Epithelial-derived interleukin-23 promotes oral mucosal immunopathology.

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.

A reappraisal of microbiome dysbiosis during experimental periodontitis.

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.

Fibrin is a critical regulator of neutrophil effector function at the oral mucosal barrier.

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 αMß2 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.

Microbial signatures of health, gingivitis, and periodontitis.

The subgingival crevice harbors diverse microbial communities. Shifts in the composition of these communities occur with the development of gingivitis and periodontitis, which are considered as successive stages of periodontal health deterioration. It is not clear, however, to what extent health- and gingivitis-associated microbiota are protective, or whether these communities facilitate the successive growth of periodontitis-associated taxa. To further our understanding of the dynamics of the microbial stimuli that trigger disruptions in periodontal homeostasis, we reviewed the available literature with the aim of defining specific microbial signatures associated with different stages of periodontal dysbiosis. Although several studies have evaluated the subgingival communities present in different periodontal conditions, we found limited evidence for the direct comparison of communities in health, gingivitis, and periodontitis. Therefore, we aimed to better define subgingival microbiome shifts by merging and reanalyzing, using unified bioinformatic processing strategies, publicly available 16S ribosomal RNA gene amplicon datasets of periodontal health, gingivitis, and periodontitis. Despite inherent methodological differences across studies, distinct community structures were found for health, gingivitis, and periodontitis, demonstrating the specific associations between gingival tissue status and the subgingival microbiome. Consistent with the concept that periodontal dysbiosis is the result of a process of microbial succession without replacement, more species were detected in disease than in health. However, gingivitis-associated communities were more diverse than those from subjects with periodontitis, suggesting that certain species ultimately become dominant as dysbiosis progresses. We identified the bacterial species associated with each periodontal condition and prevalent species that do not change in abundance from one state to another (core species), and we also outlined species co-occurrence patterns via network analysis. Most periodontitis-associated species were rarely detected in health but were frequently detected, albeit in low abundance, in gingivitis, which suggests that gingivitis and periodontitis are a continuum. Overall, we provide a framework of subgingival microbiome shifts, which can be used to generate hypotheses with respect to community assembly processes and the emergence of periodontal dysbiosis.

A cross-species interaction with a symbiotic commensal enables cell-density-dependent growth and in vivo virulence of an oral pathogen.

Recent studies describe in detail the shifts in composition of human-associated polymicrobial communities from health to disease. However, the specific processes that drive the colonization and overgrowth of pathogens within these communities remain incompletely understood. We used in vitro culture systems and a disease-relevant mouse model to show that population size, which determines the availability of an endogenous diffusible small molecule, limits the growth, colonization, and in vivo virulence of the human oral pathogen Porphyromonas gingivalis. This bacterial pathogen overcomes the requirement for an endogenous cue by utilizing a cell-density dependent, growth-promoting, soluble molecule provided by the symbiotic early colonizer Veillonella parvula, but not produced by other commensals tested. Our work shows that exchange of cell-density-dependent diffusible cues between specific early and late colonizing species in a polymicrobial community drives microbial successions, pathogen colonization and disease development, representing a target process for manipulation of the microbiome towards the healthy state.

Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome.

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted.

T Helper 17 Cells as Pathogenic Drivers of Periodontitis.

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.

A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans.

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 (TH17) cells in human periodontitis. Phenocopying humans, TH17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral TH17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)-dependent manner, periodontitis-associated expansion of TH17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. TH17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of TH17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in TH17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human TH17 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 TH17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis.

Human defects in STAT3 promote oral mucosal fungal and bacterial dysbiosis.

Studies in patients with genetic defects can provide unique insights regarding the role of specific genes and pathways in humans. Patients with defects in the Th17/IL-17 axis, such as patients harboring loss-of-function STAT3 mutations (autosomal-dominant hyper IgE syndrome; AD-HIES) present with recurrent oral fungal infections. Our studies aimed to comprehensively evaluate consequences of STAT3 deficiency on the oral commensal microbiome. We characterized fungal and bacterial communities in AD-HIES in the presence and absence of oral fungal infection compared with healthy volunteers. Analyses of oral mucosal fungal communities in AD-HIES revealed severe dysbiosis with dominance of Candida albicans (C. albicans) in actively infected patients and minimal representation of health-associated fungi and/or opportunists. Bacterial communities also displayed dysbiosis in AD-HIES, particularly in the setting of active Candida infection. Active candidiasis was associated with decreased microbial diversity and enrichment of the streptococci Streptococcus oralis (S. oralis) and S. mutans, suggesting an interkingdom interaction of C. albicans with oral streptococci. Increased abundance of S. mutans was consistent with susceptibility to dental caries in AD-HIES. Collectively, our findings illustrate a critical role for STAT3/Th17 in the containment of C. albicans as a commensal organism and an overall contribution in the establishment of fungal and bacterial oral commensal communities.

Transcriptional signature primes human oral mucosa for rapid wound healing.

Oral mucosal wound healing has long been regarded as an ideal system of wound resolution. However, the intrinsic characteristics that mediate optimal healing at mucosal surfaces are poorly understood, particularly in humans. We present a unique comparative analysis between human oral and cutaneous wound healing using paired and sequential biopsies during the repair process. Using molecular profiling, we determined that wound-activated transcriptional networks are present at basal state in the oral mucosa, priming the epithelium for wound repair. We show that oral mucosal wound-related networks control epithelial cell differentiation and regulate inflammatory responses, highlighting fundamental global mechanisms of repair and inflammatory responses in humans. The paired comparative analysis allowed for the identification of differentially expressed SOX2 (sex-determining region Y-box 2) and PITX1 (paired-like homeodomain 1) transcriptional regulators in oral versus skin keratinocytes, conferring a unique identity to oral keratinocytes. We show that SOX2 and PITX1 transcriptional function has the potential to reprogram skin keratinocytes to increase cell migration and improve wound resolution in vivo. Our data provide insights into therapeutic targeting of chronic and nonhealing wounds based on greater understanding of the biology of healing in human mucosal and cutaneous environments.

On-going Mechanical Damage from Mastication Drives Homeostatic Th17 Cell Responses at the Oral Barrier.

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.

Oral Microbiome Characterization in Murine Models.

The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.

Aggregatibacter actinomycetemcomitans-induced hypercitrullination links periodontal infection to autoimmunity in rheumatoid arthritis.

A bacterial etiology of rheumatoid arthritis (RA) has been suspected since the beginnings of modern germ theory. Recent studies implicate mucosal surfaces as sites of disease initiation. The common occurrence of periodontal dysbiosis in RA suggests that oral pathogens may trigger the production of disease-specific autoantibodies and arthritis in susceptible individuals. We used mass spectrometry to define the microbial composition and antigenic repertoire of gingival crevicular fluid in patients with periodontal disease and healthy controls. Periodontitis was characterized by the presence of citrullinated autoantigens that are primary immune targets in RA. The citrullinome in periodontitis mirrored patterns of hypercitrullination observed in the rheumatoid joint, implicating this mucosal site in RA pathogenesis. Proteomic signatures of several microbial species were detected in hypercitrullinated periodontitis samples. Among these, Aggregatibacter actinomycetemcomitans (Aa), but not other candidate pathogens, induced hypercitrullination in host neutrophils. We identified the pore-forming toxin leukotoxin A (LtxA) as the molecular mechanism by which Aa triggers dysregulated activation of citrullinating enzymes in neutrophils, mimicking membranolytic pathways that sustain autoantigen citrullination in the RA joint. Moreover, LtxA induced changes in neutrophil morphology mimicking extracellular trap formation, thereby releasing the hypercitrullinated cargo. Exposure to leukotoxic Aa strains was confirmed in patients with RA and was associated with both anticitrullinated protein antibodies and rheumatoid factor. The effect of human lymphocyte antigen-DRB1 shared epitope alleles on autoantibody positivity was limited to RA patients who were exposed to Aa These studies identify the periodontal pathogen Aa as a candidate bacterial trigger of autoimmunity in RA.

Colitis susceptibility in p47(phox-/-) mice is mediated by the microbiome.

BACKGROUND: Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91(phox) (a.k.a. Nox2), p47(phox), p67(phox), p22(phox), p40(phox)) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored. RESULTS: Although CGD mice do not spontaneously develop colitis, we demonstrate that p47(phox-/-) mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47(phox-/-) with B6Tac (wild type) mice reversed this phenotype. However, breeding p47(phox+/-) mice and standardizing the microflora between littermate p47(phox-/-) and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47(phox-/-) mice. We found similarly decreased colitis susceptibility in littermate p47(phox-/-) and B6Tac mice treated with Citrobacter rodentium. CONCLUSIONS: Our findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network.

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.

Host response mechanisms in periodontal diseases.

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.

Host response mechanisms in periodontal diseases

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.

Subgingival microbial communities in Leukocyte Adhesion Deficiency and their relationship with local immunopathology.

Leukocyte Adhesion Deficiency I (LAD-I) is a primary immunodeficiency caused by single gene mutations in the CD18 subunit of ß2 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.