RESUMO
Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60â¯cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60â¯cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.
Assuntos
Fusobacterium nucleatum/patogenicidade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Periodontite/metabolismo , Biofilmes , Sistemas CRISPR-Cas/genética , Regulação para Baixo , Células HL-60 , Histonas/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Periodontite/microbiologia , Peroxidase/metabolismo , Fagocitose , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/metabolismo , Transdução de SinaisRESUMO
AIM: The purpose of this study was to determine inflammatory and epigenetic features following induction of oral and gut dysbiosis in experimental periodontitis in order to examine the interplay between oral and systemic infection. MATERIALS AND METHODS: Periodontitis was induced in 6- to 8-week-old C57BL/6 mice by (a) Ligature placement (Lig group) (oral challenge); (b) P. gingivalis gavage (Pg group) (systemic challenge); and (c) the combination of the two models oral and systemic challenge (Pg + Lig). The duration of the experiment was 60 days, and the animals were then sacrificed for analyses. Alveolar bone loss was assessed, and a multiplex immunoassay was performed. Maxillae and gut tissues were immunostained for DNMT3b (de novo methylation marker), B and T lymphocyte attenuator (BTLA) and IL-18R1 (inflammation markers). RESULTS: Pg and Pg + Lig groups exhibited higher bone loss when compared to Sham. BAFF, VEGF, RANKL, RANTES and IP-10 were significantly higher with Pg gavage. Likewise, DNMT3b was overexpressed in both gut and maxilla after the Pg administration. The same pattern was observed for BTLA and IL-18R1 in gut tissues. CONCLUSIONS: The systemic microbial challenge either alone or in combination with local challenge leads to distinct patterns of inflammatory and epigenetic features when compared to simply locally induced experimental periodontitis.
Assuntos
Perda do Osso Alveolar , Periodontite , Animais , Modelos Animais de Doenças , Epigênese Genética , Camundongos , Camundongos Endogâmicos C57BL , Porphyromonas gingivalisRESUMO
B-lineage cells (B lymphocytes and plasma cells) predominate in the inflammatory infiltrate of human chronic periodontitis. However, their role in disease pathogenesis and the factors responsible for their persistence in chronic lesions are poorly understood. In this regard, two cytokines of the TNF ligand superfamily, a proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), are important for the survival, proliferation, and maturation of B cells. Thus, we hypothesized that APRIL and/or BLyS are upregulated in periodontitis and contribute to induction of periodontal bone loss. This hypothesis was addressed in both human and mouse experimental systems. We show that, relative to healthy controls, the expression of APRIL and BLyS mRNA and protein was upregulated in natural and experimental periodontitis in humans and mice, respectively. The elevated expression of these cytokines correlated with increased numbers of B cells/plasma cells in both species. Moreover, APRIL and BLyS partially colocalized with κ L chain-expressing B-lineage cells at the epithelial-connective tissue interface. Ligature-induced periodontitis resulted in significantly less bone loss in B cell-deficient mice compared with wild-type controls. Ab-mediated neutralization of APRIL or BLyS diminished the number of B cells in the gingival tissue and inhibited bone loss in wild-type, but not in B cell-deficient, mice. In conclusion, B cells and specific cytokines involved in their growth and differentiation contribute to periodontal bone loss. Moreover, APRIL and BLyS have been identified as potential therapeutic targets in periodontitis.
Assuntos
Perda do Osso Alveolar/metabolismo , Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Periodontite/imunologia , Periodontite/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Adulto , Idoso , Perda do Osso Alveolar/genética , Animais , Fator Ativador de Células B/genética , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Cadeias kappa de Imunoglobulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Periodontite/genética , Periodontite/patologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , RNA Mensageiro/genética , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced ß-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.
Assuntos
Actinas/metabolismo , Apoptose , Células Epiteliais/microbiologia , Porphyromonas gingivalis/patogenicidade , Adesinas Bacterianas/metabolismo , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases Gingipaínas , Humanos , Porphyromonas gingivalis/enzimologiaRESUMO
The ability of IFN-ß to induce IL-10 production from innate immune cells is important for its anti-inflammatory properties and is believed to contribute to its therapeutic value in treating multiple sclerosis patients. In this study, we identified that IFN-ß stimulates IL-10 production by activating the JAK1- and PI3K-signaling pathways. JAK1 activity was required for IFN-ß to activate PI3K and Akt1 that resulted in repression of glycogen synthase kinase 3 (GSK3)-ß activity. IFN-ß-mediated suppression of GSK3-ß promoted IL-10, because IL-10 production by IFN-ß-stimulated dendritic cells (DC) expressing an active GSK3-ß knockin was severely reduced, whereas pharmacological or genetic inhibition of GSK3-ß augmented IL-10 production. IFN-ß increased the phosphorylated levels of CREB and STAT3 but only CREB levels were affected by PI3K. Also, a knockdown in CREB, but not STAT3, affected the capacity of IFN-ß to induce IL-10 from DC. IL-10 production by IFN-ß-stimulated DC was shown to suppress IFN-γ and IL-17 production by myelin oligodendrocyte glycoprotein-specific CD4(+) T cells, and this IL-10-dependent anti-inflammatory effect was enhanced by directly targeting GSK3 in DC. These findings highlight how IFN-ß induces IL-10 production and the importance that IL-10 plays in its anti-inflammatory properties, as well as identify a therapeutic target that could be used to increase the IL-10-dependent anti-inflammatory properties of IFN-ß.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Quinase 3 da Glicogênio Sintase/fisiologia , Interferon beta/fisiologia , Interleucina-10/biossíntese , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/enzimologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Epitopos de Linfócito T/imunologia , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Interleucina-10/fisiologia , Interleucina-17/antagonistas & inibidores , Interleucina-17/biossíntese , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Janus Quinase 1/metabolismo , Janus Quinase 1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Associada a Mielina/farmacologia , Glicoproteína Mielina-Oligodendrócito , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/imunologiaRESUMO
Squamous cell carcinoma is the major form of malignancy that arises in head and neck cancer. The modest improvement in the 5year survival rate underpins its complex etiology and provides the impetus for the discovery of new therapeutics. The present study describes the discovery of an indolebased small molecule (24a) that was a potent cytotoxic agent with antiproliferative and proapoptotic properties against a pharyngeal carcinoma cell line, Detroit 562, effectively killing the cells at a halfmaximal inhibitory concentration of 0.03 µM, as demonstrated using cell proliferation studies. The antiproliferative property of 24a was demonstrated by its ability to promote G2/M blockade, as assessed by cell cycle analysis using flow cytometry and the monitoring of realtime cell cycle progression by the fluorescence ubiquitinationbased cell cycle indicator. This proapoptotic property is supported by the promotion of TUNELstaining and increase in the activities of caspases3/7 and 6, in addition to the expression of death receptors and the cleavage of poly (ADPribose) polymerase 1 protein as demonstrated by western blotting. Given that Detroit 562 lacks functional p53, it is suggested that 24a acts independently of the tumor suppressor.
Assuntos
Apoptose/efeitos dos fármacos , Citotoxinas/farmacologia , Neoplasias Faríngeas/tratamento farmacológico , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Citotoxinas/metabolismo , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/farmacologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Neoplasias Faríngeas/metabolismoRESUMO
IL-12p70 is an immunoregulatory cytokine that has been shown to induce IL-10 production from CD4+ T cells, yet the underlying cellular mechanisms controlling this process are poorly understood. In the present study, we demonstrate that IL-12p70 induces IL-10 production from human memory CD4+ T cells via a PI3K-dependent signaling mechanism. Specifically, stimulation of human memory CD4+ T cells in the presence of IL-12p70 lead to increased PI3K activity and the subsequent phosphorylation and inactivation of the downstream constitutively active serine/threonine kinase, glycogen synthase kinase-3beta (GSK3beta). Inhibition of PI3K prevented the inactivation of GSK3beta by IL-12p70, as well as the subsequent ability of IL-12p70 to augment IL-10 levels by memory CD4+ T cells. Moreover, ectopic expression of a constitutively active form of GSK3beta abrogated the ability of IL-12p70 to increase IL-10 production by TCR-stimulated CD4+ T cells. In contrast, direct inhibition of GSK3 mimicked the effect of IL-12p70 on IL-10 production by memory CD4+ T cells. Analysis of downstream transcription factors identified that the ability of IL-12p70 to inactivate GSK3beta lead to increased levels of c-jun. The ability of IL-12p70 to inactivate GSK3beta and induce c-jun levels was required for IL-12 to augment IL-10 production by human memory CD4+ T cells, since small interfering RNA-mediated gene silencing of c-jun abrogated this process. These studies identify the cellular mechanism by which IL-12 induces IL-10 production from human memory CD4+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Memória Imunológica , Interleucina-10/biossíntese , Interleucina-12/metabolismo , Proteínas Proto-Oncogênicas c-jun/fisiologia , Adulto , Linfócitos T CD4-Positivos/enzimologia , Células Cultivadas , Ativação Enzimática/imunologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Interleucina-12/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/biossíntese , Regulação para Cima/imunologiaRESUMO
DNA methylation controls several inflammatory genes affecting bone homeostasis. Hitherto, inhibition of DNA methylation in vivo in the context of periodontitis and osteoclastogenesis has not been attempted. Ligature-induced periodontitis in C57BL/6J mice was induced by placing ligature for five days with Decitabine (5-aza-2'-deoxycytidine) (1 mg/kg/day) or vehicle treatment. We evaluated bone resorption, osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) and mRNA expression of anti-inflammatory molecules using cluster differentiation 14 positive (CD14+) monocytes from human peripheral blood. Our data showed that decitabine inhibited bone loss and osteoclast differentiation experimental periodontitis, and suppressed osteoclast CD14+ human monocytes; and conversely, that it increased bone mineralization in osteoblastic cell line MC3T3-E1 in a concentration-dependent manner. In addition to increasing IL10 (interleukin-10), TGFB (transforming growth factor beta-1) in CD14+ monocytes, decitabine upregulated KLF2 (Krüppel-like factor-2) expression. Overexpression of KLF2 protein enhanced the transcription of IL10 and TGFB. On the contrary, site-directed mutagenesis of KLF2 binding site in IL10 and TFGB abrogated luciferase activity in HEK293T cells. Decitabine reduces bone loss in a mouse model of periodontitis by inhibiting osteoclastogenesis through the upregulation of anti-inflammatory cytokines via KLF2 dependent mechanisms. DNA methyltransferase inhibitors merit further investigation as a possible novel therapy for periodontitis.
RESUMO
Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and regulate innate immune responses. Our model system comprised primary human keratinocytes, which exhibited robust differences in inflammatory cytokine production (interleukin-6 and tumor necrosis factor-alpha) following specific Toll-like receptor 2 and 4 (TLR-2/TLR-4) agonist challenge. We challenged these primary cells with Porphyromonas gingivalis (a Gram-negative bacterium that triggers TLR-2 and TLR-4) and performed miRNA expression profiling. We identified miRNA (miR)-105 as a modulator of TLR-2 protein translation in human gingival keratinocytes. There was a strong inverse correlation between cells that had high cytokine responses following TLR-2 agonist challenge and miR-105 levels. Knock-in and knock-down of miR-105 confirmed this inverse relationship. In silico analysis predicted that miR-105 had complementarity for TLR-2 mRNA, and the luciferase reporter assay verified this. Further understanding of the role of miRNA in host responses may elucidate disease susceptibility and suggest new anti-inflammatory therapeutics.
Assuntos
Queratinócitos/metabolismo , MicroRNAs/fisiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Subunidade p40 da Interleucina-12/genética , Interleucina-6/genética , Queratinócitos/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Oligonucleotídeos/farmacologia , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/genéticaRESUMO
Signals induced by the TCR and CD28 costimulatory pathway have been shown to lead to the inactivation of the constitutively active enzyme, glycogen synthase kinase-3 (GSK3), which has been implicated in the regulation of IL-2 and T cell proliferation. However, it is unknown whether GSK3 plays a similar role in naive and memory CD4(+) T cell responses. Here we demonstrate a divergence in the dependency on the inactivation of GSK3 in the proliferative responses of human naive and memory CD4(+) T cells. We find that although CD28 costimulation increases the frequency of phospho-GSK3 inactivation in TCR-stimulated naive and memory CD4(+) T cells, memory cells are less reliant on GSK3 inactivation for their proliferative responses. Rather we find that GSK3beta plays a previously unrecognized role in the selective regulation of the IL-10 recall response by human memory CD4(+) T cells. Furthermore, GSK3beta-inactivated memory CD4(+) T cells acquired the capacity to suppress the bystander proliferation of CD4(+) T cells in an IL-10-dependent, cell contact-independent manner. Our findings reveal a dichotomy present in the function of GSK3 in distinct human CD4(+) T cell populations.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Quinase 3 da Glicogênio Sintase/fisiologia , Memória Imunológica , Adulto , Efeito Espectador/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Interleucina-10/antagonistas & inibidores , Interleucina-10/biossíntese , Interleucina-10/fisiologia , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Fase de Repouso do Ciclo Celular/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
AIM: The dental plaque is comprised of numerous bacterial species, which may or may not be pathogenic. Human gingival epithelial cells (HGECs) respond to perturbation by various bacteria of the dental plaque by production of different levels of inflammatory cytokines, which is a putative reflection of their virulence. The aim of the current study was to determine responses in terms of interleukin (IL)-1beta, IL-6, IL-8 and IL-10 secretion induced by Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Streptococcus gordonii in order to gauge their virulence potential. MATERIALS AND METHODS: HGECs were challenged with the four bacterial species, live or heat killed, at various multiplicity of infections and the elicited IL-1beta, IL-6, IL-8 and IL-10 responses were assayed by enzyme-linked immunosorbent assay. RESULTS: Primary HGECs challenged with live P. gingivalis produced high levels of IL-1beta, while challenge with live A. actinomycetemcomitans gave high levels of IL-8. The opportunistic pathogen F. nucleatum induces the highest levels of pro-inflammatory cytokines, while the commensal S. gordonii is the least stimulatory. CONCLUSION: We conclude that various dental plaque biofilm bacteria induce different cytokine response profiles in primary HGECs that may reflect their individual virulence or commensal status.
Assuntos
Bactérias/patogenicidade , Placa Dentária/microbiologia , Gengiva/microbiologia , Mediadores da Inflamação/análise , Interleucinas/análise , Aggregatibacter actinomycetemcomitans/patogenicidade , Técnicas Bacteriológicas , Células Cultivadas , Contagem de Colônia Microbiana , Células Epiteliais/microbiologia , Fusobacterium nucleatum/patogenicidade , Gengiva/citologia , Humanos , Interleucina-10/análise , Interleucina-1beta/análise , Interleucina-6/análise , Interleucina-8/análise , Porphyromonas gingivalis/patogenicidade , Streptococcus gordonii/patogenicidade , VirulênciaRESUMO
The role of the adaptor molecule MyD88 is thought to be independent of Toll-like receptor 3 (TLR3) signaling. In this report, we demonstrate a previously unknown role of MyD88 in TLR3 signaling in inducing endogenous ligands of TLR2 to elicit innate immune responses. Of the various TLR ligands examined, the TLR3-specific ligand polyinosinic:polycytidylic acid (poly I:C), significantly induced TNF production and the upregulation of other TLR transcripts, in particular, TLR2. Accordingly, TLR3 stimulation also led to a significant upregulation of endogenous TLR2 ligands mainly, HMGB1 and Hsp60. By contrast, the silencing of TLR3 significantly downregulated MyD88 and TLR2 gene expression and pro-inflammatory IL1ß, TNF, and IL8 secretion. The silencing of MyD88 similarly led to the downregulation of TLR2, IL1ß, TNF and IL8, thus suggesting MyD88 to somehow act downstream of TLR3. Corroborating in vitro data, Myd88-/- knockout mice downregulated TNF, CXCL1; and phospho-p65 and phospho-IRF3 nuclear localization, upon poly I:C treatment in a mouse model of skin infection. Taken together, we identified a previously unknown role for MyD88 in the TLR3 signaling pathway, underlying the importance of TLRs and adapter protein interplay in modulating endogenous TLR ligands culminating in pro-inflammatory cytokine regulation.
Assuntos
Imunidade Inata , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Feminino , Gengiva/citologia , Voluntários Saudáveis , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/genética , TransfecçãoRESUMO
In aggressive periodontitis, the dysbiotic microbial community in the subgingival crevice, which is abundant in Aggregatibacter actinomycetemcomitans, interacts with extra- and intracellular receptors of host cells, leading to exacerbated inflammation and subsequent tissue destruction. Our goal was to understand the innate immune interactions of A. actinomycetemcomitans with macrophages and human gingival epithelial cells (HGECs) on the signaling cascade involved in inflammasome and inflammatory responses. U937 macrophages and HGECs were co-cultured with A. actinomycetemcomitans strain Y4 and key signaling pathways were analyzed using real-time PCR, Western blotting and cytokine production by ELISA. A. actinomycetemcomitans infection upregulated the transcription of TLR2, TLR4, NOD2 and NLRP3 in U937 macrophages, but not in HGECs. Transcription of IL-1ß and IL-18 was upregulated in macrophages and HGECs after 1 h interaction with A. actinomycetemcomitans, but positive regulation persisted only in macrophages, resulting in the presence of IL-1ß in macrophage supernatant. Immunoblot data revealed that A. actinomycetemcomitans induced the phosphorylation of AKT and ERK1/2, possibly leading to activation of the NF-κB pathway in macrophages. On the other hand, HGEC signaling induced by A. actinomycetemcomitans was distinct, since AKT and 4EBP1 were phosphorylated after stimulation with A. actinomycetemcomitans, whereas ERK1/2 was not. Furthermore, A. actinomycetemcomitans was able to induce the cleavage of caspase-1 in U937 macrophages in an NRLP3-dependent pathway. Differences in host cell responses, such as those seen between HGECs and macrophages, suggested that survival of A. actinomycetemcomitans in periodontal tissues may be favored by its ability to differentially activate host cells.
RESUMO
BACKGROUND: The oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear. RESULTS: We demonstrate that primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main P. gingivalis proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live P. gingivalis can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism. CONCLUSION: The present study provides evidence that live, but not heat-killed, P. gingivalis can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in P. gingivalis elicited apoptosis.
Assuntos
Adesinas Bacterianas/metabolismo , Apoptose , Cisteína Endopeptidases/metabolismo , Células Epiteliais/patologia , Gengiva/citologia , Porphyromonas gingivalis/enzimologia , Caspase 3/metabolismo , Células Cultivadas , Fragmentação do DNA , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Cisteína Endopeptidases Gingipaínas , Gengiva/microbiologia , HumanosRESUMO
BACKGROUND: Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria. RESULTS: We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes. CONCLUSION: We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.
Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Apoptose , Aderência Bacteriana , Células Cultivadas , Citocinas/metabolismo , Células Epiteliais/metabolismo , Gengiva/microbiologia , HumanosRESUMO
The oral cavity is home for a plethora of bacteria and viruses. Epithelial barriers encounter these micro-organisms and recognize them via pathogen recognition receptors (PRRs) that instigate antibacterial and antiviral responses. We and others have shown that human gingival epithelial cells (HGECs) express PRRs to defend invading pathogens. Among these PRRs, TLR2, TLR3 and TLR4 are highly expressed in HGECs and appear to be important based on our previous findings. IFN-ß is one of the major type 1 interferons induced to defend viral attack. In this report, we sought to dissect TLR3 and TLR4 mediated secretion of IFN-ß in HGECs. We stimulated HGECs with ultrapure LPS (TLR4 ligand) and Poly I:C (TLR3 ligand) for 24 h and supernatant was used to determine IFN-ß secretion. We show that cells treated with Poly I:C induced IFN-ß secretion but not cells treated with LPS. In addition, silencing of TLR3 prior to Poly I:C stimulation significantly downregulated IFN-ß secretion. On the contrary, overexpression of MD2 and TLR4 in HGECs restored IFN-ß secretion. Upon further evaluation, we found that TLR3 stimulation but not TLR4 induced the phosphorylation of interferon regulatory factor 3 (IRF3), which is critical for IFN-ß secretion. We conclude that IFN-ß secretion is through TLR3 and not via TLR4 in HGECs.
Assuntos
Células Epiteliais/metabolismo , Gengiva/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Células Cultivadas , Humanos , Interferon beta/metabolismo , Poli I-C/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Periodontitis is a chronic human inflammatory disease initiated and sustained by dental plaque microorganisms. A major contributing pathogen is Porphyromonas gingivalis, a gram-negative bacterium recognized by Toll-like receptor 2 (TLR2) and TLR4, which are expressed by human gingival epithelial cells (HGECs). However, it is still unclear how these cells respond to P. gingivalis and initiate inflammatory and immune responses. We have reported previously that HGECs produce a wide range of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and IL-1beta. In this study, we show that IL-1beta has a special role in the modulation of other inflammatory cytokines in HGECs challenged with P. gingivalis. Our results show that the increased production of IL-1beta correlates with the cell surface expression of TLR4, and more specifically, TLR4-normal HGECs produce fourfold more IL-1beta than do TLR4-deficient HGECs after challenge. Moreover, blocking the IL-1beta receptor greatly reduces the production of "secondary" proinflammatory cytokines such as IL-8 or IL-6. Our data indicate that the induction of IL-1beta plays an important role in mediating the release of other proinflammatory cytokines from primary human epithelial cells following challenge with P. gingivalis, and this process may be an inflammatory enhancement mechanism adopted by epithelial cells.
Assuntos
Citocinas/biossíntese , Células Epiteliais/imunologia , Interleucina-1beta/biossíntese , Porphyromonas gingivalis/imunologia , Células Cultivadas , Inativação Gênica , Humanos , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/imunologiaRESUMO
Dental plaque, a microbial biofilm that accumulates on teeth and initiates periodontal disease, is composed of hundreds of different bacterial species within an organized structure. The biofilm bacteria and their byproducts irritate the gingival epithelium and induce an "inflammatory response". The perturbation of epithelial cells by bacteria is the first stage in the initiation of inflammatory and immune processes which eventually cause destruction of the tissues surrounding and supporting the teeth, and ultimately result in tooth loss. This review addresses the early bacterial-epithelial cell interactions and the subsequent responses of the epithelial cell. It includes discussion of how epithelial Toll-like receptors (TLRs) respond to different bacterial challenges, the variable antimicrobial peptides released and the host signaling responses which trigger release of these molecules and the overall fate of these cells in terms of survival, apoptosis, or cell lysis.
Assuntos
Células Epiteliais/microbiologia , Porphyromonas gingivalis/metabolismo , Animais , Apoptose , Biofilmes , Adesão Celular , Células Epiteliais/metabolismo , Genótipo , Humanos , Inflamação , Lipopolissacarídeos/metabolismo , Modelos Biológicos , Boca/microbiologia , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP - rs4073 (alias -251), TT genotype at the second SNP - rs2227307 (alias +396), TC or CC genotypes at the third SNP - rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma Humano , Neutrófilos/metabolismo , Polimorfismo de Nucleotídeo Único , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Células HEK293 , Humanos , Interleucina-8/biossíntese , Interleucina-8/genética , Poli I-C/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.