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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 (T TM ) 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., (©2018 The Authors. Journal of Periodontology Published by Wiley Periodicals, Inc. on behalf of American Academy of Periodontology.)

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Objective: Aggregatibacter actinomycetemcomitans is known to be a major cause of localized aggressive periodontitis. Previous research has suggested that A. actinomycetemcomitans can damage many types of host cells. There is evidence for the ability of this organism to invade endothelial and epithelial cells, but information pertaining to its potential for invading gingival fibroblasts is very limited. Internalization of bacteria is not only responsible for damaging host tissue but also a means to evade the host immune response. It was hypothesized that A. actinomycetemcomitans can invade and reside in human gingival fibroblasts (HGF)., Methods: Primary cultures of HGF were infected with A. actinomycetemcomitans at a ratio of 1:100. Bacterial internalization was determined by an antibiotic protection assay. Bacterial-fibroblast interaction was examined using phase-contrast, scanning and transmission electron microscopy., Results: It was demonstrated that A. actinomycetemcomitans was internalized into HGF at an efficiency of 0.084%. Transmission electron microscopic study showed the presence of A. actinomycetemcomitans in the cytoplasm of HGF without the surrounding membrane. Scanning electron micrographs revealed the sloughing of HGF surfaces on which A. actinomycetemcomitans adhered. Rounded cells, attachment loss and damaged cells were also observed., Conclusions: It is concluded that the attachment and invasion of A. actinomycetemcomitans into human gingival fibroblasts play a role in periodontal tissue damage and may also be a means of immune evasion., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Objective: Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs., Material and Methods: Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay., Results: The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment., Conclusion: Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

Interleukin (IL)-17 is present in inflammatory periodontal lesions, thus suggesting a role in mediating inflammation. We tested the hypothesis that IL-17, especially when combined with interferon (IFN)-gamma, may modulate the responses of human gingival fibroblasts (HGFs). IL-17 induced IL-8 and minimal intercellular adhesion molecule (ICAM)-1 expression. It had no effect on expression of HLA-DR, CD40, or the immune-suppressive enzyme indoleamine 2,3-dioxygenase (IDO). The effects of IL-17 on HGFs were compared with those of IFN-gamma. Unlike IL-17, IFN-gamma augmented the expression of HLA-DR, ICAM-1, and IDO, but not IL-8. Thus, IL-17 and IFN-gamma induce different HGF responses when administered separately. Interestingly, when IL-17 and IFN-gamma were combined, marked enhancement of ICAM-1, IL-8, and IDO expression by HGFs was observed. These findings suggest that IL-17, especially when combined with IFN-gamma, could play an important role in immune modulation through stimulation of HGFs in periodontal disease.

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.

Objectives and Background: It is well documented that in periodontitis lesions, most infiltrated gingival T cells are antigen-specific memory T cells. These cells play an important role as regulators and effector cells in the pathogenesis of periodontitis. In this study, we used dendritic cells (DCs) as antigen-presenting cells to generate human gingival T cell lines and clones specific for Porphyromonas gingivalis from periodontitis patients., Methods: Autologous DCs were derived from the patients' adherent monocytes using granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Lymphocytes were isolated from gingival biopsies using collagenase enzyme digestion and the number was increased by subsequent culturing in IL-2-containing medium. T cells were then negatively sorted using flow cytometry, cocultured with P. gingivalis-pulsed DCs and subsequently expanded in the culture medium containing IL-2. T cells were kept viable and active by periodic exposure to antigen-pulsed DCs. The specificity of the T cell lines was tested against four plaque bacteria: P. gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia and Actinomyces viscosus. The established T cell lines were then cloned. Three P. gingivalis-specific T cell lines and 12 gingival T cell clones were generated. They all showed good specificity against P. gingivalis but not to other plaque bacteria., Results: All T cell clones were positive for CD4 and the majority of them produced interferon gamma, but a minimal or negligible amount of IL-5., Conclusions: The data obtained clearly showed that monocyte-derived DCs could be used as powerful antigen-presenting cells to generate antigen-specific T cells from periodontitis tissues.

Periodontitis is an inflammatory bone disease caused by Gram-negative anaerobic bacteria, but the precise mechanism of bone destruction remains unknown. Activated T lymphocytes secrete receptor activator of NF-kappaB ligand (RANKL) and support the differentiation of monocytes into mature osteoclasts. The purpose of this study was to examine the expression of RANKL and its inhibitor, osteoprotegerin (OPG), in inflamed gingival tissue and to clarify the role of human gingival fibroblasts (HGFs) in osteoclastogenesis regulated by RANKL. HGFs and gingival mononuclear cells (GMCs) were obtained from chronic periodontitis patients during routine periodontal surgery. Expression of OPG and RANKL mRNA in gingival tissue and HGFs was examined with RT-PCR. OPG production was measured using ELISA. Expression of RANKL, CD4, CD8 and CD69 on GMCs was determined by flow-cytometry using RANK-Fc fusion protein and the respective monoclonal antibodies. Osteoclastogenesis by RANKL was assayed by counting the number of tartarate-resistant acid phosphatase (TRAP)-positive cells after culturing human peripheral blood monocytes with recombinant human RANKL and macrophage-colony stimulating factor (M-CSF) for 10 days. OPG and RANKL mRNA were expressed in 80% (16/20) and 25% (5/20) of periodontitis lesions, respectively. OPG, but not RANKL, mRNA was expressed within HGFs. OPG mRNA expression and production by HGFs was augmented by LPS stimulation. All GMC samples expressed CD69, and two of five GMC samples expressed RANKL. The culture supernatant of LPS-stimulated gingival fibroblasts significantly reduced the number of TRAP positive cells generated by culturing monocytes with RANKL and M-CSF. The present study suggests that LPS-stimulated HGFs inhibit monocyte differentiation into osteoclasts through the production of OPG.