalis as a keystone pathogen.     

Heme acquisition mechanisms of Porphyromonas gingivalis – strategies used in a polymicrobial community in a heme‐limited host environment

Porphyromonas gingivalis, a main etiologic agent and key pathogen responsible for initiation and progression of chronic periodontitis requires heme as a source of iron and protoporphyrin IX for its survival and the ability to establish an infection. Porphyromonas gingivalis is able to accumulate a defensive cell‐surface heme‐containing pigment in the form of μ‐oxo bisheme. The main sources of heme for P. gingivalis in vivo are hemoproteins present in saliva, gingival crevicular fluid, and erythrocytes. To acquire heme, P. gingivalis uses several mechanisms. Among them, the best characterized are those employing hemagglutinins, hemolysins, and gingipains (Kgp, RgpA, RgpB), TonB‐dependent outer‐membrane receptors (HmuR, HusB, IhtA), and hemophore‐like proteins (HmuY, HusA). Proteins involved in intracellular heme transport, storage, and processing are less well characterized (e.g. PgDps). Importantly, P. gingivalis may also use the heme acquisition systems of other bacteria to fulfill its own heme requirements. Porphyromonas gingivalis displays a novel paradigm for heme acquisition from hemoglobin, whereby the Fe(II)‐containing oxyhemoglobin molecule must first be oxidized to methemoglobin to facilitate heme release. This process not only involves P. gingivalis arginine‐ and lysine‐specific gingipains, but other proteases (e.g. interpain A from Prevotella intermedia) or pyocyanin produced by Pseudomonas aeruginosa. Porphyromonas gingivalis is then able to fully proteolyze the more susceptible methemoglobin substrate to release free heme or to wrest heme from it directly through the use of the HmuY hemophore.     

Identification of T‐cell epitopes of Porphyromonas gingivalis heat‐shock‐protein 60 in periodontitis

The heat shock proteins (hsp) of bacterial species are considered to be involved in regulating the autoimmune mechanism in human diseases due to the considerable homology of their sequences with human hsp. To elucidate how stress proteins contribute to the immunopathogenesis of periodontitis, mononuclear cells from gingival connective tissue of 10 periodontitis patients were simulated with Porphyromonas gingivalis hsp60. T‐cell lines reactive to P. gingivalis hsp60 were established from each patient to define T‐cell epitope specificities. Anti‐P. gingivalis IgG antibody titres were elevated in all patients. We could establish P. gingivalis hsp‐reactive T‐cell lines from gingival mononuclear cells that were mixtures of CD4⁺ and CD8⁺ cells. Of 108 overlapping synthetic peptides spanning the whole P. gingivalis hsp60 molecule, 10 peptides with epitope specificities for T‐cells were identified, and were identical to those reported be B‐cell epitopes in periodontitis.     

Porphyromonas gingivalis gingipain is involved in the detachment and aggregation of Aggregatibacter actinomycetemcomitans biofilm

Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans are major periodontal pathogens that cause several types of periodontal disease. Our previous study suggested that P. gingivalis gingipains secreted in the subgingival environment are related to the detachment of A.actinomycetemcomitans biofilms. However, it remains unclear whether arginine‐specific cysteine proteinase (Rgp) and lysine‐specific proteinase (Kgp) play different roles in the detachment of A. actinomycetemcomitans biofilm. The aim of this study was to investigate possible disruptive roles of Kgp and Rgp in the aggregation and attachment of A. actinomycetemcomitans. While P. gingivalis ATCC33277 culture supernatant has an ability to decrease autoaggregation and coaggregation of A. actinomycetemcomitans cells, neither the boiled culture supernatant of ATCC33277 nor the culture supernatant of KDP136 showed this ability. The addition of KYT‐1 and KYT‐36, specific inhibitors of Rgp and Kgp, respectively, showed no influence on the ability of P. gingivalis culture supernatant. The result of gelatin zymography suggested that other proteases processed by gingipains mediated the decrease of A. actinomycetemcomitans aggregations. We also examined the biofilm‐destructive effect of gingipains by assessing the detachment of A. actinomycetemcomitans from polystyrene surfaces. Scanning electron microscope analysis indicated that A. actinomycetemcomitans cells were detached by P. gingivalis Kgp. The quantity of A. actinomycetemcomitans in biofilm was decreased in co‐culture with P. gingivalis. However, this was not found after the addition of KYT‐36. These findings suggest that Kgp is a critical component for the detachment and decrease of A. actinomycetemcomitans biofilms.     

Establishment of Porphyromonas gingivalis–specific T‐cell lines from atherosclerosis patients

Porphyromonas gingivalis–specific T‐cell lines were established from atherosclerotic lesions from two patients. T‐cell lines were found to be a mixture of CD4⁺ and CD8⁺ T‐cells producing variable amount of interferon‐gamma, interleukin‐4 and interleukin‐10. The two patients have had a periodontal disease whose anti–P. gingivalis immunoglobulin G titers were significantly elevated, and P. gingivalis was the one of the predominantly cultivable microorganisms. This is the first report of the successful establishment of P. gingivalis–specific T‐cell lines from atherosclerotic patients.     

The role of Porphyromonas gingivalis gingipains in platelet activation and innate immune modulation

Platelets are considered to have important functions in inflammatory processes and as actors in the innate immunity. Several studies have shown associations between cardiovascular disease and periodontitis, where the oral anaerobic pathogen Porphyromonas gingivalis has a prominent role in modulating the immune response. Porphyromonas gingivalis has been found in atherosclerotic plaques, indicating spreading of the pathogen via the circulation, with an ability to interact with and activate platelets via e.g. Toll‐like receptors (TLR) and protease‐activated receptors. We aimed to evaluate how the cysteine proteases, gingipains, of P. gingivalis affect platelets in terms of activation and chemokine secretion, and to further investigate the mechanisms of platelet–bacteria interaction. This study shows that primary features of platelet activation, i.e. changes in intracellular free calcium and aggregation, are affected by P. gingivalis and that arg‐gingipains are of great importance for the ability of the bacterium to activate platelets. The P. gingivalis induced a release of the chemokine RANTES, however, to a much lower extent compared with the TLR2/1‐agonist Pam₃CSK₄, which evoked a time‐dependent release of the chemokine. Interestingly, the TLR2/1‐evoked response was abolished by a following addition of viable P. gingivalis wild‐types and gingipain mutants, showing that both Rgp and Kgp cleave the secreted chemokine. We also demonstrate that Pam₃CSK₄‐stimulated platelets release migration inhibitory factor and plasminogen activator inhibitor‐1, and that also these responses were antagonized by P. gingivalis. These results supports immune‐modulatory activities of P. gingivalis and further clarify platelets as active players in innate immunity and in sensing bacterial infections, and as target cells in inflammatory reactions induced by P. gingivalis infection.     

The roles of RgpB and Kgp in late onset gingipain activity in the vimA‐defective mutant of Porphyromonas gingivalis W83

Previous studies have shown that VimA, an acetyltransferase, can modulate gingipain biogenesis in Porphyromonas gingivalis. Inactivation of the vimA gene resulted in isogenic mutants that showed a late onset of gingipain activity that only occurred during the stationary growth phase. To further elucidate the role and contribution of the gingipains in this VimA‐dependent process, isogenic mutants defective in the gingipain genes in the vimA‐deficient genetic background were evaluated. In contrast with the wild‐type strain, RgpB and Kgp gingipain activities were absent in exponential phase in the ?rgpA::tetQ‐vimA::ermF mutant. However, these activities increased to 31 and 53%, respectively, of that of the wild‐type during stationary phase. In the ?rgpA::cat‐?kgp::tetQ‐vimA::ermF mutant, the RgpB protein was observed in the extracellular fraction but no activity was present even at the stationary growth phase. There was no gingipain activity observed in the ?rgpB::cat‐?kgp::tetQ‐vimA::ermF mutant whereas Kgp activity in ?rgpA::cat‐?rgpB::tetQ‐vimA::ermF mutant was 24% of the wild‐type at late stationary phase. In contrast to RgpA, the glycosylation profile of the RgpB catalytic domain from both W83 and P. gingivalis FLL92 (vimA::ermF) showed similarity. Taken together, the results suggest multiple gingipain activation pathways in P. gingivalis. Whereas the maturation pathways for RgpA and RgpB are different, the late‐onset gingipain activity in the vimA‐defective mutant was due to activation/maturation of RgpB and Kgp. Moreover, unlike RgpA, which is VimA‐dependent, the maturation/activation pathways for RgpB and Kgp are interdependent in the absence VimA.     

Porphyromonas gingivalis induces autophagy in THP‐1‐derived macrophages

Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome‐dependent degradation pathway and is a possible mechanism in inflammatory disease. Periodontitis is an inflammatory disease caused by periodontal pathogens. Porphyromonas gingivalis, an important periodontal pathogen, activates cellular autophagy to provide a replicative niche while suppressing apoptosis in endothelial cells. However, the molecular basis for a causal relationship between P. gingivalis and autophagy is unclear. This research examines the involvement of P. gingivalis in autophagy through light chain 3 (LC3) and autophagic proteins, and the role of P. gingivalis‐induced autophagy in the clearance of P. gingivalis and inflammation. To investigate the molecular mechanism of autophagy induced by P. gingivalis, PMA‐differentiated THP‐1‐derived macrophages were infected with live P. gingivalis. The P. gingivalis increased the formation of autophagosomes in a multiplicity of infection‐dependent manner, as well as autophagolysosomes. Porphyromonas gingivalis activated LC3‐I/LC3‐II conversion and increased the conjugation of autophagy‐related 5 (ATG5) –ATG12 and the expression of Beclin1. The expressions of Beclin1, ATG5–ATG12 conjugate, and LC3‐II were significantly inhibited by the presence of 3‐methyladenine, an autophagy inhibitor. Interestingly, 3‐methyladenine increased the survival of P. gingivalis and proinflammatory cytokine interleukin‐1β production. The data indicate that P. gingivalis induces autophagy in PMA‐differentiated THP‐1‐derived macrophages and in turn, macrophages eliminate P. gingivalis through an autophagic response, which can lead to the restriction of an excessive inflammatory response by downregulating interleukin‐1β production. The induction of autophagy by P. gingivalis may play an important role in the periodontal inflammatory process and serve as a target for the development of new therapies.     

Immunoprotective effects of a hemin‐binding peptide derived from hemagglutinin‐2 against infection with Porphyromonas gingivalis

The principal etiologic agent in periodontal disease, Porphyromonas gingivalis, generates cysteine proteases that bind heme with domains such as hemagglutinin‐2 (HA2). High‐affinity HA2–hemin binding supplies the porphyrin and ferric iron needed for growth and virulence. The DHYAVMISK peptide, recently identified at the hemin‐binding site of HA2, inhibits hemin binding. We now evaluate the protective effect of vaccination with DGFPGDHYAVMISK (termed DK) against P. gingivalis using a rat infection model. Rats immunized with DK generated anti‐peptide serum IgGs and salivary sIgAs (as measured by ELISA). In a subcutaneous abscess model, the protective effect of immunization was then investigated by measuring abscess size following subcutaneous injection with P. gingivalis. In an oral infection model, a ligature inoculated with P. gingivalis was used to induce periodontitis. The degree of bone erosion, ordinarily provoked by infection, was then evaluated by micro‐computed tomography. We found that anti‐peptide antibody titers of serum IgGs and salivary sIgAs for rats immunized with DK and adjuvant were significantly higher than for sham‐immunized rats (injected with adjuvant/PBS alone; P < .05). In the subcutaneous abscess model, the DK + adjuvant‐vaccinated rats recovered faster than sham‐vaccinated animals, with their abscess sizes significantly smaller (P < .05). Further, in the experimental periodontitis model, bone loss at the molar palatal side for DK + adjuvant‐vaccinated rats was significantly lower than for sham‐vaccinated animals (P < .05). Collectively, these data demonstrate the potential of (DK) peptide immunization in terms of eliciting an immunoprotective effect against infection with P. gingivalis.     

The role of phagocytosis,oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram‐negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild‐type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild‐type strain ATCC33277, whereas the other wild‐type strain W50 was less effective. Quantitative real‐time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria.     

Functional characteristics of antibodies induced by Arg‐gingipain (HRgpA) and Lys‐gingipain (Kgp) from Porphyromonas gingivalis

Arginine‐specific gingipain (HRgpA) and lysine‐specific gingipain (Kgp), enzymes produced by Porphyromonas gingivalis, may be candidates for an anti–P. gingivalis vaccine. The purpose of our study was to determine whether HRgpA and Kgp have opsonic target sites and whether these sites are available and accessible on intact P. gingivalis cells. Rabbits were used to generate polyclonal antibodies to both proteins. Animals were immunized and immunoglobulin G (IgG) fractions were isolated from preimmune and immune sera. Functional characteristics of the antibodies were assessed by determining antibody titers by enzyme‐linked immunosorbent assay (ELISA), generating Western immunoblots, and measuring antibody enhancement of P. gingivalis opsonization, phagocytosis and killing by polymorphonuclear leukocytes (PMN) of intact cells of strains of P. gingivalis representative of the four serotypes. Strains studied included 33277 (serotype A), A7A1–28 (serotype B), W50 (serotype C) and 381 (serotype D). Both HRgpA and Kgp induced high titers of IgG antibody. Anti‐HRgpA and anti‐Kgp bound to both HRgpA and Kgp demonstrating a large proportion of shared antigenic epitopes. The two antibodies bound equally well to all four P. gingivalis serotypes with titers ranging from 77 to 205 ELISA units when compared to preimmune IgG set at 1 ELISA unit. The immunoblot patterns of binding of the two antibodies to HRgpA and Kgp and to sonicates of the four P. gingivalis serotypes were virtually identical. Both antibodies detected components in HRgpA at 27, 35 and 45 kDa and in Kgp at 27, 32, 35, 40 and 55 kDa. The antibodies also detected components at or near these same positions in addition to multiple high molecular mass components in the cell sonicates of P. gingivalis. Both proteins induced antibodies that significantly enhanced opsonization as assessed by chemiluminescence, with values ranging from 130 mV to 375 mV for anti‐HRgpA IgG and from 240 mV to 475 mV for anti‐Kgp IgG. Both antibodies significantly enhanced PMN‐mediated bacterial killing of the four P. gingivalis serotypes, although the percentage of killing varied among the serotypes (24–81% for anti‐HRgpA and 37–89% for anti‐Kgp). Thus, both HRgpA and Kgp express opsonic target sites and induce high titers of antibodies that opsonize and enhance killing of all four serotypes of P. gingivalis. These two proteins appear to be potential candidate antigens for an anti–P. gingivalis vaccine.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

Potential role for Streptococcus gordonii‐derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co‐aggregates with many oral species including Porphyromonas gingivalis. It causes α‐hemolysis on blood agar, a process mediated by H₂O₂ and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K‐gingipain protease and/or the HmuY hemophore‐like protein. The ability of S. gordonii to mediate Hb oxidation may potentially aid heme capture during co‐aggregation with P. gingivalis. Hemoglobin derived from zones of S. gordonii α‐hemolysis was found to be metHb. Generation of metHb from oxyHb by S. gordonii cells was inhibited by catalase, and correlated with levels of cellular H₂O₂ production. Generation of metHb by S. gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P. gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S. gordonii α‐hemolysis and from metHb generated by the action of S. gordonii cells on isolated oxyHb. The rate of HmuY‐Fe(III)heme complex formation from S. gordonii‐mediated metHb was greater than from an equivalent concentration of auto‐oxidized metHb. It is concluded that S. gordonii may potentially aid heme acquisition by P. gingivalis by facilitating metHb formation in the presence of oxyHb.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.     

Serotype b of Aggregatibacter actinomycetemcomitans increases osteoclast and memory T‐lymphocyte activation

During periodontitis, alveolar bone resorption is associated with activation of T helper type 17 (Th17) lymphocytes and receptor activator of nuclear factor‐κB ligand (RANKL) ‐induced osteoclasts. We previously reported that serotype b of Aggregatibacter actinomycetemcomitans has a higher capacity to trigger Th17‐type differentiation and function in activated T lymphocytes and its lipopolysaccharide is a more potent immunogen compared with the other serotypes. This study aimed to investigate whether serotype b of A. actinomycetemcomitans induces higher Th17‐associated RANKL production, RANKL‐induced osteoclast activation, and antigen‐specific memory T lymphocyte proliferation. On naive CD4⁺ T lymphocytes stimulated with autologous dendritic cells primed with different A. actinomycetemcomitans serotypes, RANKL production, T‐bet, GATA‐3, RORC2 and Foxp3 expression, RORC2/RANKL intracellular double‐expression, TRAP⁺ osteoclast activation, and bone resorption were quantified. The frequency of proliferating memory T lymphocytes in response to A. actinomycetemcomitans serotypes was determined in periodontitis and healthy subjects. Naive CD4⁺ T lymphocytes stimulated by serotype b‐primed dendritic cells elicited higher levels of RANKL, RORC2, TRAP⁺ osteoclasts, and bone resorption than the same cells stimulated with the other serotypes. RANKL positively correlated and co‐expressed with RORC2. Memory T lymphocytes responding to serotype b were more frequently detected in periodontitis patients than healthy subjects. These results indicate that serotype b of A. actinomycetemcomitans is associated with higher production of RANKL and these increased levels are associated with Th17 lymphocyte induction, osteoclast activation, and bone resorption.     

Lipoprotein modifications by gingipains of Porphyromonas gingivalis

Background and Objective

Several studies have shown an association between periodontitis and cardiovascular disease (CVD). Atherosclerosis is the major cause of CVD, and a key event in the development of atherosclerosis is accumulation of lipoproteins within the arterial wall. Bacteria are the primary etiologic agents in periodontitis and Porphyromonas gingivalis is the major pathogen in the disease. Several studies support a role of modified low‐density lipoprotein (LDL) in atherogenesis; however, the pathogenic stimuli that induce the changes and the mechanisms by which this occur are unknown. This study aims to identify alterations in plasma lipoproteins induced by the periodontopathic species of bacterium, P. gingivalis, in vitro.

Material and Methods

Plasma lipoproteins were isolated from whole blood treated with wild‐type and gingipain‐mutant (lacking either the Rgp‐ or Kgp gingipains) P. gingivalis by density/gradient‐ultracentrifugation and were studied using 2‐dimensional gel electrophoresis followed by matrix‐assisted laser desorption/ionization mass spectrometry. Porphyromonas gingivalis‐induced lipid peroxidation and antioxidant levels were measured by thiobarbituric acid‐reactive substances and antioxidant assay kits, respectively, and lumiaggregometry was used for measurement of reactive oxygen species (ROS) and aggregation.

Results

Porphyromonas gingivalis exerted substantial proteolytic effects on the lipoproteins. The Rgp gingipains were responsible for producing 2 apoE fragments, as well as 2 apoB‐100 fragments, in LDL, and the Kgp gingipain produced an unidentified fragment in high‐density lipoproteins. Porphyromonas gingivalis and its different gingipain variants induced ROS and consumed antioxidants. Both the Rgp and Kgp gingipains were involved in inducing lipid peroxidation.

Conclusion

Porphyromonas gingivalis has the potential to change the expression of lipoproteins in blood, which may represent a crucial link between periodontitis and CVD.     

Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis

Takahashi N, Honda T, Domon H, Nakajima T, Tabeta K, Yamazaki K. Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis. J Periodont Res 2010; 45: 512–519. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies have revealed that negative regulatory molecules, including interleukin‐1 receptor‐associated kinase‐M (IRAK‐M), control the overactivation of Toll‐like receptor (TLR) signaling. The role of IRAK‐M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK‐M on interleukin‐8 and macrophage chemoattractant protein‐1 (MCP‐1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. Material and Methods: Primary HGECs and an SV40 T‐antigen‐immortalized HGEC line (epi 4) were stimulated with live or heat‐killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM₃CSK₄, and subsequent expression of IRAK‐M, interleukin‐8 and MCP‐1 was evaluated at the mRNA and protein levels. The effects of IRAK‐M on interleukin‐8 and MCP‐1 expressions were evaluated by IRAK‐M‐specific RNA interference (RNAi)‐based loss‐of‐function assay. Results: All tested stimulants up‐regulated the expression of IRAK‐M in HGECs. The P. gingivalis lipopolysaccharide or PAM₃CSK₄ increased MCP‐1 expression, whereas live P. gingivalis down‐regulated the MCP‐1 expression in HGECs. However, IRAK‐M RNAi increased the expression of MCP‐1 irrespective of up‐ or down‐regulation mediated by the respective stimulants. Interleukin‐8 gene expression, up‐regulated by all tested stimulants, was further enhanced by IRAK‐M RNAi. In contrast, IRAK‐M RNAi had no effect on the interleukin‐8 protein levels, irrespective of the stimulant, indicating that post‐translational modification, not IRAK‐M, controls interleukin‐8 protein expression. Conclusion: Interleukin‐1 receptor‐associated kinase‐M appeared to have distinct regulatory roles on the interleukin‐8 and MCP‐1 produced by HGECs, further suggesting an important role for interleukin‐8 in the immune reponse to periodontopathic bacteria.     

Role of MyD88‐dependent and MyD88‐independent signaling in Porphyromonas gingivalis‐elicited macrophage foam cell formation

Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. Toll‐like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and TRIF during macrophage foam cell formation in response to P. gingivalis. In the presence of human low‐density lipoprotein (LDL) mouse bone‐marrow‐derived macrophages (BMφ) cultured with P. gingivalis responded with significant reduction in tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). The BMφ stained strongly with oil red O, regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P. gingivalis stimulated foam cell formation in a similar way to live bacteria. The BMφ from MyD88‐knockout and Lps2 mice revealed a significant role for MyD88, and a minor role for TRIF in P. gingivalis‐elicited foam cell formation. Porphyromonas gingivalis‐elicited TNF‐α and IL‐6 were affected by MyD88 ablation and to a lesser extent by TRIF status. These data indicate that LDL affects the TNF‐α and IL‐6 response of macrophages to P. gingivalis challenge and that MyD88 and TRIF play important roles in P. gingivalis‐elicited foam cell formation.