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.

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.

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.

Infection of human gingival fibroblasts with Aggregatibacter actinomycetemcomitans: An in vitro study.

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.

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.