Targeting Atp6v1c1 Prevents Inflammation and Bone Erosion Caused by Periodontitis and Reveals Its Critical Function in Osteoimmunology

Periodontal disease (Periodontitis) is a serious disease that affects a majority of adult Americans and is associated with other systemic diseases, including diabetes, rheumatoid arthritis, and other inflammatory diseases. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this pervasive and destructive disease. In this study, we utilized novel adeno-associated virus (AAV)-mediated Atp6v1c1 knockdown gene therapy to treat bone erosion and inflammatory caused by periodontitis in mouse model. Atp6v1c1 is a subunit of the V-ATPase complex and regulator of the assembly of the V0 and V1 domains of the V-ATPase complex. We demonstrated previously that Atp6v1c1 has an essential function in osteoclast mediated bone resorption. We hypothesized that Atp6v1c1 may be an ideal target to prevent the bone erosion and inflammation caused by periodontitis. To test the hypothesis, we employed AAV RNAi knockdown of Atp6v1c1 gene expression to prevent bone erosion and gingival inflammation simultaneously. We found that lesion-specific injection of AAV-shRNA-Atp6v1c1 into the periodontal disease lesions protected against bone erosion (>85%) and gingival inflammation caused by P. gingivalis W50 infection. AAV-mediated Atp6v1c1 knockdown dramatically reduced osteoclast numbers and inhibited the infiltration of dendritic cells and macrophages in the bacteria-induced inflammatory lesions in periodontitis. Silencing of Atp6v1c1 expression also prevented the expressions of osteoclast-related genes and pro-inflammatory cytokine genes. Our data suggests that AAV-shRNA-Atp6v1c1 treatment can significantly attenuate the bone erosion and inflammation caused by periodontitis, indicating the dual function of AAV-shRNA-Atp6v1c1 as an inhibitor of bone erosion mediated by osteoclasts, and as an inhibitor of inflammation through down-regulation of pro-inflammatory cytokine expression. This study demonstrated that Atp6v1c1 RNAi knockdown gene therapy mediated by AAV-shRNA-Atp6v1c1 is a promising novel therapeutic approach for the treatment of bone erosion and inflammatory related diseases, such as periodontitis and rheumatoid arthritis.     

Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1''s expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.     

Porphyromonas gingivalis GroEL Induces Osteoclastogenesis of Periodontal Ligament Cells and Enhances Alveolar Bone Resorption in Rats

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.     

Active Invasion of Oral and Aortic Tissues by Porphyromonas gingivalis in Mice Causally Links Periodontitis and Atherosclerosis

Atherosclerotic vascular disease is a leading cause of myocardial infarction and cerebrovascular accident, and independent associations with periodontal disease (PD) are reported. PD is caused by polymicrobial infections and aggressive immune responses. Genomic DNA of Porphyromonas gingivalis, the best-studied bacterial pathogen associated with severe PD, is detected within atherosclerotic plaque. We examined causal relationships between chronic P. gingivalis oral infection, PD, and atherosclerosis in hyperlipidemic ApoE^null mice. ApoE^null mice (n = 24) were orally infected with P. gingivalis for 12 and 24 weeks. PD was assessed by standard clinical measurements while the aorta was examined for atherosclerotic lesions and inflammatory markers by array. Systemic inflammatory markers serum amyloid A, nitric oxide, and oxidized low-density lipoprotein were analyzed. P. gingivalis infection elicited specific antibodies and alveolar bone loss. Fluorescent in situ hybridization detected viable P. gingivalis within oral epithelium and aorta, and genomic DNA was detected within systemic organs. Aortic plaque area was significantly increased in P. gingivalis-infected mice at 24 weeks (P<0.01). Aortic RNA and protein arrays indicated a strong Th2 response. Chronic oral infection with P. gingivalis results in a specific immune response, significant increases in oral bone resorption, aortic inflammation, viable bacteria in oral epithelium and aorta, and plaque development.     

Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.     

Lactobacillus helveticus SBT2171 upregulates the expression of β‐defensin and ameliorates periodontal disease caused by Porphyromonas gingivalis

Antimicrobial peptides play important roles in the innate immune system of various organisms, and they may also be considered to prevent the organisms from infections. In particular, β‐defensins, mainly produced in epithelial cells, are recognized as one of the major antimicrobial peptides in mammals, including humans. In this study, we showed that Lactobacillus helveticus SBT2171 (LH2171), one of the several species of lactic acid bacteria, upregulates the production of β‐defensins in oral epithelial cells in vitro. Moreover, LH2171 reduced the increase of proinflammatory cytokine expression, induced by Porphyromonas gingivalis stimulation, in gingival epithelial cells. These data suggested that LH2171 suppresses P. gingivalis‐induced inflammation by upregulating the expression of β‐defensins in gingival epithelial cells. We subsequently investigated the effects of LH2171 in vivo and revealed that β‐defensin expression was increased in the oral cavities of LH2171‐fed mice. Furthermore, LH2171 decreased alveolar bone loss, gingival inflammation, and amounts of P. gingivalis‐specific 16S ribosomal RNA in the gingiva of P. gingivalis‐inoculated mice. Taken together, our results showed that LH2171 upregulates the expression of β‐defensins in oral cavity, thereby decreasing the number of P. gingivalis consequently ameliorating the experimental periodontal disease.     

Neutrophil Mobilization by Surface-Glycan Altered Th17-Skewing Bacteria Mitigates Periodontal Pathogen Persistence and Associated Alveolar Bone Loss

Alveolar bone (tooth-supporting bone) erosion is a hallmark of periodontitis, an inflammatory disease that often leads to tooth loss. Periodontitis is caused by a select group of pathogens that form biofilms in subgingival crevices between the gums and teeth. It is well-recognized that the periodontal pathogen Porphyromonas gingivalis in these biofilms is responsible for modeling a microbial dysbiotic state, which then initiates an inflammatory response destructive to the periodontal tissues and bone. Eradication of this pathogen is thus critical for the treatment of periodontitis. Previous studies have shown that oral inoculation in mice with an attenuated strain of the periodontal pathogen Tannerella forsythia altered in O-glycan surface composition induces a Th17-linked mobilization of neutrophils to the gingival tissues. In this study, we sought to determine if immune priming with such a Th17-biasing strain would elicit a productive neutrophil response against P. gingivalis. Our data show that inoculation with a Th17-biasing T. forsythia strain is effective in blocking P. gingivalis-persistence and associated alveolar bone loss in mice. This work demonstrates the potential of O-glycan modified Tannerella strains or their O-glycan components for harnessing Th17-mediated immunity against periodontal and other mucosal pathogens.     

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE^null) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE^null mice.     

Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis

Introduction

Clinical studies suggest a direct influence of periodontal disease (PD) on serum inflammatory markers and disease assessment of patients with established rheumatoid arthritis (RA). However, the influence of PD on arthritis development remains unclear. This investigation was undertaken to determine the contribution of chronic PD to immune activation and development of joint inflammation using the collagen-induced arthritis (CIA) model.

Methods

DBA1/J mice orally infected with Porphyromonas gingivalis were administered with collagen II (CII) emulsified in complete Freund’s adjuvant (CFA) or incomplete Freund’s adjuvant (IFA) to induce arthritis. Arthritis development was assessed by visual scoring of paw swelling, caliper measurement of the paws, mRNA expression, paw micro-computed tomography (micro-CT) analysis, histology, and tartrate resistant acid phosphatase for osteoclast detection (TRAP)-positive immunohistochemistry. Serum and reactivated splenocytes were evaluated for cytokine expression.

Results

Mice induced for PD and/or arthritis developed periodontal disease, shown by decreased alveolar bone and alteration of mRNA expression in gingival tissues and submandibular lymph nodes compared to vehicle. P. gingivalis oral infection increased paw swelling and osteoclast numbers in mice immunized with CFA/CII. Arthritis incidence and severity were increased by P. gingivalis in mice that received IFA/CII immunizations. Increased synovitis, bone erosions, and osteoclast numbers in the paws were observed following IFA/CII immunizations in mice infected with P gingivalis. Furthermore, cytokine analysis showed a trend toward increased serum Th17/Th1 ratios when P. gingivalis infection was present in mice receiving either CFA/CII or IFA/CII immunizations. Significant cytokine increases induced by P. gingivalis oral infection were mostly associated to Th17-related cytokines of reactivated splenic cells, including IL-1β, IL-6, and IL-22 in the CFA/CII group and IL-1β, tumor necrosis factor-α, transforming growth factor-β, IL-6 and IL-23 in the IFA/CII group.

Conclusions

Chronic P. gingivalis oral infection prior to arthritis induction increases the immune system activation favoring Th17 cell responses, and ultimately accelerating arthritis development. These results suggest that chronic oral infection may influence RA development mainly through activation of Th17-related pathways.     

Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway

Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well‐known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11‐ and RalA‐positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell‐to‐cell spreading.     

Fusobacterium nucleatum Alters Atherosclerosis Risk Factors and Enhances Inflammatory Markers with an Atheroprotective Immune Response in ApoEnull Mice

The American Heart Association supports an association between periodontal disease (PD) and atherosclerotic vascular disease (ASVD) but does not as of yet support a causal relationship. Recently, we have shown that major periodontal pathogens Porphyromonas gingivalis and Treponema denticola are causally associated with acceleration of aortic atherosclerosis in ApoE^null hyperlipidemic mice. The aim of this study was to determine if oral infection with another significant periodontal pathogen Fusobacterium nucleatum can accelerate aortic inflammation and atherosclerosis in the aortic artery of ApoE^null mice. ApoE^null mice (n = 23) were orally infected with F. nucleatum ATCC 49256 and euthanized at 12 and 24 weeks. Periodontal disease assessments including F. nucleatum oral colonization, gingival inflammation, immune response, intrabony defects, and alveolar bone resorption were evaluated. Systemic organs were evaluated for infection, aortic sections were examined for atherosclerosis, and inflammatory markers were measured. Chronic oral infection established F. nucleatum colonization in the oral cavity, induced significant humoral IgG (P=0.0001) and IgM (P=0.001) antibody response (12 and 24 weeks), and resulted in significant (P=0.0001) alveolar bone resorption and intrabony defects. F. nucleatum genomic DNA was detected in systemic organs (heart, aorta, liver, kidney, lung) indicating bacteremia. Aortic atherosclerotic plaque area was measured and showed a local inflammatory infiltrate revealed the presence of F4/80⁺ macrophages and CD3⁺ T cells. Vascular inflammation was detected by enhanced systemic cytokines (CD30L, IL-4, IL-12), oxidized LDL and serum amyloid A, as well as altered serum lipid profile (cholesterol, triglycerides, chylomicrons, VLDL, LDL, HDL), in infected mice and altered aortic gene expression in infected mice. Despite evidence for systemic infection in several organs and modulation of known atherosclerosis risk factors, aortic atherosclerotic lesions were significantly reduced after F. nucleatum infection suggesting a potential protective function for this member of the oral microbiota.     

Association between Polycystic Ovary Syndrome,Oral Microbiota and Systemic Antibody Responses

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.     

Adrm1 interacts with Atp6v0d2 and regulates osteoclast differentiation

Bone homeostasis is tightly regulated by matrix-producing osteoblasts and bone-resorbing osteoclasts. During osteoclast development, mononuclear preosteoclasts derived from myeloid cells fuse together to form multinucleated, giant cells. Previously, we reported that the d2 isoform of the vacuolar (H⁺) ATPase V0 domain (Atp6v0d2) plays an important role in osteoclast maturation and bone formation. To understand how Atp6v0d2 controls osteoclast maturation, we have performed a yeast two-hybrid screen using full-length Atp6v0d2 as the bait, and identified adhesion-regulating molecule 1 protein (Adrm1) as a potential functional partner of Atp6v0d2. The interaction between Atp6v0d2 and Adrm1 was confirmed in yeast and invivo using immunoprecipitation assays. We also show that Adrm1 is required for cell migration and osteoclast maturation.     

Implication of Toll/IL-1 receptor domain containing adapters in Porphyromonas gingivalis-induced inflammation

Periodontitis is induced by periodontal dysbiosis characterized by the predominance of anaerobic species. TLRs constitute the classical pathway for cell activation by infection. Interestingly, the Toll/IL-1 receptor homology domain adapters initiate signaling events, leading to the activation of the expression of the genes involved in the host immune response. The aim of this study was to evaluate the effects of Porphyromonas gingivalis on the expression and protein-protein interactions among five TIR adapters (MAL, MyD88, TRIF, TRAM and SARM) in gingival epithelial cells and endothelial cells. It was observed that P. gingivalis is able to modulate the signaling cascades activated through its recognition by TLR4/2 in gingival epithelial cells and endothelial cells. Indeed, MAL-MyD88 protein-protein interactions associated with TLR4 was the main pathway activated by P. gingivalis infection. When transient siRNA inhibition was performed, cell viability, inflammation, and cell death induced by infection decreased and such deleterious effects were almost absent when MAL or TRAM were targeted. This study emphasizes the role of such TIR adapter proteins in P. gingivalis elicited inflammation and the precise evaluation of TIR adapter protein interactions may pave the way for future therapeutics in both periodontitis and systemic disease with a P. gingivalis involvement, such as atherothrombosis.     

Thrombospondin-1 Production Is Enhanced by Porphyromonas gingivalis Lipopolysaccharide in THP-1 Cells

Periodontitis is a chronic inflammatory disease caused by gram-negative anaerobic bacteria. Monocytes and macrophages stimulated by periodontopathic bacteria induce inflammatory mediators that cause tooth-supporting structure destruction and alveolar bone resorption. In this study, using a DNA microarray, we identified the enhanced gene expression of thrombospondin-1 (TSP-1) in human monocytic cells stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS). TSP-1 is a multifunctional extracellular matrix protein that is upregulated during the inflammatory process. Recent studies have suggested that TSP-1 is associated with rheumatoid arthritis, diabetes mellitus, and osteoclastogenesis. TSP-1 is secreted from neutrophils, monocytes, and macrophages, which mediate immune responses at inflammatory regions. However, TSP-1 expression in periodontitis and the mechanisms underlying TSP-1 expression in human monocytic cells remain unknown. Here using real-time RT-PCR, we demonstrated that TSP-1 mRNA expression level was significantly upregulated in inflamed periodontitis gingival tissues and in P. gingivalis LPS-stimulated human monocytic cell line THP-1 cells. TSP-1 was expressed via Toll-like receptor (TLR) 2 and TLR4 pathways. In P. gingivalis LPS stimulation, TSP-1 expression was dependent upon TLR2 through the activation of NF-κB signaling. Furthermore, IL-17F synergistically enhanced P. gingivalis LPS-induced TSP-1 production. These results suggest that modulation of TSP-1 expression by P. gingivalis plays an important role in the progression and chronicity of periodontitis. It may also contribute a new target molecule for periodontal therapy.     

Tetra- and Penta-Acylated Lipid A Structures of Porphyromonas gingivalis LPS Differentially Activate TLR4-Mediated NF-κB Signal Transduction Cascade and Immuno-Inflammatory Response in Human Gingival Fibroblasts

Background

Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS_1435/1449) and penta-acylated (LPS₁₆₉₀) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.

Methodology/Principal Findings

This study systematically investigated the effects of P. gingivalis LPS_1435/1449 and LPS₁₆₉₀ on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS_1435/1449 and LPS₁₆₉₀ differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS₁₆₉₀ but not by LPS_1435/1449. In addition, LPS₁₆₉₀ induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS₁₆₉₀ while down-regulated by LPS_1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS₁₆₉₀-induced expression of IL-6 and IL-8 in HGFs.

Conclusions/Significance

The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.     

Endogenous acid ceramidase protects epithelial cells from Porphyromonas gingivalis-induced inflammation in vitro

Ceramidases are a group of enzymes that degrade pro-inflammatory ceramide by cleaving a fatty acid to form anti-inflammatory sphingosine lipid. Thus far, acid, neutral and alkaline ceramidase isozymes have been described. However, the expression patterns of ceramidase isoforms as well as their role in periodontal disease pathogenesis remain unknown. In this study, expression patterns of ceramidase isoforms were quantified by real-time PCR and immunohistochemistry in gingival samples of patients with periodontitis and healthy subjects, as well as in EpiGingivalTM-3D culture and OBA-9 gingival epithelial cells both of which were stimulated with or without the presence of live Porphyromonas gingivalis (ATCC 33277 strain). A significantly lower level of acid ceramidase expression was detected in gingival tissues from periodontal patients compared to those from healthy subjects. In addition, acid-ceramidase expression in EpiGingival? 3D culture and OBA-9?cells was suppressed by stimulation with P. gingivalis in vitro. No significant fluctuation was detected for neutral or alkaline ceramidases in either gingival samples or cell cultures. Next, to elucidate the role of acid ceramidase in P. gingivalis-induced inflammation in vitro, OBA-9?cells were transduced with adenoviral vector expressing the human acid ceramidase (Ad-ASAH1) gene or control adenoviral vector (Ad-control). In response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9?cells showed significantly lower mRNA expressions of caspase-3 as well as the percentage of Annexin V-positive cells, when compared with OBA-9?cells transduced with Ad-control vector. Furthermore, in response to stimulation with P. gingivalis, ASAH1-over-expressing OBA-9?cells produced less TNF-α, IL-6, and IL1β pro-inflammatory cytokines than observed in OBA-9?cells transduced with Ad-control vector. Collectively, our data show the novel discovery of anti-inflammatory and anti-apoptotic effects of acid ceramidase in host cells exposed to periodontal bacteria, and the attenuation of the expression of host-protective acid ceramidase in periodontal lesions.