RESUMO
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.
Assuntos
Imunidade nas Mucosas , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Neutrófilos/citologia , Adulto , Células Epiteliais/citologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Gengiva/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Microbiota , Células Mieloides/citologia , Periodontite/genética , Periodontite/imunologia , Periodontite/patologia , Análise de Célula Única , Células Estromais/citologia , Linfócitos T/citologiaRESUMO
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.
Assuntos
Interleucina-23 , Periodontite , Humanos , Células Epiteliais , Inflamação , Receptor 5 Toll-Like/metabolismoRESUMO
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.
Assuntos
Gengiva/imunologia , Imunidade nas Mucosas/imunologia , Vigilância Imunológica/imunologia , Mucosa Bucal/imunologia , Células Th17/imunologia , Animais , Citometria de Fluxo , Gengiva/microbiologia , Humanos , Mastigação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Mucosa Bucal/microbiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/imunologia , Leucócitos/imunologia , Periodontite/microbiologia , Porphyromonas gingivalis , Animais , DNA Bacteriano/genética , DNA Bacteriano/imunologia , Placa Dentária/genética , Humanos , Interleucina-23/metabolismo , Síndrome da Aderência Leucocítica Deficitária/metabolismo , Síndrome da Aderência Leucocítica Deficitária/terapia , Camundongos , Microbiota/imunologia , RNA Ribossômico 16S/genéticaRESUMO
Single-cell RNA sequencing and flow cytometry approaches have been instrumental in understanding cellular states within various tissues and organs. However, tissue dissociation methods can potentially alter results and create bias due to preferential recovery of particular cell types. Here we present efforts to optimize methods for dissociation of murine oral mucosal tissues and provide three different protocols that can be utilized to isolate major cell populations in the oral mucosa. These methods can be used both in health and in states of inflammation, such as periodontitis. The optimized protocols use different enzymatic approaches (collagenase II, collagenase IV and the Miltenyi whole skin dissociation kit) and yield preferential recovery of immune, stromal and epithelial cells, respectively. We suggest choosing the dissociation method based on the cell population of interest to study, while understanding the limitations of each approach.
Assuntos
Mucosa Bucal , Periodontite , Animais , Camundongos , Citometria de Fluxo/métodos , Colagenases/metabolismo , InflamaçãoRESUMO
We investigated whether interferon-inducible genes (IFIGs) with known anti-human immunodeficiency virus (HIV) activity in vitro were associated with in vivo virological response in HIV infection. Nine untreated HIV-1-infected volunteers were treated for 12 weeks with peginterferon alfa-2a. A subset of IFIGs (23 of 47) increased compared with baseline through 6 weeks beyond therapy, and 10 of the 23 IFIGs significantly inversely correlated (r = -0.7; P < .05) with virological response. The strength of peginterferon alfa-2a-induced IFIG response significantly correlated with declines in HIV load during treatment (r(2) = 0.87, p = .003). This study links HIV virological response to a specific IFIG subset, a potential prognostic indicator in peginterferon alfa-2a-treated patients with HIV infection.
Assuntos
Antivirais/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/genética , Interferon-alfa/administração & dosagem , Polietilenoglicóis/administração & dosagem , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , Humanos , Leucócitos Mononucleares/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , Proteínas Recombinantes/administração & dosagem , Carga ViralRESUMO
Neutrophil infiltration is a hallmark of periodontitis, a prevalent oral inflammatory condition in which Th17-driven mucosal inflammation leads to destruction of tooth-supporting bone. Herein, we document that neutrophil extracellular traps (NETs) are early triggers of pathogenic inflammation in periodontitis. In an established animal model, we demonstrate that neutrophils infiltrate the gingival oral mucosa at early time points after disease induction and expel NETs to trigger mucosal inflammation and bone destruction in vivo. Investigating mechanisms by which NETs drive inflammatory bone loss, we find that extracellular histones, a major component of NETs, trigger upregulation of IL-17/Th17 responses, and bone destruction. Importantly, human findings corroborate our experimental work. We document significantly increased levels of NET complexes and extracellular histones bearing classic NET-associated posttranslational modifications, in blood and local lesions of severe periodontitis patients, in the absence of confounding disease. Our findings suggest a feed-forward loop in which NETs trigger IL-17 immunity to promote immunopathology in a prevalent human inflammatory disease.
Assuntos
Armadilhas Extracelulares , Periodontite , Animais , Humanos , Histonas , Interleucina-17 , Inflamação/patologia , Periodontite/patologia , Neutrófilos/patologiaRESUMO
Oral mucosal tissue is composed of several cell types that are difficult to dissociate while maintaining high cell viability. We describe a protocol for the preparation and dissociation of human buccal and gingival oral mucosal tissue to a high-viability single-cell suspension composed of heterogeneous cell types. This heterogeneous cell suspension can subsequently be used for cytometric analyses or to generate single-cell RNA sequencing libraries. For complete details on the use and execution of this protocol, please refer to Williams et al. (2021).
Assuntos
Mucosa Bucal/citologia , Análise de Célula Única/métodos , Adulto , Sobrevivência Celular , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Fibrina/metabolismo , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Periodontite/genética , Plasminogênio/genética , Perda do Osso Alveolar , Animais , Armadilhas Extracelulares/metabolismo , Feminino , Fibrina/química , Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Fibrinólise , Microbioma Gastrointestinal/fisiologia , Gengiva/imunologia , Humanos , Imunidade nas Mucosas , Antígeno de Macrófago 1/metabolismo , Masculino , Camundongos , Mucosa Bucal/microbiologia , Periodontite/imunologia , Plasminogênio/deficiência , Plasminogênio/metabolismo , Polimorfismo de Nucleotídeo Único , RNA-Seq , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Disbiose , Síndrome de Job/imunologia , Microbiota/imunologia , Mucosa Bucal/microbiologia , Fator de Transcrição STAT3/metabolismo , Adulto , Candida albicans , Candidíase , Cárie Dentária/microbiologia , Feminino , Humanos , Interleucina-17 , Síndrome de Job/genética , Masculino , Microbiota/genética , Pessoa de Meia-Idade , Mutação , RNA Ribossômico 16S , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Streptococcus mutans , Streptococcus oralis , Células Th17 , Adulto JovemRESUMO
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.
Assuntos
Disbiose/imunologia , Disbiose/microbiologia , Microbiota , Mucosa Bucal/imunologia , Mucosa Bucal/patologia , Células Th17/imunologia , Animais , Bactérias/metabolismo , Reabsorção Óssea/microbiologia , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Diferenciação Celular , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Camundongos , Neutrófilos/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Periodontite/imunologia , Periodontite/microbiologia , Periodontite/patologiaRESUMO
The oral mucosa is a barrier site constantly exposed to rich and diverse commensal microbial communities, yet little is known of the immune cell network maintaining immune homeostasis at this interface. We have performed a detailed characterization of the immune cell subsets of the oral cavity in a large cohort of healthy subjects. We focused our characterization on the gingival interface, a particularly vulnerable mucosal site, with thin epithelial lining and constant exposure to the tooth adherent biofilm. In health, we find a predominance of T cells, minimal B cells, a large presence of granulocytes/neutrophils, a sophisticated network of professional antigen-presenting cells (APCs), and a small population of innate lymphoid cells (ILCs) policing the gingival barrier. We further characterize cellular subtypes in health and interrogate shifts in immune cell populations in the common oral inflammatory disease periodontitis. In disease, we document an increase in neutrophils and an upregulation of interleukin-17 (IL-17) responses. We identify the main source of IL-17 in health and Periodontitis within the CD4(+) T-cell compartment. Collectively, our studies provide a first view of the landscape of physiologic oral immunity and serve as a baseline for the characterization of local immunopathology.