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Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)–6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut- Stat3 mice). Compared with controls, mut- Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut- Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss. [ABSTRACT FROM AUTHOR]

Background: Matrix metalloproteinase (MMP)-8 is a central mediator in chronic periodontitis. MMP-8 can be activated by the cooperative action of other MMPs such as MMP-14, reactive oxygen species, and microbial proteases. The aim of this study is to associate the levels, molecular forms, isoenzyme distribution, and degree of activation of MMP-8 and -14, myeloperoxidase (MPO), and tissue inhibitor of MMP (TIMP)-1 in gingival crevicular fluid (GCF) from patients with progressive periodontitis at baseline and after periodontal therapy.Methods: In this longitudinal study, GCF samples from active (n = 25) and inactive (n = 25) sites of subjects with periodontitis were screened at baseline for GCF levels of MMP-8 by immunofluorometric assay, of MMP-14 by specific activity assay, and of MPO and TIMP-1 by enzyme-linked immunosorbent assay. MMP-8 and MPO were also measured after periodontal treatment. Molecular forms were determined by immuno-Western blot analyses and subjected to densitometric scanning and statistical analyses.Results: High MMP-8 and MPO levels and a strong MPO/MMP-8 positive correlation were found in active and inactive sites at baseline. After treatment, decreases in MPO and MMP-8 were seen, except for active sites in which MMP-8 differences were not significant (P <0.05).Conclusions: We present initial data that show that GCF levels and associations between MPO and MMP-8 are related to progression episodes and treatment responses in patients with chronic periodontitis. Our results suggest an interaction between the MPO oxidative pathway and MMP-8 activation, and this cascade might be useful as a potential biomarker for treatment outcomes. [ABSTRACT FROM AUTHOR]

Aim: T regulatory (Treg) cells have been detected in periodontitis lesions, and forkhead box P3 (Foxp3) expression has been negatively correlated to receptor activator of nuclear factor- [kappa] B ligand (RANKL). The aim of this study was to correlate T-helper type 1 (Th1), Th2, Th17 and Treg transcription factor expressions, in gingival tissues from patients undergoing active periodontal tissue destruction, with bone loss-associated cytokines. Materials and Methods: In 10 chronic periodontitis patients undergoing disease progression, the mRNA expressions of T-bet, GATA-3, Foxp3, RORC2, interleukin (IL)-1 [beta], IL-10, IL-17, RANKL, interferon (IFN)- [gamma] and transforming growth factor (TGF)- [beta]1 were quantified using real-time reverse transcription-polymerase chain reaction. The levels of these markers were compared between active and inactive periodontal lesions. Results: In active periodontal lesions, Foxp3, T-bet, RANKL, IL-17, IL-1 [beta] and IFN- [gamma] were significantly over-expressed compared with inactive lesions. The expression of IFN- [gamma] was the highest within the active periodontal lesions, similar to that of TGF- [beta]1 within the inactive ones. There was a positive correlation between RANKL and IL-17. Additionally, RANKL and IL-17 were positively correlated with RORC2, but no correlation was detected with Foxp3. Conclusions: These results lead us to speculate that Foxp3+ cells that do not have a regulatory function might have a role in the pathogenesis of active periodontal lesions by down-regulating TGF- [beta]1 and IL-10 synthesis that lead to the over-expression of Th17-associated cytokines RANKL and IL-17. [ABSTRACT FROM AUTHOR]

BACKGROUND AND AIMS: Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. MATERIAL AND METHOD: Fifty-six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test and chi(2) tests were used. RESULTS: Higher RANK-L, IL-1beta and MMP-13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION: The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth. [ABSTRACT FROM AUTHOR]

At mucosal surfaces, epithelial cells provide a structural barrier and an immune defense system. However, dysregulated epithelial responses can contribute to disease states. Here, we demonstrated that epithelial cell-intrinsic production of interleukin-23 (IL-23) triggers an inflammatory loop in the prevalent oral disease periodontitis. Epithelial IL-23 expression localized to areas proximal to the disease-associated microbiome and was evident in experimental models and patients with common and genetic forms of disease. Mechanistically, flagellated microbial species of the periodontitis microbiome triggered epithelial IL-23 induction in a TLR5 receptor-dependent manner. Therefore, unlike other Th17-driven diseases, non-hematopoietic-cell-derived IL-23 served as an initiator of pathogenic inflammation in periodontitis. Beyond periodontitis, analysis of publicly available datasets revealed the expression of epithelial IL-23 in settings of infection, malignancy, and autoimmunity, suggesting a broader role for epithelial-intrinsic IL-23 in human disease. Collectively, this work highlights an important role for the barrier epithelium in the induction of IL-23-mediated inflammation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: To characterize the distribution of macrophages, neutrophils, NK cells, and blood vessels in peri-implantitis compared to healthy aged gingiva samples., Materials and Methods: This observational study included eight gingival samples from peri-implantitis and eight from periodontally healthy individuals. By immunofluorescence were identified neutrophils, NK cells, macrophages, and their pro-inflammatory or pro-healing phenotypes, and blood vessels. Two ROIs were designated as zone 1, connective tissue closest to the epithelium and zone 2, connective tissue over 200 microns from the rete ridges. Immune cells and vascular structures were quantified and characterized according to their distribution in both zones., Results: Two peri-implantitis zones were characterized by unique macrophage phenotypes and blood vessel architecture. Blood vessels were larger in zone 2 in peri-implantitis. A greater number of NK cells and macrophages were found in peri-implantitis compared to healthy aged samples. A higher presence of pro-inflammatory macrophages was found in zone 1 compared to zone 2. A similar proportion of pro-inflammatory and pro-healing macrophages were found in zone 2., Conclusion: A specific distribution for pro-inflammatory macrophages and vascular architecture is observed in peri-implantitis. TNF-α colocalizes with macrophages in the connective tissue near rete ridges. NK cells are more abundant in peri-implantitis than in healthy samples., (© 2024 Wiley Periodicals LLC.)

Periodontitis is a chronic inflammatory disease associated with the presence of dysbiotic microbial communities. Several studies interrogating periodontitis pathogenesis have utilized the murine ligature-induced periodontitis (LIP) model and have further examined the ligature-associated microbiome relying on 16S rRNA-based sequencing techniques. However, it is often very challenging to compare microbial profiles across studies due to important differences in bioinformatic processing and databases used for taxonomic assignment. Thus, our study aim was to reanalyze microbiome sequencing datasets from studies utilizing the LIP model through a standardized bioinformatic analysis pipeline, generating a comprehensive overview of microbial dysbiosis during experimental periodontitis.We conducted a reanalysis of 16S rDNA gene sequencing datasets from nine published studies utilizing the LIP model. Reads were grouped according to the hypervariable region of the 16S rDNA gene amplified (V1-V3 and V4), preprocessed, binned into operational taxonomic units and classified utilizing relevant databases. Alpha- and beta-diversity analyses were conducted, along with relative abundance profiling of microbial communities. Our findings revealed similar microbial richness and diversity across studies and determined shifts in microbial community structure determined by periodontitis induction and study of origin. Clear variations in the relative abundance of bacterial taxa were observed starting on day 5 after ligation and onward, consistent with a distinct microbial composition during health and experimental periodontitis. We also uncovered differentially represented bacterial taxa across studies, dominating periodontal health and LIP-associated communities. Collectively, this reanalysis provides a unified overview of microbial dysbiosis during the LIP model, providing new insights that aim to inform further studies dedicated to unraveling oral host-microbial interactions., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the α M β 2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG , encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Puel and Casanova and Kisand et al . challenge our conclusions that interferonopathy and not IL-17/IL-22 autoantibodies promote candidiasis in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy. We acknowledge that conclusive evidence for causation is difficult to obtain in complex human diseases. However, our studies clearly document interferonopathy driving mucosal candidiasis with intact IL-17/IL-22 responses in Aire -deficient mice, with strong corroborative evidence in patients.

The oral mucosa remains an understudied barrier tissue. This is a site of rich exposure to antigens and commensals, and a tissue susceptible to one of the most prevalent human inflammatory diseases, periodontitis. To aid in understanding tissue-specific pathophysiology, we compile a single-cell transcriptome atlas of human oral mucosa in healthy individuals and patients with periodontitis. We uncover the complex cellular landscape of oral mucosal tissues and identify epithelial and stromal cell populations with inflammatory signatures that promote antimicrobial defenses and neutrophil recruitment. Our findings link exaggerated stromal cell responsiveness with enhanced neutrophil and leukocyte infiltration in periodontitis. Our work provides a resource characterizing the role of tissue stroma in regulating mucosal tissue homeostasis and disease pathogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Human monogenic disorders have revealed the critical contribution of type 17 responses in mucosal fungal surveillance. We unexpectedly found that in certain settings, enhanced type 1 immunity rather than defective type 17 responses can promote mucosal fungal infection susceptibility. Notably, in mice and humans with AIRE deficiency, an autoimmune disease characterized by selective susceptibility to mucosal but not systemic fungal infection, mucosal type 17 responses are intact while type 1 responses are exacerbated. These responses promote aberrant interferon-γ (IFN-γ)- and signal transducer and activator of transcription 1 (STAT1)-dependent epithelial barrier defects as well as mucosal fungal infection susceptibility. Concordantly, genetic and pharmacologic inhibition of IFN-γ or Janus kinase (JAK)-STAT signaling ameliorates mucosal fungal disease. Thus, we identify aberrant T cell-dependent, type 1 mucosal inflammation as a critical tissue-specific pathogenic mechanism that promotes mucosal fungal infection susceptibility in mice and humans., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

T helper 17 (Th17) cells were first described as a T helper subset involved in the pathogenesis of experimental autoimmune inflammation. Since then, these cells have been described as orchestrators of immunopathology in several human inflammatory conditions including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. More recently, the crucial role of Th17 cells in the regulation of immunity and protection of barrier sites has been unveiled. In the present work, we review the available evidence regarding Th17 cells in health and disease with a focus on the oral mucosa and their role in periodontitis pathogenesis. Recent mechanistic studies in animal models have demonstrated that interleukin-17A (IL-17A) and Th17 cells are critical mediators for alveolar bone destruction during periodontal inflammation. Observations in a cohort of patients with naturally occurring impaired Th17 cell differentiation supported these findings. However, interventional studies are needed to conclusively implicate Th17 cells in the immunopathogenesis of human alveolar bone and tissue destruction that characterize periodontitis.

Periodontitis is one of the most common human inflammatory diseases, yet the mechanisms that drive immunopathology and could be therapeutically targeted are not well defined. Here, we demonstrate an expansion of resident memory T helper 17 (T H 17) cells in human periodontitis. Phenocopying humans, T H 17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral T H 17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)-dependent manner, periodontitis-associated expansion of T H 17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. T H 17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of T H 17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in T H 17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human T H 17 cell defects were associated with diminished periodontal inflammation and bone loss, despite increased prevalence of recurrent oral fungal infections. Our study highlights distinct functions of T H 17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A patient with leukocyte adhesion deficiency type 1 (LAD1) had severe periodontitis and an intractable, deep, nonhealing sacral wound. We had previously found a dominant interleukin-23-interleukin-17 signature at inflamed sites in humans with LAD1 and in mouse models of the disorder. Blockade of this pathway in mouse models has resulted in resolution of the immunopathologic condition. We treated our patient with ustekinumab, an antibody that binds the p40 subunit of interleukin-23 and interleukin-12 and thereby blocks the activity of these cytokines, inhibiting interleukin-23-dependent production of interleukin-17. After 1 year of therapy, our patient had resolution of his inflammatory lesions without serious infections or adverse reactions. Inhibition of interleukin-23 and interleukin-17 may have a role in the management of LAD1. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

Virulence factors on the surface of Porphyromonas gingivalis constitute the first line of interaction with host cells and contribute to immune modulation and periodontitis progression. In order to characterize surface virulence factors present on P. gingivalis, we obtained clinical isolates from healthy and periodontitis subjects and compared them with reference strains. Colony morphology, aggregation in liquid medium, surface charge, membrane permeability to bactericidal compounds, novobiocin and polymyxin B resistance, capsule presence and lipopolysaccharide (LPS) profiles were evaluated. By comparing isolates from healthy and periodontitis subjects, differences in colony morphology and aggregation in liquid culture were found; the latter being similar to two reference strains. These differences were not a consequence of variations in bacterial surface charge. Furthermore, isolates also presented differences in polymyxin B and novobiocin resistance; isolates from healthy subjects were susceptible to polymyxin B and resistant to novobiocin and, in contrast, isolates from periodontitis subjects were resistant to polymyxin B and susceptible to novobiocin. These changes in antimicrobial resistance levels correlate with variations in LPS profiles, since -unlike periodontitis isolates-isolates from healthy samples synthesize LPS molecules lacking both O-antigen moieties and anionic polysaccharide. Additionally, this phenotype correlated with the absence of O-antigen ligase activity. Altogether, our results reveal novel variations on surface components of P. gingivalis isolates obtained from healthy and periodontitis subjects that could be associated with differences in bacterial virulence and periodontitis progression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Periodontal diseases usually refer to common inflammatory disorders known as gingivitis and periodontitis, which are caused by a pathogenic microbiota in the subgingival biofilm, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola that trigger innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually in tissue, bone and finally, tooth loss. The innate immune response constitutes a homeostatic system, which is the first line of defense, and is able to recognize invading microorganisms as non-self, triggering immune responses to eliminate them. In addition to the innate immunity, adaptive immunity cells and characteristic cytokines have been described as important players in the periodontal disease pathogenesis scenario, with a special attention to CD4+ T-cells (T-helper cells). Interestingly, the T cell-mediated adaptive immunity development is highly dependent on innate immunity-associated antigen presenting cells, which after antigen capture undergo into a maturation process and migrate towards the lymph nodes, where they produce distinct patterns of cytokines that will contribute to the subsequent polarization and activation of specific T CD4+ lymphocytes. Skeletal homeostasis depends on a dynamic balance between the activities of the bone-forming osteoblasts (OBLs) and bone-resorbing osteoclasts (OCLs). This balance is tightly controlled by various regulatory systems, such as the endocrine system, and is influenced by the immune system, an osteoimmunological regulation depending on lymphocyte- and macrophage-derived cytokines. All these cytokines and inflammatory mediators are capable of acting alone or in concert, to stimulate periodontal breakdown and collagen destruction via tissue-derived matrix metalloproteinases, a characterization of the progression of periodontitis as a stage that presents a significantly host immune and inflammatory response to the microbial challenge that determine of susceptibility to develop the destructive/progressive periodontitis under the influence of multiple behavioral, environmental and genetic factors.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of β2 integrins which result in defective transmigration of neutrophils into the tissues. Affected patients suffer from recurrent life threatening infections and severe oral disease (periodontitis). Microbial communities in the local environment (subgingival plaque) are thought to be the triggers for inflammatory periodontitis, yet little is known regarding the microbial communities associated with LAD-I periodontitis. Here we present the first comprehensive characterization of the subgingival communities in LAD-I, using a 16S rRNA gene-based microarray, and investigate the relationship of this tooth adherent microbiome to the local immunopathology of periodontitis. We show that the LAD subgingival microbiome is distinct from that of health and Localized Aggressive Periodontitits. Select periodontitis-associated species in the LAD microbiome included Parvimonas micra, Porphyromonas endodontalis, Eubacterium brachy and Treponema species. Pseudomonas aeruginosa, a bacterium not typically found in subgingival plaque is detected in LAD-I. We suggest that microbial products from LAD-associated communities may have a role in stimulating the local inflammatory response. We demonstrate that bacterial LPS translocates into the lesions of LAD-periodontitis potentially triggering immunopathology. We also show in in vitro assays with human macrophages and in vivo in animal models that microbial products from LAD-associated subgingival plaque trigger IL-23-related immune responses, which have been shown to dominate in patient lesions. In conclusion, our current study characterizes the subgingival microbial communities in LAD-periodontitis and supports their role as triggers of disease pathogenesis.

We have previously reported a novel method for the production of tumour-antigen-presenting cells (referred to as TAPCells) that are currently being used in cancer therapy, using an allogeneic melanoma-derived cell lysate (referred to as TRIMEL) as an antigen provider and activation factor. It was recently demonstrated that TAPCell-based immunotherapy induces T-cell-mediated immune responses resulting in improved long-term survival of stage IV melanoma patients. Clinically, dendritic cell (DC) migration from injected sites to lymph nodes is an important requirement for an effective anti-tumour immunization. This mobilization of DCs is mainly driven by the C-C chemokine receptor type 7 (CCR7), which is up-regulated on mature DCs. Using flow cytometry and immunohistochemistry, we investigated if TRIMEL was capable of inducing the expression of the CCR7 on TAPCells and enhancing their migration in vitro, as well as their in vivo relocation to lymph nodes in an ectopic xenograft animal model. Our results confirmed that TRIMEL induces a phenotypic maturation and increases the expression of surface CCR7 on melanoma patient-derived DCs, and also on the monocytic/macrophage cell line THP-1. Moreover, in vitro assays showed that TRIMEL-stimulated DCs and THP-1 cells were capable of migrating specifically in the presence of the CCR7 ligand CCL19. Finally, we demonstrated that TAPCells could migrate in vivo from the injection site into the draining lymph nodes. This work contributes to an increased understanding of the biology of DCs produced ex vivo allowing the design of new strategies for effective DC-based vaccines for treating aggressive melanomas., (© 2014 John Wiley & Sons Ltd.)

Background: Macrophages account for 5% to 30% of the inflammatory infiltrate in periodontitis and are activated by the classic and alternative pathways. These pathways are identified by indirect markers, among which interferon (IFN)-γ and interleukin-6 (IL)-6 of the classic pathway and IL-4 of the alternative pathway have been studied widely. Recently, factor XIII-A (FXIII-A) was reported to be a good marker of alternative pathway activation. The aim of this study is to determine the macrophage activation pathways involved in chronic periodontitis (CP) by the detection of the indirect markers IFN-γ, IL-6, FXIII-A, and IL-4., Methods: Biopsies were taken from patients with CP (n = 10) and healthy individuals (n = 10) for analysis of IFN-γ, IL-6, IL-4, and FXIII-A by Western blot (WB), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). The same biopsies of healthy and diseased gingival tissue were used, and the expressions of these markers were compared between healthy individuals and those with CP., Results: The presence of macrophages was detected by CD68+ immunohistochemistry and their IFN-γ, IL-6, IL-4, and FXIII-A markers by WB, IHC, and ELISA in all samples of healthy and diseased tissue. IL-6, IL-4, and FXIII-A were significantly higher in patients with CP, whereas FXIII-A was higher in healthy individuals., Conclusion: The presence of IFN-γ, IL-6, IL-4, and FXIII-A in healthy individuals and in patients with CP suggests that macrophages may be activated by both classic and alternative pathways in health and in periodontal disease.

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β₂ integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

Background: Interleukin-21 (IL-21) controls the differentiation of T-helper Th17 cells and induces the production of IL-17 in this T-cell subtype. The aim of this study is to determine the relative expression of IL-21 in gingival tissues of chronic periodontitis patients and correlate/associate this expression with proinflammatory cytokines and clinical parameters of disease., Methods: Samples of gingival biopsies were collected from chronic periodontitis patients (n = 10) and controls (n = 8). The mRNA expressions of IL-21, IL-1β, IL-6, IL-17, IL-23, IL-10, and transforming growth factor-β1 (TGF-β1) were quantified using real-time reverse transcription-polymerase chain reaction. IL-21 levels were compared between chronic periodontitis and healthy gingival tissues and correlated with cytokine and clinical parameters of tissue destruction., Results: A significant overexpression of IL-21, IL-1β, IL-6, IL-17, and IL-23p19 was detected in periodontal disease-affected tissues compared to healthy gingival tissues. IL-10 and TGF-β1 were, however, downregulated in periodontal lesions. IL-21 yielded significant positive correlations with probing depth, clinical attachment level, IL-1β, and IL-6. In addition, IL-21 was negatively correlated with IL-10 and TGF-β1., Conclusions: IL-21 was overexpressed in chronic periodontitis gingival tissues and correlated with clinical parameters of periodontal destruction and with proinflammatory cytokines. Therefore, IL-21 might play a role in the tissue destruction that characterizes chronic periodontal disease.

Background: A growing body of evidence suggested that interleukin (IL)-21 enhances the effector phase during T-cell responses. The aim of our study is to determine the levels of IL-21 in periodontal sites from patients with chronic periodontitis and controls., Methods: The population studied consisted of 34 patients (15 with chronic periodontitis and 19 healthy patients). Twenty samples (10 gingival crevicular fluid [GCF] and 10 gingival biopsies) were collected from each group before the patients with periodontitis received periodontal treatment. Total protein concentrations were measured in all samples; the presence of IL-21 was confirmed by immunohistochemistry and Western blot, and IL-21 levels were quantified through an enzyme-linked immunosorbent assay. Statistical analyses were performed using statistical software. Data were expressed as patient means ± SDs or medians (interquartile ranges) by using the χ(2), Student t, and Mann-Whitney U tests., Results: GCF IL-21 was mainly detected in patients with chronic periodontitis (P <0.05). Levels of IL-21 in gingival tissues were significantly higher in patients with chronic periodontitis compared to healthy individuals (P <0.05). The Western blot and immunohistochemical staining confirmed the presence of IL-21 in periodontal tissues and GCF., Conclusion: IL-21 was highly expressed in patients with chronic periodontitis, especially in gingival biopsies; therefore, IL-21 might play a role in the T-cell response.

Background: Chemokines are central in the activation and direction of leukocyte subsets to target tissues. However, the monocyte chemoattractant protein-3 (MCP-3) has not been associated with chronic periodontitis. Chronic periodontitis is an infection showing episodic supporting tissue destruction. The aim of this study is to determine the levels and expression of MCP-3 in periodontal sites characterized by active periodontal connective tissue destruction., Methods: The study population consisted of 15 patients with a progression of periodontitis (15 of 56 patients), 18 patients with chronic periodontitis, and 10 healthy subjects without periodontal disease. As determined by the tolerance method, the 15 patients with moderate to advanced chronic periodontitis showed a progression of periodontitis over a 4-month period. Periodontitis was characterized by at least six sites with a probing depth >or=5 mm, clinical attachment level >or=3 mm, and radiographic bone loss. Gingival crevicular fluid was collected using a paper strip. The total protein concentration was determined. An enzyme-linked immunosorbent assay was performed to determine the total amount of MCP-3, and an immunoWestern blot was conducted to assess molecular MCP-3 forms. To determine the MCP-3 expression by immunohistochemistry, gingival biopsies were obtained from patients with chronic periodontitis and healthy subjects during third-molar extraction surgery. Statistical analyses were performed using statistical software. Data were expressed as subject means +/- SD, using the chi(2) and Student t tests., Results: The total amount and concentration of chemokine MCP-3 were significantly higher in patients with chronic periodontitis than in healthy subjects (8.25 pg versus 0.53 pg, P = 0.006 and 2.95 pg/microl versus 0.45 pg/microl, P = 0.04, respectively). Active sites showed a significantly higher total amount and concentration of MCP-3 than inactive sites (11.12 versus 2.88 pg, P value = 0.005 and 3.95 versus 1.02, P value = 0.005, respectively). Western blot and immunohistochemical staining confirmed the presence of MCP-3 in periodontal disease, with observable differences between patients with chronic periodontitis and healthy subjects., Conclusions: MCP-3 was highly expressed in patients with chronic periodontitis, particularly in those with progressive periodontal lesions. MCP-3 could be involved in the recruitment of inflammatory cells toward periodontal tissues during the progression of the disease.

Aim: Matrix metalloproteinases (MMP)-13 can initiate bone resorption and activate proMMP-9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP-13 activity and TIMP-1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy-terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP-13 could potentiate gelatinase activation in diseased gingival tissue., Materials and Methods: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP-13 activity and TIMP-1. Diseased gingival explants were cultured, treated or not with MMP-13 with or without adding CL-82198, a synthetic MMP-13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis., Results: Active sites demonstrated increased ICTP levels and MMP-13 activity (p<0.05) in progression subjects. The MMP-9 activation rate was elevated in MMP-13-treated explants (p<0.05) and MMP-13 inhibitor prevented MMP-9 activation., Conclusions: MMP-13 could be implicated in the degradation of soft and hard supporting tissues and proMMP-9 activation during progression of chronic periodontitis. MMP-13 and -9 can potentially form an activation cascade overcoming the protective TIMP-1 shield, which may become useful for diagnostic aims and a target for drug development.

Background: Periodontitis is an infection with an episodic pattern of tissue-support destruction. During the generation of a primary CD4(+) T helper 1 (Th1) response, interferon-gamma (IFN-gamma) acts as a positive regulator by selectively inducing Th1 differentiation through increased transcription of T-bet. The aims of this work were to determine IFN-gamma levels in samples of gingival crevicular fluid (GCF) and to determine IFN-gamma and transcription factor T-bet expression in gingival tissue from patients undergoing the progression of chronic periodontitis., Methods: One hundred six patients with moderate or advanced chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth >or=5 mm, clinical attachment loss >or=3 mm, and radiographic bone loss. Periodontitis progression was determined by the tolerance method. GCF was collected using a paper strip, and enzyme-linked immunosorbent assay was performed to determine the total amount of IFN-gamma. Gingival biopsies were obtained from patients for real-time reverse transcription-polymerase chain reaction to determine IFN-gamma and T-bet expression. Statistical analysis was performed using statistical software. Data were expressed as subject means +/- SD. The chi(2) and Student t tests were used., Results: The total amount and concentration of cytokine IFN-gamma were significantly higher in active sites than in inactive sites (99.90 versus 68.90 pg; P = 0.03; 106.62 pg/mg versus 75.64 pg/mg, P = 0.04, respectively). Active sites showed a significantly lower Delta cycle threshold (Ct) of IFN-gamma than inactive sites (P = 0.04), whereas the expression of transcription factor T-bet was increased 1.42-fold in active sites compared to inactive sites., Conclusion: The total amount and concentration of cytokine IFN-gamma in GCF samples and transcription factor T-bet expression were increased in progressive periodontal lesions in patients with chronic periodontitis.

Background: Chronic periodontitis is an infectious disease characterized by alveolar bone destruction and teeth loss. Receptor activator of nuclear factor-kappa B ligand (RANKL) is an osteoclastogenic cytokine, a central regulatory factor in the osteoclast's lifespan, and a participant in physiological and pathological bone resorption. Gingival T cells synthesize RANKL, contributing to molecular local imbalance that entails the alveolar bone resorption seen in periodontitis. Our study was aimed at associating the levels of RANKL with the CD4(+) T-cell activity present in gingival tissues of chronic periodontitis patients., Methods: Gingival biopsies were obtained from 33 chronic periodontitis patients and 20 healthy controls. Specimens were either formalin fixed and paraffin embedded for real-time reverse transcription-polymerase chain reaction (RT-PCR) and histologic analysis or tissue digestion processed for cell culture and flow-cytometry analysis. RANKL mRNA and protein levels were determined by quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) in gingival-cell culture supernatants. Gingival leukocytes were quantified by flow cytometry. RANKL and CD4 immunoreactivity were analyzed by flow cytometry and confocal microscopy., Results: RANKL mRNA levels were higher in patients with periodontitis than in healthy subjects, and spontaneous and lipopolysaccharide (LPS)- and phytohemagglutinin (PHA)-stimulated RANKL synthesis were higher also in patients than controls. CD4(+) T lymphocytes were the predominant infiltrate cell subset present in gingival tissues of periodontitis patients. Furthermore, an association between RANKL and CD4(+) T cells was determined by double-staining flow cytometry and confocal microscopy., Conclusion: Taken together, these data demonstrate that gingival CD4(+) T cells are the main cells responsible for higher levels of RANKL observed in human chronic periodontitis patients.