Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 17 de 17
Filtrar
1.
Artigo em Inglês | MEDLINE | ID: mdl-37302095

RESUMO

BACKGROUND: Toll-like receptor 9 (TLR9), located in the endosomal compartment, is known to play a role in inflammation by recognizing oligonucleotides that contain CpG motive (CpG-ODN). Signaling by TLR9 leads to the production of proinflammatory cytokines and can trigger cell death. OBJECTIVE: This study aims to investigate the molecular mechanism of pyroptosis induced by ODN1826 in the mouse macrophage cell line (Raw264.7). METHODS: The protein expression and the amount of lactate dehydrogenase (LDH) of ODN1826-treated cells were determined by immunoblotting and LDH assay, respectively. In addition, the level of cytokine production was observed by ELISA assay and the ROS production was determined by flow cytometry. RESULTS: Our results showed that ODN1826 induced pyroptosis as judged by LDH releases. Furthermore, caspase-11 and gasdermin D activation, which are the key molecules in pyroptosis, were also observed in ODN1826-activated cells. Moreover, we also demonstrated that Reactive Oxygen Species (ROS) production by ODN1826 is essential for caspase-11 activation and gasdermin D release, which leads to pyroptosis. CONCLUSIONS: ODN1826 induces pyroptosis in Raw264.7 cells via caspase-11 and GSDMD activation. Moreover, the production of ROS by this ligand plays an essential role in the regulation of caspase-11 and GSDMD activation, which then controls pyroptosis in TLR9 activation.

2.
Artigo em Inglês | MEDLINE | ID: mdl-36278779

RESUMO

BACKGROUND: NLRP12 has been shown to play an essential role as a negative regulator in several bacterial infection. OBJECTIVE: The purpose of this study is to elucidate the role of NLRP12 in B. pseudomallei-infected RAW264.7 macrophages. METHODS: The protein expression and the level of TNF-α production were determined by immunoblotting and ELISA assay, respectively. RESULTS: The results demonstrated that unlike the LPS-mutant strain which lacks O antigenic polysaccharide, the wild-type B. pseudomallei was able to upregulate NLRP12 protein expression in RAW264.7 macrophages. NLRP12 expression also correlated with the suppression of TNF-α production as demonstrated in wild-type B. pseudomallei-infected Nlrp12-depleted macrophages when compared to that of the control siRNA-transfected cells. The expression of NLRP12 was also inhibited in cytochalasin D treated cells. CONCLUSIONS: Our findings showed that wild-type B. pseudomallei can activate NLRP12 expression leading to the suppression of TNF-α production. It is possible that the regulation of NLRP12 may contribute to the pathogenesis of B. pseudomallei infection in melioidosis patients.

3.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31818963

RESUMO

Melioidosis is an infectious disease with a high mortality rate responsible for community-acquired sepsis in Southeast Asia and Northern Australia. The causative agent of this disease is Burkholderia pseudomallei, a Gram-negative bacterium that resides in soil and contaminated natural water. After entering into host cells, the bacteria escape into the cytoplasm, which has numerous cytosolic sensors, including the noncanonical inflammatory caspases. Although the noncanonical inflammasome (caspase-11) has been investigated in a murine model of B. pseudomallei infection, its role in humans, particularly in lung epithelial cells, remains unknown. We, therefore, investigated the function of caspase-4 (ortholog of murine caspase-11) in intracellular killing of B. pseudomallei The results showed that B. pseudomallei induced caspase-4 activation at 12 h postinfection in human alveolar epithelial A549 cells. The number of intracellular B. pseudomallei bacteria was increased in the absence of caspase-4, suggesting its function in intracellular bacterial restriction. In contrast, a high level of caspase-4 processing was observed when cells were infected with lipopolysaccharide (LPS) mutant B. pseudomallei The enhanced bacterial clearance in LPS-mutant-infected cells is also correlated with a higher degree of caspase-4 activation. These results highlight the susceptibility of the LPS mutant to caspase-4-mediated intracellular bacterial killing.


Assuntos
Células Epiteliais Alveolares/fisiologia , Burkholderia pseudomallei/patogenicidade , Caspases Iniciadoras/fisiologia , Melioidose/imunologia , Animais , Burkholderia pseudomallei/fisiologia , Melioidose/microbiologia , Camundongos
4.
Inflamm Res ; 68(10): 841-844, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31292668

RESUMO

OBJECTIVE: The aim of the present study is to investigate the participation of NLRP12 in Porphyromonas gingivalis LPS-activated mouse macrophages. METHODS: NLRP12-depleted mouse macrophages were stimulated with P. gingivalis LPS (1 µg/ml.). At indicated time points, the treated cells were lysed and the supernatant from treated cells was collected. Gene and protein expression of NLRP12 and iNOS were determined by RT-PCR and immunoblotting, respectively. The level of TNF-α production in the supernatant of the activated cells was determined by ELISA. RESULTS AND CONCLUSION: NLRP12 was upregulated in response to stimulation with P. gingivalis LPS. In addition, when NLRP12 was depleted in P. gingivalis LPS-treated macrophages, an increase in TNF-α production and iNOS expression were observed when compared to those of the control cells, indicating that NLRP12 downregulates the inflammatory cytokine and antimicrobial molecule production in the macrophages.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Porphyromonas gingivalis , Células RAW 264.7
5.
Inflamm Res ; 67(9): 723-726, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29980803

RESUMO

OBJECTIVE: The aim of this study is to investigate the involvement of TLR9 in the regulation of iNOS expression and nitric oxide (NO) production in Porphyromonas gingivalis LPS-treated mouse macrophages. METHODS: Mouse macrophage cell line (RAW264.7) was transfected with siRNAs against TLR9 and then stimulated with P. gingivalis LPS. At indicated time points, the activated cells were lysed. Gene and protein expression of iNOS were determined by RT-PCR and immunoblotting, respectively. The level of nitric oxide (NO) production in the supernatant of the activated cells was determined by Griess reaction assay. RESULTS AND CONCLUSION: Depletion of TLR9 in mouse macrophages demonstrated the markedly decreased iNOS gene and protein expression by P. gingivalis LPS compared to those of the wild-type or control siRNA transfected cells. In consistent with these results, the level of NO secretion was also significantly diminished in TLR9-depleted cells after challenged with P. gingivalis LPS. These results indicate that TLR9 involves in the regulation of the iNOS expression and the NO secretion in P. gingivalis LPS-treated macrophages.


Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Indução Enzimática/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/genética , Porphyromonas gingivalis , Células RAW 264.7 , RNA Mensageiro/metabolismo
6.
Inflamm Res ; 66(10): 843-853, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28593434

RESUMO

OBJECTIVE: The aim of this study was to investigate the involvement of TLR adaptor molecules, such as TRIF, MyD88, and TBK1 in the induction of iNOS and nitric oxide (NO) production in Pam2CSK4 and Pam3CSK4-treated mouse macrophages. METHOD: Mouse macrophage cell line (RAW264.7) was transfected with trif, myd88, and tbk1 siRNAs before stimulated with Pam2CSK4 and Pam3CSK4. The iNOS gene and protein expression were determined by RT-PCR and immunoblotting, respectively. The NO production was determined by Griess reaction assay. RESULTS: The results showed that the induction of iNOS expression and NO production by Pam2CSK4 and Pam3CSK4 were diminished in tbk1 and myd88-depleted mouse macrophages but not trif-depleted cells. CONCLUSION: These results suggested that the TBK1 and MyD88 molecules were essential for the induction of iNOS expression and NO production by both Pam2CSK4 and Pam3CSK4 via TLR2 signaling.


Assuntos
Lipopeptídeos/imunologia , Fator 88 de Diferenciação Mieloide/biossíntese , Óxido Nítrico Sintase Tipo II/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Adaptadoras de Transporte Vesicular/efeitos dos fármacos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Expressão Gênica/efeitos dos fármacos , Ligantes , Lipopeptídeos/farmacologia , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Proteínas Serina-Treonina Quinases/genética , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo
7.
Inflamm Res ; 66(12): 1099-1105, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28889202

RESUMO

INTRODUCTION: We aimed to investigate the involvement of surface TLRs and endosomal TLRs in the regulation of SARM expression by TLR2 ligands (Pam2CSK4 and Pam3CSK4). MATERIALS AND METHODS: Mouse macrophage cell line (RAW264.7) was treated with either Pam2CSK4 or Pam3CSK4 (TLR2 ligands) at a concentration of 100 ng/ml. At indicated time points, the treated cells were lysed. The gene and protein expression of SARM were determined by RT-PCR and immunoblotting, respectively. For silencing of TLR9 function, the cells were transfected with TLR9 siRNAs before stimulation by these two TLR2 ligands RESULTS: The SARM expression was upregulated at both transcriptional and translational levels in time-dependent manner during activation of Pam2CSK4 and Pam3CSK4 in mouse macrophages. Blocking of ligand internalization by cytochalasin D showed interference effect with SARM expression. Moreover, our results also demonstrated that endosomal acidification and TLR9 were required for SARM expression suggesting the essential role of endosomal compartment acidification and TLR9 in regulating SARM expression. CONCLUSION: Our findings suggested the collaboration of TLR2-TLR9 at least in the regulation of SARM expression. However, the underlying mechanism that participated in these two TLRs cooperation is underinvestigated.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Proteínas do Domínio Armadillo/genética , Proteínas do Citoesqueleto/genética , Ligantes , Lipopeptídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células RAW 264.7 , RNA Interferente Pequeno/genética , Receptor 2 Toll-Like/imunologia , Receptor Toll-Like 9/genética
8.
PLoS Pathog ; 10(3): e1003986, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24626296

RESUMO

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1ß and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1ß secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1ß, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.


Assuntos
Infecções por Burkholderia/imunologia , Burkholderia pseudomallei/imunologia , Inflamassomos/imunologia , Macrófagos/microbiologia , Transdução de Sinais/imunologia , Animais , Sistemas de Secreção Bacterianos/fisiologia , Western Blotting , Infecções por Burkholderia/metabolismo , Burkholderia pseudomallei/metabolismo , Caspase 1/metabolismo , Morte Celular/fisiologia , Modelos Animais de Doenças , Citometria de Fluxo , Células HEK293 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Transfecção
9.
Med Microbiol Immunol ; 205(3): 255-60, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26563410

RESUMO

Burkholderia pseudomallei, a gram-negative intracellular bacterium, is a causative agent of melioidosis. The bacterium has been shown to induce the innate immune response, particularly pro-inflammatory cytokine production in several of both mouse and human cell types. In the present study, we investigate host immune response in B. pseudomallei-infected primary human monocytes. We discover that wild-type B. pseudomallei is able to survive and multiply inside the primary human monocytes. In contrast, B. pseudomallei LPS mutant, a less virulent strain, is susceptible to host killing during bacterial infection. Moreover, microarray result showed that wild-type B. pseudomallei but not B. pseudomallei LPS mutant is able to activate gene expression of IL-23 as demonstrated by the up-regulation of p19 and p40 subunit expression. Consistent with gene expression analysis, the secretion of IL-23 analyzed by ELISA also showed that wild-type B. pseudomallei induces a significantly higher level of IL-23 secretion than that of B. pseudomallei LPS mutant. These results implied that IL-23 may be an important cytokine for the innate immune response during B. pseudomallei infection. The regulation of IL-23 production may drive the different host innate immune responses between patients and may relate to the severity of melioidosis.


Assuntos
Burkholderia pseudomallei/imunologia , Imunidade Inata , Subunidade p19 da Interleucina-23/biossíntese , Monócitos/imunologia , Monócitos/microbiologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Humanos , Subunidade p19 da Interleucina-23/metabolismo , Análise em Microsséries , Viabilidade Microbiana
10.
J Oral Sci ; 66(1): 5-8, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-37853618

RESUMO

PURPOSE: This study investigated the efficacy of various antiseptics for disinfection of rubber dams used during endodontic treatment, the duration of disinfection effectiveness, and the disinfection protocol employed by dental schools in Thailand. METHODS: The efficacy of 10% povidone-iodine, 1.5% tincture iodine and 70% ethyl alcohol in eliminating Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans) on the rubber dam was investigated. Time duration of disinfection was evaluated at 0, 30, 60, and 120 min. The two-step disinfection method adopted at Thai dental school was examined. Independent t-test or Kruskal-Wallis followed by a Dunnett's test was used for statistical analysis. RESULTS: Among the three antiseptics, 10% povidone and 1.5% tincture iodine eradicated the microorganisms completely, whereas 70% ethyl alcohol did not achieve a statistically significant decrease. The duration of sterilization effectiveness was 120 min for 10% povidone-iodine, but bacteria were eliminated only at 0 min by 1.5% tincture iodine. The results also indicated that the two-step protocol scarcely eliminated the microorganisms. CONCLUSION: The best antiseptic for rubber dam disinfection is 10% povidone-iodine, which remains effective for 120 min. The two-step protocol typically practiced in Thai dental schools needs to be updated. The use of 10% povidone-iodine alone is adequate for complete eradication of E. faecalis and C. albicans.


Assuntos
Anti-Infecciosos Locais , Iodo , Anti-Infecciosos Locais/farmacologia , Povidona-Iodo/farmacologia , Diques de Borracha , Desinfecção/métodos , Etanol
11.
PLoS Negl Trop Dis ; 18(7): e0012368, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39042701

RESUMO

Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1ß and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients.


Assuntos
Burkholderia pseudomallei , Burkholderia , Melioidose , Monócitos , Piroptose , Sepse , Humanos , Burkholderia pseudomallei/imunologia , Burkholderia pseudomallei/fisiologia , Monócitos/imunologia , Monócitos/microbiologia , Melioidose/microbiologia , Melioidose/imunologia , Burkholderia/patogenicidade , Sepse/microbiologia , Sepse/imunologia , Citocinas/metabolismo , Células THP-1 , Imunidade Inata , Células Cultivadas
12.
PLoS One ; 18(11): e0292340, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38011122

RESUMO

BACKGROUND: Cleistanthin A (CA), extracted from Phyllanthus taxodiifolius Beille, was previously reported as a potential V-ATPase inhibitor relevant to cancer cell survival. In the present study, ECDD-S16, a derivative of cleistanthin A, was investigated and found to interfere with pyroptosis induction via V-ATPase inhibition. OBJECTIVE: This study examined the ability of ECDD-S16 to inhibit endolysosome acidification leading to the attenuation of pyroptosis in Raw264.7 macrophages activated by both surface and endosomal TLR ligands. METHODS: To elucidate the activity of ECDD-S16 on pyroptosis-induced inflammation, Raw264.7 cells were pretreated with the compound before stimulation with surface and endosomal TLR ligands. The release of lactate dehydrogenase (LDH) was determined by LDH assay. Additionally, the production of cytokines and the expression of pyroptosis markers were examined by ELISA and immunoblotting. Moreover, molecular docking was performed to demonstrate the binding of ECDD-S16 to the vacuolar (V-)ATPase. RESULTS: This study showed that ECDD-S16 could inhibit pyroptosis in Raw264.7 cells activated with surface and endosomal TLR ligands. The attenuation of pyroptosis by ECDD-S16 was due to the impairment of endosome acidification, which also led to decreased Reactive Oxygen Species (ROS) production. Furthermore, molecular docking also showed the possibility of inhibiting endosome acidification by the binding of ECDD-S16 to the vacuolar (V-)ATPase in the region of V0. CONCLUSION: Our findings indicate the potential of ECDD-S16 for inhibiting pyroptosis and prove that vacuolar H+ ATPase is essential for pyroptosis induced by TLR ligands.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Piroptose , Simulação de Acoplamento Molecular , Inflamação
13.
Polymers (Basel) ; 14(16)2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-36015520

RESUMO

The antimicrobial properties, cell cytotoxicity and surface hardness of soft lining materials (GC soft liner, Viscogel and Coe comfort) incorporated with various concentrations of Litsea cubeba essential oil (LCEO) were evaluated. The minimum inhibitory concentrations of LCEO against Candida albicans and Streptococcus mutans were 1.25% v/v and 10% v/v, respectively. However, when LCEO was incorporated into the three soft lining materials (GC soft liner, Viscogel and Coe comfort), 10% v/v and 30% v/v of LCEO could inhibit the growth of C. albicans and S. mutans, respectively. The extracts of soft lining materials with 10% and 30% v/v LCEO, 2% chlorhexidine, 30% v/v nystatin and no additive were used for cytotoxicity tests on a human gingival fibroblast cell line. There was no significant difference in cell viability in all groups with additives compared to the no additive group (p > 0.05). Surface hardness increased significantly between 2 h and 7 day incubation times in all groups, including the controls (p < 0.05). A higher LCEO concentration had a dose-dependent effect on the surface hardness of all soft lining materials (p < 0.05). However, the surface hardness of materials with additive remained in accordance with ISO 10139-1. LCEO could be used as a natural product against oral pathogens, without having a negative impact on soft lining materials.

14.
Microbiol Spectr ; 10(5): e0348822, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36194127

RESUMO

Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-ß, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention. IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-ß, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.


Assuntos
Burkholderia pseudomallei , Melioidose , Camundongos , Animais , Burkholderia pseudomallei/metabolismo , Receptor Toll-Like 9/metabolismo , Receptor 2 Toll-Like/metabolismo , Piroptose , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/metabolismo , Melioidose/metabolismo , Melioidose/microbiologia , Macrófagos , Linhagem Celular , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Citocinas/metabolismo , Caspases/metabolismo , Lactato Desidrogenases/metabolismo
16.
Pathog Dis ; 71(1): 69-72, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24376210

RESUMO

Burkholderia pseudomallei is a Gram-negative intracellular bacterium and the causative agent of melioidosis. Innate immune mechanisms against this pathogen, which might contribute to outcomes of melioidosis, are little known. We demonstrated here that B. pseudomallei could activate NADPH oxidase in primary human monocytes as judged by production of reactive oxygen species (ROS) and p40(phox) phosphorylation after infection. However, as similar to other intracellular bacteria, this bacterium was able to resist and multiply inside monocytes despite being able to activate NADPH oxidase. In the presence of NADPH oxidase inhibitor, diphenyleneiodonium or apocynin, intracellular multiplication of B. pseudomallei was significantly increased, suggesting that NADPH oxidase-mediated ROS production is essential in suppressing intracellular multiplication of B. pseudomallei. Additionally, interferon-γ (IFN-γ)-mediated intracellular killing of B. pseudomallei requires NADPH oxidase activity, even though ROS level was not detected at higher levels in IFN-γ-treated infected monocytes. Altogether, these results imply that the activation of NADPH plays an essential role in suppressing intracellular multiplication of B. pseudomallei in human monocytes, although this enzyme is not sufficient to stop intracellular multiplication.


Assuntos
Burkholderia pseudomallei/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Monócitos/enzimologia , Monócitos/imunologia , NADPH Oxidases/metabolismo , Burkholderia pseudomallei/imunologia , Células Cultivadas , Humanos , Proteínas I-kappa B/metabolismo , Monócitos/microbiologia , Inibidor de NF-kappaB alfa , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
17.
Innate Immun ; 17(6): 532-40, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21088051

RESUMO

Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular Gram-negative bacterium that can survive and multiply inside the macrophages. Toll-like receptors are one class of pattern recognition receptors (PRRs) that have been documented to play significant role in B. pseudomallei infection. In the present study, we investigated a potential role of nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1 and NOD2), cytoplasmic pattern recognition receptors, in B. pseudomallei-infected mouse macrophage cell line RAW 264.7. Both live and heat-killed B. pseudomallei were able to up-regulate NOD1 and NOD2 expression in a time-dependent manner. Marked reduction of a negative regulator, suppressor of cytokine signaling 3 (SOCS3), expression was observed only in B. pseudomallei-infected NOD2-depleted macrophages and not in NOD1-depleted macrophages. The decrease in SOCS3 expression also led to an increase in IFN-γ responsiveness as judged by an enhanced STAT-1 phosphorylation on tyrosine 701 in the B. pseudomallei-infected macrophages. Together, these results suggested that, in addition to using other PRRs to evade macrophage defense, B. pseudomallei may also use NOD2 to regulate a negative regulator like SOCS3.


Assuntos
Burkholderia pseudomallei/fisiologia , Macrófagos/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Bacteriólise , Linhagem Celular , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Temperatura Alta , Macrófagos/microbiologia , Camundongos , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Tempo , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA