RESUMO
Whereas type I interferons (IFNs-I) were proposed to be elevated in human periodontitis, their role in the disease remains elusive. Using a bacterial-induced model of murine periodontitis, we revealed a prolonged elevation in IFN-I expression. This was due to the downregulation of TAM signaling, a major negative regulator of IFN-I. Further examination revealed that the expression of certain TAM components was reduced as a result of prolonged degradation of MYD88 by the infection. As a result of such prolonged IFN-I production, innate immunological functions of the gingiva were disrupted, and CD4+ T cells were constitutively primed by dendritic cells, leading to elevated RANKL expression and, subsequently, alveolar bone loss (ABL). Blocking IFN-I signaling restored proper immunological function and prevented ABL. Importantly, a loss of negative regulation on IFN-I expression by TAM signaling was also evident in periodontitis patients. These findings thus suggest a role for IFN-I in the pathogenesis of periodontitis.
Assuntos
Interferon Tipo I/biossíntese , Fator 88 de Diferenciação Mieloide/metabolismo , Porphyromonas gingivalis/fisiologia , Proteólise , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Perda do Osso Alveolar/complicações , Perda do Osso Alveolar/imunologia , Perda do Osso Alveolar/patologia , Animais , Infecções por Bacteroidaceae/complicações , Infecções por Bacteroidaceae/imunologia , Infecções por Bacteroidaceae/microbiologia , Reabsorção Óssea/complicações , Reabsorção Óssea/imunologia , Reabsorção Óssea/patologia , Células Dendríticas/imunologia , Gengiva/microbiologia , Gengiva/patologia , Humanos , Interferon Tipo I/metabolismo , Leucócitos/patologia , Linfonodos/patologia , Camundongos , Mucosa Bucal/microbiologia , Mucosa Bucal/patologia , Periodontite/imunologia , Periodontite/microbiologia , Periodontite/patologiaRESUMO
The eukaryotic cell cycle is comprised of different phases that take place sequentially once, and normally only once, every division cycle. Such a dynamic process is best viewed in real time in living dividing cells. The insights that can be gained from such methods are considerably larger than any alternative technique that only generates snapshots. A great number of studies can gain from live cell imaging; however this method often feels somewhat intimidating to the novice. The purpose of this chapter is to demonstrate that imaging cell cycle phases in living cells from yeast to human is relatively easy and can be performed with equipment that is available in most research institutes. We present the different approaches, review different types of reporters, and discuss in depth all the aspects to be considered to obtain optimal results. We also describe our latest cell cycle markers, which afford unprecedented "sub"-phase temporal resolution.
Assuntos
Ciclo Celular , Imagem Molecular/métodos , Saccharomycetales/citologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Células NIH 3T3RESUMO
Langerhans cells (LCs) populate the mucosal epithelium, a major entry portal for pathogens, yet their ontogeny remains unclear. We found that, in contrast to skin LCs originating from self-renewing radioresistant embryonic precursors, oral mucosal LCs derive from circulating radiosensitive precursors. Mucosal LCs can be segregated into CD103(+)CD11b(lo) (CD103(+)) and CD11b(+)CD103(-) (CD11b(+)) subsets. We further demonstrated that similar to non-lymphoid dendritic cells (DCs), CD103(+) LCs originate from pre-DCs, whereas CD11b(+) LCs differentiate from both pre-DCs and monocytic precursors. Despite this ontogenetic discrepancy between skin and mucosal LCs, the transcriptomic signature and immunological function of oral LCs highly resemble those of skin LCs but not DCs. These findings, along with the epithelial position, morphology, and expression of the LC-associated phenotype strongly suggest that oral mucosal LCs are genuine LCs. Collectively, in a tissue-dependent manner, murine LCs differentiate from at least three distinct precursors (embryonic, pre-DC, and monocytic) in steady state.
Assuntos
Diferenciação Celular , Células Dendríticas/imunologia , Células de Langerhans/imunologia , Monócitos/imunologia , Mucosa Bucal/imunologia , Animais , Antígenos CD/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Circulação Sanguínea , Antígeno CD11b/metabolismo , Células Cultivadas , Epitélio/imunologia , Cadeias alfa de Integrinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Pele/imunologia , Transcriptoma/imunologiaRESUMO
We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously (1). Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.
Assuntos
Técnicas Citológicas/métodos , Gengiva/citologia , Análise de Célula Única/métodos , Animais , Citometria de Fluxo , CamundongosRESUMO
Memory CD8(+) T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8(+) T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8(+) T-cell expansion.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos , Memória Imunológica , Lentivirus/genética , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/citologia , Proliferação de Células , Cinética , Lentivirus/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Excessive bone resorption is frequently associated with chronic infections and inflammatory diseases. Whereas T cells were demonstrated to facilitate osteoclastogenesis in such diseases, the role of dendritic cells, the most potent activators of naive T cells, remains unclear. Using a model involving inflammation-driven alveolar bone loss attributable to infection, we showed that in vivo ablation of Langerhans cells (LCs) resulted in enhanced bone loss. An increased infiltration of B and T lymphocytes into the tissue surrounding the bone was observed in LC-ablated mice, including receptor activator of NF-κB ligand (RANKL)-expressing CD4(+) T cells with known capabilities of altering bone homeostasis. In addition, the absence of LCs significantly reduced the numbers of CD4(+)Foxp3(+) T-regulatory cells in the tissue. Further investigation revealed that LCs were not directly involved in presenting antigens to T cells. Nevertheless, despite their low numbers in the tissue, the absence of LCs resulted in an elevated activation of CD4(+) but not CD8(+) T cells. This activation involved elevated production of IFN-γ but not IL-17 or IL-10 cytokines. Our data, thus, reveal a protective immunoregulatory role for LCs in inflammation-induced alveolar bone resorption, by inhibiting IFN-γ secretion and excessive activation of RANKL(+)CD4(+) T cells with a capability of promoting osteoclastogenesis.