Primary Human Gingival Epithelial Cells Research Articles

Antagonistic effect of Candida albicans and IFNγ on E-cadherin expression and production by human primary gingival epithelial cells

Caused mainly by Candida albicans, oropharyngeal candidiasis is the most common oral complication associated with HIV disease worldwide. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral mucosa is the first line of defense in the form of a physical barrier against C. albicans invasion, and since epithelial cells are involved in anti-Candida innate immunity through different cytokines, we wanted to determine whether C. albicans alters E-cadherin expression and production, and whether interferon-γ (INFγ), a TH1 cytokine, is involved in the anti-Candida defense. Using primary human gingival epithelial cells, we demonstrated that C. albicans significantly decreased E-cadherin mRNA expression and protein production. This effect was basically obtained at later infective periods (24 and 48h). Interestingly, when IFNγ was added to C. albicans infected epithelial cell cultures, it prevented the side effect of C. albicans on E-cadherin mRNA expression and protein production and deposition. All together, these results suggested concomitant interactions between oral epithelial cells and IFNγ against C. albicans infection.

Identification by DNA microarray of genes involved in Candida albicans‐treated gingival epithelial cells

Oral epithelial cells significantly influence host inflammatory responses against Candida albicans in oropharyngeal candidiasis. Pro-inflammatory cytokines function as an early innate immune system mediator during C. albicans infection in oral epithelial cells. We sought to elucidate the pattern of the molecular mechanisms governing the human gingival epithelial cells (HGECs) to C. albicans infection likely involve multiple converging signal transduction pathways. Primary HGECs were cultured with C. albicans ATCC90029. Total RNA was extracted after 8 h of infection and monitored mRNA levels using Affymetrix GeneChip (Human Genome U133 plus 2.0 Array, 48 000 genes). GeneChip data was analyzed by GeneSpring software and Ingenuity Pathway Analysis system. Reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR and immunohistochemistry were used to investigate gene expression changes. The differentially expressed genes represented functions as diverse as immune response and inflammatory disease. IL-8, ICAM-1 and Cox-2 showed a greater than two fold change in expression relative to those in control cells. Altered mRNA levels in GeneChip analysis were confirmed by RT-PCR and real-time RT-PCR. Stronger immunoreactivity against ICAM-1 and Cox-2 was also observed in the infection with C. albicans in rat gingival epithelium. We have identified differential gene expression up-regulated or down-regulated with the up-regulation of IL-8 in C. albicans-treated cells. These findings indicate that the molecular mechanisms underlying the IL-8 response of HGECs to C. albicans infection likely involve multiple converging signal transduction pathways.

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 β-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.

J022033 培養組織の球圧子押込試験による柔さ計測に関する研究

In this study, in order to measure the softness of cultured tissue, ball indentation tests were performed. As first step, the principle of ball indentation test was applied to the thin silicone rubber sheet (t=0.1mm), which achieved results confirm the validation of the measurement result. Next step, Young's modulus of monolayer cultured nerve cell (PCI2) was measured, and the validation of the measured results was confirmed by comparing with previous studies. The Young's modulus of the cell body is 36.5kPa, and distal, middle and proximal of neuritis are 50.2kPa, 21.8kPa and 51.5kPa, respectively. In addition, Young's modulus of three-dimensional gel-culture system consisting of primary human gingival fibroblasts (GF) and gingival epithelial (GE) cells in collagen gels were measured. The Young's modulus of whole gel and gingival epithelial cells are 3.46kPa and 81.9kPa, respectively. The results were shown the possibility of measuring the Young's modulus of each layer of cultured tissue in the case of laminate structure.

TGF-β Signaling in Gingival Fibroblast-Epithelial Interaction

The underlying mechanism and the therapeutic regimen for the transition of reversible gingivitis to irreversible periodontitis are unclear. Since transforming growth factor (TGF)-β has been implicated in differentially regulated gene expression in gingival fibroblasts, we hypothesized that TGF-β signaling is activated in periodontitis-affected gingiva, along with enhanced collagen degradation, that is reversed by TGF-β inhibition. A novel three-dimensional (3D) gel-culture system consisting of primary human gingival fibroblasts (GF) and gingival epithelial (GE) cells in collagen gels was applied. GF populations from patients with severe periodontitis degraded collagen gels, which was reduced by TGF-β-receptor kinase inhibition. Up-regulation of TGF-β-responsive genes was evident in GF/GE co-cultures. Furthermore, the TGF-β downstream transducer Smad3C was highly phosphorylated in periodontitis-affected gingiva and 3D cultures. These results imply that TGF-β signaling is involved in fibroblast-epithelial cell interaction in periodontitis, and suggest that the 3D culture system is a useful in vitro model for therapeutic drug screening for periodontitis.

Epithelial to Mesenchymal Transition in Gingival Overgrowth

Epithelial to mesenchymal transition (EMT) occurs normally in development. In pathology, EMT drives cancer and fibrosis. Medication with phenytoin, nifedipine, and cyclosporine-A often causes gingival overgrowth. Based partly on the histopathology of gingival overgrowth, the present study investigates the hypothesis that EMT could contribute to its development. We found that phenytoin-induced human gingival overgrowth tissues, the most fibrotic drug-induced variety, contain diminished epithelial E-cadherin expression, whereas fibroblast-specific protein-1 (FSP-1) and alphavbeta6 integrin levels are up-regulated. In connective tissue stroma, fibronectin and alternatively spliced fibronectin extra type III domain A (FN-ED-A) levels are increased in overgrowth lesions. Transforming growth factor (TGF)-beta1 treatment of primary human gingival epithelial cells cultured in transwell plates resulted in inhibited barrier function as determined by reduced electrical resistance, paracellular permeability assays, and cell surface E-cadherin expression. Moreover, TGF-beta1 altered the expression of other markers of EMT determined at the mRNA and protein levels: E-cadherin decreased, whereas SLUG, fibronectin, matrix metalloproteinase (MMP)2, MMP9, and MMP13 increased. Nifedipine- and cyclosporine A-induced gingival overgrowth tissues similarly contain diminished E-cadherin and elevated levels of FSP-1 and fibronectin, but normal levels of alphavbeta6 integrin. In summary, data in vitro support that human gingival epithelial cells undergo functional and gene expression changes consistent with EMT in response to TGF-beta1, and in vivo studies show that important EMT markers occur in clinical gingival overgrowth tissues. These findings support the hypothesis that EMT likely occurs in drug-induced gingival overgrowth.

In vitromodeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells

BackgroundMicrobial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.ResultsWe describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.ConclusionWe developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism

BackgroundThe oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear.ResultsWe demonstrate that primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main P. gingivalis proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live P. gingivalis can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism.ConclusionThe present study provides evidence that live, but not heat-killed, P. gingivalis can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in P. gingivalis elicited apoptosis.

Sphingosine 1-Phosphate 1 and TLR4 Mediate IFN-β Expression in Human Gingival Epithelial Cells

IFN-beta production is a critical step in human innate immune responses and is primarily controlled at the transcription level by highly ordered mechanisms. IFN-beta can be induced by pattern-recognition receptors such as the TLR4. S1P1 is a G protein-coupled receptor, which has a high affinity for sphingosine 1-phosphate (S1P). Although many of the receptors and signaling pathways leading to the expression of IFN-beta have been identified and characterized, it is still unclear how IFN-beta is regulated in primary human gingival epithelial cells (HGECs). In this study, we demonstrate that S1P1 and TLR4, acting in unison, play an important role in IFN-beta expression at the protein and mRNA level in HGECs. We demonstrate that the expression of both IFN-beta and IFN-inducible protein-10 (CXCL-10) is significantly up-regulated by LPS and S1P or LPS and a specific S1P1 agonist. This enhanced innate immune response is attenuated in HGECs by small interfering RNA knockdown of either TLR4 or S1P1. Moreover, we show that triggering of TLR4 results in the increased expression of S1P1 receptors. Furthermore, we found that IFN-regulatory factor 3 activation was maximized by LPS and S1P through PI3K. Our data show that triggering TLR4 increases S1P1, such that both TLR4 and S1P1 acting through PI3K enhancement of IFN-regulatory factor 3 activation increase IFN-beta expression in epithelial cells. The functional association between TLR4 and the S1P1 receptor demonstrates a novel mechanism in the regulation of IFN-beta and CXCL-10 in human primary gingival epithelial cells.

Differential Activation of Human Gingival Epithelial Cells and Monocytes by Porphyromonas gingivalis Fimbriae

Humans develop periodontitis in response to challenge by microbial dental plaque. Inflammation begins after perturbation of gingival epithelial cells by subgingival bacteria interacting through pattern-recognition receptors, including the Toll-like receptors (TLR). Porphyromonas gingivalis is a major periodontopathogen that interacts with epithelial cells through its cell surface fimbriae (FimA), leading to colonization and/or invasion. Previous work by our group has established membrane CD14 as an essential coreceptor for TLR2-mediated activation of transfected cell lines by P. gingivalis FimA. We have shown that gingival epithelial cells express TLR2 but not CD14 on their cell surfaces. We thus speculated that P. gingivalis FimA does not readily activate epithelial innate immune responses but rather functions to promote P. gingivalis colonization in the absence of a vigorous FimA-induced response. This hypothesis was verified by the findings that primary human gingival epithelial cells responded poorly to FimA in terms of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha responses, in stark contrast to the marked response to other TLR2 agonists (Pam3Cys, FSL-1) that are not strictly dependent on CD14. On the other hand, CD14-expressing human primary monocytes responded with high levels of the same cytokines to both FimA and the control TLR2 agonists. The gingival epithelial cells failed to respond to FimA even in the presence of exogenously added soluble CD14. These data indicate that the gingival epithelial cell hyporesponsiveness to FimA is attributable to the lack of membrane-expressed but not soluble CD14. In conclusion, P. gingivalis FimA differentially activates human monocytes and epithelial cells, perhaps reflecting different tactics used by P. gingivalis when interacting with different host cell types or a host strategy to limit inflammation.

In vitro mechanisms of interleukin-8-mediated responses of human gingival epithelial cells to Candida albicans infection

Oral epithelial cells significantly influence host inflammatory responses against Candida albicans in oropharyngeal candidiasis. We sought to elucidate the pattern of interleukin-8 (IL-8) expression by oral epithelial cells, which may function as an early innate immune system mediator during C. albicans infection. Primary human gingival epithelial cells (HGECs) were co-cultured with either viable or heat-killed C. albicans or fungal-derived substances, such as fungal secretion, fungal extracted proteins, and α-mannan. In vitro cell injury due to viable C. albicans was detectable by an adenosine triphosphate-based assay after 12 h of infection. Prior to the detection of cell injury, HGECs clearly increased production of interleukin-1 alpha (IL-1 α) and IL-8 in response to C. albicans infection, as determined by enzyme-linked immunosorbent assay and real-time reverse transcription PCR. High concentrations of a suspension of heat-killed yeast and all fungal-derived substances examined also stimulated IL-8 production by HGECs. Incubation with neutralizing anti-IL-1 α or anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) significantly inhibited C. albicans-induced IL-8 production. Use of mAbs against both IL-1 α and ICAM-1 produced a more significant combined inhibitory effect on the IL-8 production than either mAb alone. These findings indicate that HGECs synthesize increased levels of IL-1 α and IL-8 in response to viable C. albicans before cell injury is manifested. Fungal cell-wall components, α-mannan, and fungal protein extracts are all sufficient to increase IL-8 production. The molecular mechanisms governing the IL-8 response of HGECs to C. albicans infection likely involve multiple converging signal transduction pathways, including those mediated by IL-1 α and ICAM-1 activation.

Porphyromonas gingivalis enhances FasL expression via up-regulation of NFκB-mediated gene transcription and induces apoptotic cell death in human gingival epithelial cells

The interaction between epithelial cells and micro-organisms is often a crucial initiating event in infectious diseases. Infection with Porphyromonas gingivalis, a Gram-negative anaerobe, is strongly associated with severe periodontal disease. This bacterium possesses an array of virulence factors, some of which can induce apoptosis. The tumour necrosis factor (TNF) receptor family is involved in the regulation of cellular homeostasis, cell surface molecules involved in phagocytosis, Fas ligand (L) expression and activation of the caspase cascade resulting in DNA fragmentation and cell blebbing. The current study examined the role of nuclear factor-kappaB (NFkappaB) in FasL-mediated apoptotic cell death in primary human gingival epithelial cells (HGEC) induced by heat-killed P. gingivalis, probably through TLR signalling pathways. A marked up-regulation of TLR2 and Fas-FasL was detected in HGEC stimulated with P. gingivalis. Activation of NFkappaB by P. gingivalis in HGEC was demonstrated by an NFkappaB promoter luciferase assay as well as by phosphorylation of p65 as detected by Western blotting. Activation of cleaved caspase-3 and caspase-8 resulted in apoptotic cell death of HGEC. The survival proteins c-IAP-1/c-IAP-2 were decreased in HGEC exposed to P. gingivalis. HGEC apoptosis induced by P. gingivalis was inhibited by an anti-human FasL monoclonal antibody. Blockade of NFkappaB by helenalin resulted in down-regulation of FasL whereas a caspase-8 inhibitor did not decrease FasL. Taken together, these studies show that P. gingivalis can induce epithelial cell apoptosis through Fas-FasL up-regulation and activation of caspase-3 and caspase-8.

Capacity of human β-defensin expression in gene-transduced and cytokine-induced cells

The purpose of this study was to determine the capacity of cells transduced with human β-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9–99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFα. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.

Culturing primary human gingival epithelial cells: comparison of two isolation techniques

Background: Cultured epithelial cells offer many potential clinical applications. There have generally been two techniques that have been used to cultivate oral keratinocytes, which include the direct explant technique and the enzymatic method. Little work has been done comparing these two techniques and their capacity to isolate and cultivate oral keratinocytes. Objectives: The objectives of this study were to (1) investigate the difference in the percentage of keratinocyte isolation between the direct explant technique and the enzymatic method of human gingival epithelial cell culture and (2) to examine the effect of age and sex of the subjects providing the tissue samples on (a) the success in cultivation and (b) the growth patterns of gingival keratinocytes. Material and Methods: Gingival tissue was obtained from healthy human subjects and was used for keratinocyte isolation using the direct explant technique or the enzymatic method. Epithelial cell cultures from each of the two culture techniques were selected randomly for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted. Results: The success rate for cultivation from the direct explant technique was higher (82%) than in the enzymatic method (57.9%). The success rate of both methods was not significantly associated with either age or sex of the subjects providing the tissue. From flow cytometry, the average percentage of cells that was positive to anti-pan cytokeratin was nearly the same for both methods at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentages of cells that were positive to anti-pan cytokeratin only. Conclusion: Both the direct explant technique and the enzymatic method can be used for isolating and culturing human oral keratinocytes. The direct explant technique appeared to be more successful in culturing human oral keratinocytes than the enzymatic method, although there were limitations found with both methods. The age and sex of the subjects providing the gingival samples did not appear to be a factor influencing the success rate in culturing the keratinocytes. However, contamination by oral microbiological flora from the gingival tissue samples remained an ever present problem. Further studies are needed in the investigation of clinical applications of these two epithelial cell isolation methods.