In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

AIM: Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. METHODOLOGY: DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. RESULTS: The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1ß and TGF-ß1, whereas IL-6 and NFκB1 expression was significantly reduced. CONCLUSION: The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

Alveolar Bone Loss in a Ligature-Induced Periodontitis Model in Rat Using Different Ligature Sizes.

OBJECTIVES: Ligature-induced periodontitis model has been widely used as a preclinical stage for investigating new treatment modalities. However, the effect of different ligature sizes on alveolar bone loss has never been studied. Therefore, we examined alveolar bone loss in this rat model using different sizes of silk ligatures, as well as healing after ligature removal. MATERIALS AND METHODS: Left maxillary second molars of Sprague-Dawley rats were ligated with 3-0, 4-0, or 5-0 silk ligatures (n = 4-5/group) for 14 days before harvested maxillae and gingival tissues. For subsequent experiment, animals were ligated for 14 days using the ligature size that induced the most alveolar bone loss before ligature removal and sacrificed at 0, 7 and 14 days (n = 5-6/group). All maxillae and gingival tissues were harvested to evaluate alveolar bone level, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) levels. STATISTICAL ANALYSIS: Data was analyzed using SPSS Statistics 23.0 software (SPSS Inc., Chicago, Illinois, United States). Data from all experiments were tested for normality using Shapiro-Wilk test. Data between ligatured and nonligatured teeth were compared using Student's t-test or Wilcoxon signed-rank test. Differences among different ligature sizes were analyzed by analysis of variance followed by multiple comparisons with post-hoc test. A p-value less than 0.05 was considered statistically significant. RESULTS: The alveolar bone loss of ligated teeth was substantially higher than that of control after 14 days of ligation. While 3-0 and 4-0 resulted in significantly greater bone loss than 5-0 silk, the 3-0 group had the lowest rate of ligature loss. Therefore, alveolar bone healing postligature removal was investigated further using 3-0 silk. The results showed no significant bone level change at 2 weeks after ligature removal. In term of IL-1ß and TNF-α levels, there was no statistically significant difference in IL-1ß level between groups at any time point, while TNF-α was undetectable. CONCLUSION: These data showed that 3-0 silk was the most effective ligature size in promoting alveolar bone loss comparing with 4-0 and 5-0 silk. During the 2-week period following ligature removal, spontaneous bone healing was not observed.

Characteristics of mesenchymal stem cells from supracrestal gingival connective tissue.

BACKGROUND: Although periodontal ligament-derived mesenchymal stem cells (PDL-MSCs) have been recognized as the best MSC choice for periodontal regeneration, using PDL-MSCs requires tooth extraction for cell isolation. The supracrestal gingival (SG) connective tissue is a part of the gingiva which is located close to the PDL. SG-MSCs might have similar characteristic to the PDL-MSCs and serve as a good MSC candidate for periodontal regeneration. This study aimed to investigate and compare the characteristics of human MSCs isolated from SG tissue (hSG-MSCs), marginal gingival tissue (hMG-MSCs), and PDL (hPDL-MSCs) in terms of MSCs properties and differential gene expression profile. METHODS: Human periodontal tissue from five healthy subjects, including SG, MG, and PDL, was harvested. The primary cells of the hSG-MSCs, hMG-MSCs, and hPDL-MSCs were isolated and expanded to assess MSCs markers by flow cytometry, colony-forming ability, differentiation potential, RNA sequencing, and real-time polymerase chain reaction (PCR). RESULTS: Of the three cell types, the hSG-MSCs demonstrated the highest colony-forming ability. The number of alizarin red S positive colonies produced by the hSG-MSCs was higher than the hMG-MSCs but lower than the hPDL-MSCs. RNA sequencing revealed that the hSG-MSCs had a more similar gene expression profile to the hPDL-MSCs than the hMG-MSCs. Pathway enrichment analysis found no significant differences in the differentially expressed genes between the hSG-MSCs and hPDL-MSCs; however, there were significantly enriched pathways between the hPDL-MSCs and hMG-MSCs for the extracellular matrix (ECM) organization and ECM-receptor interaction pathways. CONCLUSION: This study demonstrated a close relationship between hSG-MSCs and hPDL-MSCs. hSG-MSCs could be a potential MSC source for periodontal tissue engineering.

Reduced ELANE and SLPI expression compromises dental pulp cell activity.

BACKGROUND: Patients with ELANE variants and severe congenital neutropenia (SCN) commonly develop oral complications. Whether they are caused only by low neutrophil count or the combination of neutropenia and aberrant dental cells is unknown. METHODS: Genetic variant was identified with exome sequencing. Dental pulp cells isolated from the SCN patient with an ELANE mutation were investigated for gene expression, enzyme activity, proliferation, colony formation, wound healing, apoptosis, ROS, attachment, spreading and response to lipopolysaccharide. RESULTS: ELANE cells had diminished expression of ELANE and SLPI and reduced neutrophil elastase activity. Moreover, ELANE cells exhibited impaired proliferation, colony forming, migration, attachment and spreading; and significantly increased ROS formation and apoptosis, corresponding with increased Cyclin D1 and MMP2 levels. The intrinsic levels of TGF-ß1 and TNF-α were significantly increased; however, IL-6, IL-8 and NF-kB1 were significantly decreased in ELANE cells compared with those in controls. After exposure to lipopolysaccharide, ELANE cells grew larger, progressed to more advanced cell spreading stages and showed significantly increased SLPI, TNF-α and NF-kB1 and tremendously increased IL-6 and IL-8 expression, compared with controls. CONCLUSION: This study, for the first time, suggests that in addition to neutropenia, the aberrant levels and functions of ELANE, SLPI and their downstream molecules in pulp cells play an important role in oral complications in SCN patients. In addition, pulp cells with diminished neutrophil elastase and SLPI are highly responsive to inflammation.

Human dental pulp stem cell responses to different dental pulp capping materials.

BACKGROUND: Direct pulp capping is a vital pulp therapy for a pin-point dental pulp exposure. Applying a pulp capping material leads to the formation of a dentin bridge and protects pulp vitality. The aim of this study was to compare the effects of four dental materials, DyCal®, ProRoot® MTA, Biodentine™, and TheraCal™ LC in vitro. METHODS: Human dental pulp stem cells (hDPs) were isolated and characterized. Extraction medium was prepared from the different pulp capping materials. The hDP cytotoxicity, proliferation, and migration were examined. The odonto/osteogenic differentiation was determined by alkaline phosphatase, Von Kossa, and alizarin red s staining. Osteogenic marker gene expression was evaluated using real-time polymerase chain reaction. RESULTS: ProRoot® MTA and Biodentine™ generated less cytotoxicity than DyCal® and TheraCal™ LC, which were highly toxic. The hDPs proliferated when cultured with the ProRoot® MTA and Biodentine™ extraction media. The ProRoot® MTA and Biodentine™ extraction medium induced greater cell attachment and spreading. Moreover, the hDPs cultured in the ProRoot® MTA or Biodentine™ extraction medium migrated in a similar manner to those in serum-free medium, while a marked reduction in cell migration was observed in the cells cultured in DyCal® and TheraCal™ LC extraction media. Improved mineralization was detected in hDPs maintained in ProRoot® MTA or Biodentine™ extraction medium compared with those in serum-free medium. CONCLUSION: This study demonstrates the favorable in vitro biocompatibility and bioactive properties of ProRoot® MTA and Biodentine™ on hDPs, suggesting their superior regenerative potential compared with DyCal® and TheraCal™.

Effects of lipopolysaccharide on proliferation, migration and osteogenic differentiation of apical papilla cells from early and late stage of root development.

The aim of this study was to investigate the effects of lipopolysaccharide on cell proliferation, migration and osteogenic differentiation of apical papilla cells from early and late stage of root development. After challenging with various lipopolysaccharide concentrations to apical papilla cells from both stages of root development for 168 h, cell proliferation and migration were investigated. Osteogenic differentiation was examined by Alizarin red staining, and gene expressions of bone/cementum or dentin-related genes were examined by polymerase chain reaction. Lipopolysaccharide did not affect cell proliferation and migration in both groups. Lipopolysaccharide at 1 and 5 µg mL-1 increased Alizarin red staining in apical papilla cells from early-stage but not the late-stage cells. Bone sialoprotein (bone/cementum marker) gene expression increased in both early and late stage of root development at 5 µg mL-1 . These results might explain bone/cementum generation in regenerative endodontic procedures.

Gene expression profiling of Jagged1-treated human periodontal ligament cells.

OBJECTIVE: Jagged1 regulates several biological functions in human periodontal ligament cells (hPDLs). The present study aimed to evaluate mRNA expression profiling of Jagged1-treated hPDLs using microarray technique. METHODS: Notch ligands, Jagged1, were indirectly immobilized on tissue culture surface. Subsequently, hPDLs were seeded on Jagged1 immobilized surface and maintained in growth medium for 48 hr. Total RNA was collected and processed. Gene expression profiling was examined using microarray technique. Real-time polymerase chain reaction and immunofluorescence staining were employed to determine mRNA and protein expression levels, respectively. Cell proliferation and colony-forming unit assay were performed. Cell cycle was evaluated using propidium iodide staining and flow cytometry analysis. RESULTS: The isolated cells demonstrated fibroblast-like morphology and exhibited the co-expression of CD44, CD90, and CD105 surface markers. After stimulated with Jagged1, the total of 411 genes was differentially expressed, consisting both coding and non-coding genes. For coding genes, 165 and 160 coding genes were upregulated and downregulated, respectively. Pathway analysis revealed that the upregulated genes were mainly involved in cellular interactions, signal transduction, and collagen formation and degradation while the downregulated genes were in the events and phases in cell cycle. Jagged1 significantly decreased cell proliferation, reduced colony-forming unit ability, and induced G0/G1 cell cycle arrest in hPDLs. CONCLUSION: Jagged1 regulates various biological pathways in hPDLs. This gene expression profiling could help to understand the mechanisms potentially involved in the Notch signaling regulation in periodontal homeostasis.

Jagged1 promotes mineralization in human bone-derived cells.

OBJECTIVES: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. METHODS: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. RESULTS: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. CONCLUSION: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

Cell wall mannan of Candida krusei mediates dendritic cell apoptosis and orchestrates Th17 polarization via TLR-2/MyD88-dependent pathway.

Dendritic cells (DCs) abundantly express diverse receptors to recognize mannans in the outer surface of Candida cell wall, and these interactions dictate the host immune responses that determine disease outcomes. C. krusei prevalence in candidiasis worldwide has increased since this pathogen has developed multidrug resistance. However, little is known how the immune system responds to C. krusei. Particularly, the molecular mechanisms of the interplay between C. krusei mannan and DCs remain to be elucidated. We investigated how C. krusei mannan affected DC responses in comparison to C. albicans, C. tropicalis and C. glabrata mannan. Our results showed that only C. krusei mannan induced massive cytokine responses in DCs, and led to apoptosis. Although C. krusei mannan-activated DCs underwent apoptosis, they were still capable of initiating Th17 response. C. krusei mannan-mediated DC apoptosis was obligated to the TLR2 and MyD88 pathway. These pathways also controlled Th1/Th17 switching possibly by virtue of the production of the polarizing cytokines IL-12 and IL-6 by the C. krusei mannan activated-DCs. Our study suggests that TLR2 and MyD88 pathway in DCs are dominant for C. krusei mannan recognition, which differs from the previous reports showing a crucial role of C-type lectin receptors in Candida mannan sensing.

Memory T cell subsets in healthy gingiva and periodontitis tissues.

BACKGROUND: In the gingival sulcus, effective and balanced innate and adaptive immune responses against subgingival plaque microbiome are crucial to maintain immune homeostasis. In this study, we investigated the memory T cell subsets in healthy gingiva and periodontitis tissues. METHODS: Anatomical localization of T cells (CD3+ , CD4+ , and CD8+ ) in healthy gingiva and periodontitis tissues were examined immunohistochemically. Subsets of memory T cells from isolated gingival cells were analyzed by flow cytometry using a cocktail of monoclonal antibodies (anti-CD69, anti-CD103, anti-CD45RA, anti-CCR7, anti-CD28, and anti-CD95). Intracellular cytokine staining of interleukin (IL)-17 and interferon (IFN)-γ expression on memory T cells in periodontitis tissues was also investigated. RESULTS: We found that healthy gingiva contains two memory T cell populations; a CD69- recirculating population and a CD69+ gingiva-resident memory T cell population. CD4+ T cells with transitional memory (TTM ) phenotype (CD45RA- CCR7- CD28+ CD95+ ) constitute the major subset within these two populations. A significant increase in the proportion of CD4+ CD69+ CD103- memory T cells was observed in periodontitis tissues compared with healthy gingiva. CD4+ memory T cells from periodontitis tissues produced either IL-17 or IFN-γ whereas CD8+ memory T cells produced only IFN-γ. CONCLUSIONS: Our findings suggest that recirculating and gingiva-resident memory T cells could represent an important part of the immune surveillance network in the connective tissue, maintaining periodontal homeostasis. Imbalance of subgingival bacterial communities could damage gingival barrier allowing bacterial antigens to get access to the deeper connective tissue where they activate memory T cells leading to deleterious inflammation; a hallmark of periodontitis.

The activation of B cells enhances DC-SIGN expression and promotes susceptibility of B cells to HPAI H5N1 infection.

The interplay between highly pathogenic avian influenza (HPAI) H5N1 virus and immune cells has been extensively studied for years, as host immune components are thought to play significant roles in promoting the systemic spread of the virus and responsible for cytokine storm. Previous studies suggested that the interaction of B cells and monocytes could promote HPAI H5N1 infection by enhancing avian influenza virus receptor expression. In this study, we further investigate the relationship between the HPAI H5N1 virus, activated B cells, and DC-SIGN expression. DC-SIGN has been described as an important factor for mediating various types of viral infection. Here, we first demonstrate that HPAI H5N1 infection could induce an activation of B cells, which was associated with DC-SIGN expression. Using CD40L and recombinant IL-4 for B cell stimulation, we determined that DC-SIGN expressed on activated B cells was able to enhance its susceptibility to HPAI H5N1 infection. Our findings uncover the interplay between this H5N1 virus and B cells and provide important information in understanding how the virus overcomes our immune system, contributing to its unusual immunopathogenesis.

Human Memory B Cells in Healthy Gingiva, Gingivitis, and Periodontitis.

The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19(+)CD27(+)CD38(-) memory B cells, the major B cell component in periodontitis was CD19(+)CD27(+)CD38(+)CD138(+)HLA-DR(low) plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell-activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis.

The presence of monocytes enhances the susceptibility of B cells to highly pathogenic avian influenza (HPAI) H5N1 virus possibly through the increased expression of α2,3 SA receptor.

The highly pathogenic avian influenza (HPAI) H5N1 virus causes severe systemic infection in avian and mammalian species, including humans by first targeting immune cells. This subsequently renders the innate and adaptive immune responses less active, thus allowing dissemination of the virus to systemic organs. To gain insight into the pathogenesis of H5N1, this study aims to determine the susceptibility of human PBMCs to the H5N1 virus and explore the factors which influence this susceptibility. We found that PBMCs were a target of H5N1 infection, and that monocytes and B cells were populations which were clearly the most susceptible. Analysis of PBMC subpopulations showed that isolated monocytes and monocytes residing in whole PBMCs had comparable percentages of infection (28.97 ± 5.54% vs 22.23 ± 5.14%). In contrast, isolated B cells were infected to a much lower degree than B cells residing in a mixture of whole PBMCs (0.88 ± 0.34% vs 34.87 ± 4.63%). Different susceptibility levels of B cells for these tested conditions spurred us to explore the B cell-H5N1 interaction mechanisms. Here, we first demonstrated that monocytes play a crucial role in the enhancement of B cell susceptibility to H5N1 infection. Although the actual mechanism by which this enhancement occurs remains in question, α2,3-linked sialic acid (SA), known for influenza virus receptors, could be a responsible factor for the greater susceptibility of B cells, as it was highly expressed on the surface of B cells upon H5N1 infection of B cell/monocyte co-cultures. Our findings reveal some of the factors involved with the permissiveness of human immune cells to H5N1 virus and provide a better understanding of the tropism of H5N1 in immune cells.

Differential inflammasome activation by Porphyromonas gingivalis and cholesterol crystals in human macrophages and coronary artery endothelial cells.

OBJECTIVE: Observational evidence suggests association between periodontitis and atherosclerotic vascular disease (ASVD), however the cause-effect remains unclear. In this study, we investigated the mechanistic link of the two diseases by measuring production of interleukin (IL)-1ß, a potent inflammatory cytokine, induced via inflammasome activation by a key periodontal pathogen--Porphyromonas gingivalis LPS and cholesterol crystals (CC). METHODS: An in vitro model of primary human monocyte-derived macrophages (M1 and M2 macrophages) and coronary artery endothelial cells (HCAEC) was employed as a source of inflammasome product-IL-1ß. Both cell types are essential in initial inflammatory process of ASVD. As inflammasome activation requires 2 signals, P. gingivalis LPS was used as a signal1 and CC as a signal2. RESULTS: We found markedly release of IL-1ß from P. gingivalis LPS-primed M1 and M2 macrophages treated with CC. Unlike macrophages, HCAEC showed no release of IL-1ß in response to P. gingivalis LPS priming and subsequent treatment with either CC or extracellular danger molecule adenosine-5'-triphosphate (signal2). However, HCAEC, which were primed with pro-inflammatory cytokine TNF-α (signal1) and treated with adenosine-5'-triphosphate, consistently secreted minimal IL-1ß. The amount of IL-1ß released from activated HCAEC was much lower than that from M1 or M2 macrophages. CONCLUSIONS: P. gingivalis LPS and CC induced a differential activation of the inflammasome between human macrophages and HCAEC. The mechanistic role of periodontal infection in inflammasome activation as a cause of ASVD requires further investigation.

MxA expression induced by α-defensin in healthy human periodontal tissue.

Although periodontal tissue is continually challenged by microbial plaque, it is generally maintained in a healthy state. To understand the basis for this, we investigated innate antiviral immunity in human periodontal tissue. The expression of mRNA encoding different antiviral proteins, myxovirus resistance A (MxA), protein kinase R (PKR), oligoadenylate synthetase (OAS), and secretory leukocyte protease inhibitor (SLPI) were detected in both healthy tissue and that with periodontitis. Immunostaining data consistently showed higher MxA protein expression in the epithelial layer of healthy gingiva as compared with tissue with periodontitis. Human MxA is thought to be induced by type I and III interferons (IFNs) but neither cytokine type was detected in healthy periodontal tissues. Treatment in vitro of primary human gingival epithelial cells (HGECs) with α-defensins, but not with the antimicrobial peptides ß-defensins or LL-37, led to MxA protein expression. α-defensin was also detected in healthy periodontal tissue. In addition, MxA in α-defensin-treated HGECs was associated with protection against avian influenza H5N1 infection and silencing of the MxA gene using MxA-targeted-siRNA abolished this antiviral activity. To our knowledge, this is the first study to uncover a novel pathway of human MxA induction, which is initiated by an endogenous antimicrobial peptide, namely α-defensin. This pathway may play an important role in the first line of antiviral defense in periodontal tissue.

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10.

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-alpha mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-alpha led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-alpha and IFN-alpha also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-alpha and TNF-alpha may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.

Indoleamine 2,3-dioxygenase expression and regulation in chronic periodontitis.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-oxidizing enzyme with immunosuppressive characteristics. Its expression and regulation in periodontal tissues are unknown. The aim of this study was to determine IDO expression in healthy gingiva and chronic periodontitis lesions. In addition, the effect of inflammatory cytokines and bacterial products on the expression and activity of DOI in human gingival fibroblasts (HGFs) was assessed. METHODS: Human gingival tissue samples were obtained from patients who underwent periodontal surgery. IDO expression in healthy gingiva and periodontitis lesions was determined by immunohistochemistry. HGF cells were treated with interferon-gamma (IFN-gamma), interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides from Porphyromonas gingivalis (PgLPS). IDO mRNA expression was determined by reverse transcription-polymerase chain reaction. The IDO enzymatic activity was determined by measuring the kynurenine level using a colorimetric method. RESULTS: In gingival tissues, IDO expression was detected in epithelial cells, fibroblasts, endothelial cells, and inflammatory mononuclear cells. IDO expression was higher in periodontitis lesions than in healthy gingiva. HGFs did not constitutively express IDO. IFN-gamma strongly induced IDO expression and activity in HGFs, in a dose-dependent manner. IL-1beta, TNF-alpha, and PgLPS were also able to induce IDO expression in HGF cells. IFN-gamma in combination with IL-1beta, TNF-alpha, or PgLPS showed enhanced IDO expression. CONCLUSIONS: IDO was expressed in human gingiva, and the expression was upregulated in chronic periodontitis. The increased IDO expression in periodontitis lesions may be due, in part, to the activation of HGFs by inflammatory cytokines and bacterial products.

IL-8 and IDO expression by human gingival fibroblasts via TLRs.

Human gingival fibroblasts (HGFs), a predominant cell type in tooth-supporting structure, are presently recognized for their active role in the innate immune response. They produce a variety of inflammatory cytokines in response to microbial components such as LPS from the key periodontal pathogen, Porphyromonas gingivalis. In this study, we demonstrated that HGFs expressed mRNA of TLRs 1, 2, 3, 4, 5, 6, and 9, but not TLRs 7, 8, and 10. Stimulation of HGFs with highly purified TLR2 ligand (P. gingivalis LPS), TLR3 ligand (poly(I:C)), TLR4 ligand (Escherichia coli LPS), and TLR5 ligand (Salmonella typhimurium flagellin) led to expression of IL-8 and IDO. A potent TLR 9 ligand, CpG oligodeoxynucleotide 2006 had no effect, although HGFs showed a detectable TLR9 mRNA expression. No significant enhancement on IL-8 or IDO expression was observed when HGFs were stimulated with various combinations of TLR ligands. Surprisingly, the TLR9 ligand CpG oligodeoxynucleotide 2006 was able to specifically inhibit poly(I:C)-induced IL-8 and IDO expression. TNF-alpha enhanced TLR ligand-induced IL-8 production in HGFs, whereas IFN-gamma enhanced TLR ligand-induced IDO expression. HGF production of IDO in response to P. gingivalis LPS, IFN-gamma, or the two in combination inhibited T cell proliferation in MLRs. The observed T cell inhibition could be reversed by addition of either 1-methyl-dl-tryptophan or l-tryptophan. Our results suggest an important role of HGFs not only in orchestrating the innate immune response, but also in dampening potentially harmful hyperactive inflammation in periodontal tissue.

The effects of Porphyromonas gingivalis LPS and Actinobacillus actinomycetemcomitans LPS on human dendritic cells in vitro, and in a mouse model in vivo.

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.