RESUMO
Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a pronounced inflammatory response associated with ulcer development. Monocytes/macrophages, the main cells harboring parasites, are largely responsible for parasite control. Toll-like receptor (TLR) signaling leads to the transcription of inflammatory mediators, such as IL-1ß and TNF during innate immune response. TLR antagonists have been used in the treatment of inflammatory disease. The neutralization of these receptors may attenuate an exacerbated inflammatory response. We evaluated the ability of TLR2 and TLR4 antagonists to modulate host immune response in L. braziliensis-infected monocytes and cells from CL patient skin lesions. Following TLR2 and TLR4 neutralization, decreased numbers of infected cells and internalized parasites were detected in CL patient monocytes. In addition, reductions in oxidative burst, IL-1ß, TNF and CXCL9 production were observed. TNF production by cells from CL lesions also decreased after TLR2 and TLR4 neutralization. The attenuation of host inflammatory response after neutralizing these receptors suggests the potential of TLR antagonists as immunomodulators in association with antimonial therapy in human cutaneous leishmaniasis.
Assuntos
Leishmaniose Cutânea/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Adolescente , Adulto , Células Cultivadas , Feminino , Humanos , Inflamação/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Carga Parasitária , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Adulto JovemRESUMO
Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.
Assuntos
Lipopeptídeos/farmacologia , Periodontite/etiologia , Periodontite/patologia , Receptor 2 Toll-Like/antagonistas & inibidores , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/patologia , Animais , Modelos Animais de Doenças , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengivite/etiologia , Gengivite/patologia , Masculino , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Periodontite/microbiologia , Distribuição Aleatória , Fosfatase Ácida Resistente a Tartarato , Fatores de Tempo , Microtomografia por Raio-XRESUMO
Abstract Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.
Assuntos
Animais , Masculino , Periodontite/etiologia , Periodontite/patologia , Receptor 2 Toll-Like/antagonistas & inibidores , Lipopeptídeos/farmacologia , Osteoclastos/efeitos dos fármacos , Periodontite/microbiologia , Fatores de Tempo , Distribuição Aleatória , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Modelos Animais de Doenças , Microtomografia por Raio-X , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/patologia , Fosfatase Ácida Resistente a Tartarato , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengivite/etiologia , Gengivite/patologia , Camundongos Endogâmicos C57BLRESUMO
Abstract Lipoproteins are important bacterial immunostimulating molecules capable of inducing receptor activator of nuclear factor-κB (RANKL) and osteoclast formation in vitro and in vivo . Although these molecules are present in periodontopathogenic bacteria, their role in periodontitis is not known. In this study, we used Pam2CSK4 (PAM2), a synthetic molecule that mimics bacterial lipoprotein, to investigate the effects of lipoproteins on periodontitis in mice. C57BL/6 male mice were randomly divided into three experimental groups: 1) Negative control group: animals received vehicle injection; 2) Positive control group: animals received injection of Escherichia coli lipopolysaccharide (LPS); 3) PAM2 group: animals received PAM2 injection. All the injections were performed bilaterally every other day into the palatal mucosa between first and second molars. After twenty-four days, the animals were euthanized to assess alveolar bone volume (micro-CT), cellular and extracellular composition in the gingiva (stereometric analysis), and osteoclast numbers (TRAP staining). Treatment with either PAM2 or LPS induced gingival inflammation, as demonstrated by increased infiltration of inflammatory cells and enhanced angiogenesis, associated with a smaller number of fibroblasts and decreased extracellular matrix. Importantly, treatment not only with LPS but also with PAM2 resulted in a larger number of TRAP+ multinucleated osteoclasts and significant loss of alveolar bone. Collectively, our data demonstrate that PAM2 can induce gingival inflammation and bone loss in mice, broadening the avenues of investigation into the role of lipoproteins in the pathogenesis of periodontal disease.
Assuntos
Animais , Masculino , Periodontite/etiologia , Periodontite/patologia , Receptor 2 Toll-Like/antagonistas & inibidores , Lipopeptídeos/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Periodontite/microbiologia , Fatores de Tempo , Distribuição Aleatória , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/patologia , Modelos Animais de Doenças , Microtomografia por Raio-X , Processo Alveolar/efeitos dos fármacos , Processo Alveolar/patologia , Fosfatase Ácida Resistente a Tartarato , Gengiva/efeitos dos fármacos , Gengiva/patologia , Gengivite/etiologia , Gengivite/patologia , Camundongos Endogâmicos C57BLRESUMO
The nitrone spin trap 5,5dimethyl1pyrroline Noxide (DMPO) dampens endotoxin-induced and TLR4-driven priming of macrophages, but the mechanism remains unknown. The available information suggests a direct binding of DMPO to the TIR domain, which is shared between TLRs. However, TLR2-TIR domain is the only TLR that have been crystallized. Our in silico data show that DMPO binds to four specific residues in the BB-loop within the TLR2-TIR domain. Our functional analysis using hTLR2.6-expressing HEKs cells showed that DMPO can block zymosan-triggered-TLR2-mediated NF-κB activation. However, DMPO did not affect the overall TLR2-MyD88 protein-protein interaction. DMPO binds to the BB-loop in the TIR-domain and dampens downstream signaling without affecting the overall TIR-MyD88 interaction. These data encourage the use of DMPO-derivatives as potential mechanism-based inhibitors of TLR-triggered inflammation.
Assuntos
Óxidos N-Cíclicos/metabolismo , Inflamação/metabolismo , Óxidos de Nitrogênio/metabolismo , Transdução de Sinais , Marcadores de Spin , Receptor 2 Toll-Like/metabolismo , Animais , Óxidos N-Cíclicos/química , Células HEK293 , Humanos , Inflamação/imunologia , Camundongos , Simulação de Dinâmica Molecular , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Óxidos de Nitrogênio/química , Ligação Proteica , Domínios Proteicos , Células RAW 264.7 , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/químicaRESUMO
Quercetin, a dietary flavonoid abundant in fruits, vegetables, and herbs, presents various pharmacological effects. This study aimed to investigate the anti-inflammatory effect and the underlying mechanism of quercetin in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs). Cell viability was measured by the Cell Counting Kit-8 assay. The mRNA expression of Toll-like receptor 2 (TLR2) was assessed by quantitative real-time polymerase chain reaction. Inflammatory cytokine secretions and nuclear factor (NF)-kB levels were analyzed by enzyme-linked immunosorbent assay. Our findings showed that quercetin significantly reduced LPS-induced cytotoxicity in human PBMCs. Quercetin suppressed the secretion of tumor necrosis factor-a, interleukin (IL)-1b, and IL-6 in LPS-stimulated human PBMCs. Moreover, quercetin reduced the LPS-induced increase in the expression of TLR2 mRNA and decreased the NF-kB concentration in LPS-stimulated human PBMCs. The data indicates that quercetin plays an important role in LPS-induced inflammation in human PBMCs via suppression of the TLR2-NF-kB pathway.
Assuntos
Inflamação/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Quercetina/farmacologia , Receptor 2 Toll-Like/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Inflamação/sangue , Interleucina-1beta/genética , Interleucina-6/genética , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/farmacologia , NF-kappa B/sangue , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
Vitamin D is an immunomodulator that exerts anti-inflammatory effects. In this work, the effects of cholecalciferol, a vitamin D precursor, on the inflammatory response of bovine mammary epithelial cells (bMECs) during the internalization of Staphylococcus aureus were analyzed. Cholecalciferol and S. aureus inhibited TLR2 mRNA expression, but cholecalciferol differentially modulated the TLR2 membrane abundance. In fact, 50 nM cholecalciferol inhibited the TLR2 membrane abundance in bMECs infected with S. aureus, and this concentration also exerted the highest inhibitory effect on internalization. Cholecalciferol down-regulated the mRNA expression of TNF-α and IL-1ß and up-regulated that of RANTES and IL-10 but did not modify IL-6 and IL-8 expression. S. aureus strongly induced the mRNA expression of TNF-α, RANTES and IL-10 and inhibited IL-8 expression. Interestingly, cholecalciferol pre-treatments inhibited the bacterial-induced expression of TNF-α, IL-1ß, RANTES and IL-10. In conclusion, cholecalciferol differentially regulates the inflammatory response of bMECs during S. aureus internalization and may be an effective innate immunity modulator in mammary gland tissues.
Assuntos
Colecalciferol/metabolismo , Endocitose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Fatores Imunológicos/metabolismo , Staphylococcus aureus/imunologia , Staphylococcus aureus/fisiologia , Animais , Bovinos , Células Cultivadas , Citocinas/biossíntese , Células Epiteliais/imunologia , Células Epiteliais/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor 2 Toll-Like/análise , Receptor 2 Toll-Like/antagonistas & inibidoresRESUMO
UNLABELLED: Toll-like receptor (TLR)2, TLR4 and CD36 are central in inflammation and the development of atherosclerosis. Oxidized low-density lipoprotein (oxLDL) plays a critical role in this disease through its involvement in the formation of foam cells and the activation of leukocytes. The aim of this research was to analyze the role of TLR2, TLR4 and CD36 in foam cell differentiation and macrophage activation. METHODS: Human macrophages were incubated with monoclonal antibodies specific for TLR2, TLR4 and CD36 prior to stimulation with oxLDL. Subsequently, we analyzed foam cell formation, cytokine secretion, histocompatibility complex (MHC) class II molecules and CD86 expression and T cell proliferation. RESULTS: The stimulation of macrophages with oxLDL induced foam cell formation, cytokine secretion, HLA-DR and CD86 expression and T cell proliferation. The blockage of TLR2, TLR4 and CD36 reduced the secretion of IL-1ß, IL-6 and IL-8, the expression of HLA-DR and CD86, T cell proliferation and foam cell formation. However, the blockage of TLR2 did not affect the formation of foam cells. CONCLUSION: Our study demonstrates that TLR2, TLR4 and CD36 participate in the immune response to oxLDL by inducing an increase in pro-inflammatory cytokines, the expression HLA-DR and CD86 and the proliferation of T cells. However, TLR2 does not participate in the formation of foam cells, while TLR4 and CD36 play a relevant role in this process. These findings suggest that the activation of these receptors by oxLDL contributes to the pathogenesis of atherosclerosis.
Assuntos
Antígenos CD36/metabolismo , Células Espumosas , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Anticorpos Monoclonais/farmacologia , Antígeno B7-2/metabolismo , Antígenos CD36/antagonistas & inibidores , Células Cultivadas , Citocinas/biossíntese , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Voluntários Saudáveis , Humanos , Lipoproteínas LDL/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Masculino , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Adulto JovemRESUMO
Ischemia and reperfusion injury (IR) is an antigen independent inflammatory process that causes tissue damage. After IR, kidneys up-regulate leukocyte adhesion molecules and toll-like receptors (TLRs). Moreover, injured kidneys can also secrete factors (i.e. heat shock protein) which bind to TLRs and trigger intracellular events culminating with the increase in the gene expression of inflammatory cytokines. FTY720 is an immunomodulatory compound and protects at least in part kidneys submitted to IR. The mechanisms associated with FTY720's beneficial effects on kidneys after IR remain elusive. We investigated whether FTY720 administration in mice submitted to kidney IR is associated with modulation of TLR2 and TLR4 expression. C57BL/6 mice submitted to 30min of renal pedicles clamp were evaluated for serum parameters (creatinine, urea and nitric oxide), kidney histology, spleen and kidney infiltrating cells expression of TLR2 and TLR4, resident kidney cells expression of TLR2 and TLR4 and IL-6 protein expression in kidney. FTY720-treated mice presented decrease in serum creatinine, urea and nitric oxide, diminished expression of TLR2 and TLR4 both in spleen and kidney infiltrating cells, and reduced kidney IL-6 protein expression in comparison with IR non-treated mice. However, acute tubular necrosis was present both in IR non-treated and IR+FTY720-treated groups. Also, FTY720 did not prevent TLR2 and TLR4 expression in kidney resident cells. In conclusion, FTY720 can promote kidney function recovery after IR by reducing the inflammatory process. Further studies are needed in order to establish whether TLR2 and TLR4 down regulation should be therapeutically addressed as protective targets of renal function and structure after IR.
Assuntos
Propilenoglicóis/farmacologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Esfingosina/análogos & derivados , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/genética , Animais , Creatinina/sangue , Cloridrato de Fingolimode , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/sangue , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Esfingosina/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismo , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Ureia/sangueRESUMO
Alcohol has a variety of short- and long-term effects on cell-mediated and humoral immune response. Herein, we have characterized the impact of high-dose EtOH administration on phenotypic and functional features of murine APC subsets, including dendritic cell (DC), macrophages and B cells. Impaired cytokine synthesis and Leishmania-phagocytosis was observed in peritoneal macrophages following EtOH administration. Moreover, EtOH exposure led to decreased levels of splenic myeloid DC and increased percentage of macrophages with no changes in splenic lymphoid DC and B cells. Adverse effects of short-term EtOH administration also resulted in impaired OVA-endocytosis by DC and macrophages. In contrast, EtOH consumption upregulates OVA-internalization by B cells. These changes on APC hierarchy may play a role shifting the fate of the immune response after EtOH ingestion. In addition to an overall downregulation of Toll-like receptor-TLR-4 expression by splenic APC, a downregulation of TLR-2 expression in macrophages was observed. Moreover, EtOH exposure altered the expression of co-signalling molecules on splenic APC, downregulating CD40 on macrophages and upregulating CD80 on B cells, with no impact on DC subsets. The net result of changes in TLR-mediated and co-stimulatory signals may determine the altered immunological status induced by acute consumption of alcohol. A direct impact of high-dose EtOH administration in the activation status of splenic CD4(+) T cells was observed. Together, our results demonstrated that short-term high-dose EtOH administration has differential impact on APC populations, downregulating splenic macrophages and DC activity but up-regulating B lymphocyte function as APC, and ultimately yielding a micro-environment that led to increased activation of CD4(+) T cells.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Etanol/administração & dosagem , Macrófagos/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos B/efeitos dos fármacos , Antígeno B7-1/efeitos dos fármacos , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/efeitos dos fármacos , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Feminino , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
The aims of the present study were to investigate the expression of toll-like receptor 2 (TLR2) in muscle and white adipose tissue (WAT) of diet-induced obesity (DIO) mice, and also the effects of its inhibition, with the use of TLR2 antisense oligonucleotide (ASON), on insulin sensitivity and signaling. The expression of TLR2 was increased in muscle and WAT of DIO mice, compared with those that received standard chow. Inhibition of TLR2 in DIO mice, by TLR2 ASON, improved insulin sensitivity and signaling in muscle and WAT. In addition, data show that the inhibition of TLR2 expression prevents the activation of IKBKB, MAPK8, and serine phosphorylation of IRS1 in DIO mice, suggesting that TLR2 is a key modulator of the crosstalk between inflammatory and metabolic pathways. We, therefore, suggest that a selective interference with TLR2 presents an attractive opportunity for the treatment of insulin resistance in obesity and type 2 diabetes.