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1.
EMBO Rep ; 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39363059

RESUMO

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

2.
Nucleic Acids Res ; 48(22): 12833-12844, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33275131

RESUMO

RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2'-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.


Assuntos
Imunidade Inata/genética , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Receptor 7 Toll-Like/genética , Escherichia coli/genética , Regulação da Expressão Gênica/genética , Guanosina/genética , Guanosina/imunologia , Humanos , Interferons/genética , Interferons/imunologia , Metilação , Processamento Pós-Transcricional do RNA/imunologia , RNA de Transferência/imunologia , Receptor 7 Toll-Like/imunologia
3.
RNA ; 25(7): 869-880, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31019095

RESUMO

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.


Assuntos
Endossomos/metabolismo , Células Eucarióticas/imunologia , Guanosina/química , Imunidade Inata/imunologia , Células Procarióticas/imunologia , RNA de Transferência/metabolismo , Receptores Toll-Like/metabolismo , Células Eucarióticas/metabolismo , Humanos , Metilação , Células Procarióticas/metabolismo , RNA de Transferência/genética , Receptores Toll-Like/genética , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
4.
Nat Immunol ; 10(3): 241-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19221555

RESUMO

The inflammasome is a multiprotein complex that mediates the activation of caspase-1, which promotes secretion of the proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18, as well as 'pyroptosis', a form of cell death induced by bacterial pathogens. Members of the Nod-like receptor family, including NLRP1, NLRP3 and NLRC4, and the adaptor ASC are critical components of the inflammasome that link microbial and endogenous 'danger' signals to caspase-1 activation. Several diseases are associated with dysregulated activation of caspase-1 and secretion of IL-1beta. Thus, understanding inflammasome pathways may provide insight into disease pathogenesis that might identify potential targets for therapeutic intervention.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Caspase 1/imunologia , Interleucina-1beta/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Humanos , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas NLR , Domínios e Motivos de Interação entre Proteínas , Ácido Úrico/imunologia , Ácido Úrico/metabolismo
5.
Nucleic Acids Res ; 46(18): 9764-9775, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30102387

RESUMO

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.


Assuntos
Imunidade Inata/genética , Ácidos Nucleicos/imunologia , RNA de Transferência/imunologia , Receptor 7 Toll-Like/genética , Guanosina/química , Guanosina/imunologia , Humanos , Hidrogênio/química , Interferons/genética , Leucócitos Mononucleares/química , Leucócitos Mononucleares/imunologia , Metilação , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , RNA de Transferência/genética , Timidina/análogos & derivados , Timidina/química , Timidina/genética , Receptor 7 Toll-Like/imunologia
6.
RNA ; 23(9): 1344-1351, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28576825

RESUMO

Bacterial RNA serves an important function as activator of the innate immune system. In humans bacterial RNA is sensed by the endosomal receptors TLR7 and TLR8. Differences in the posttranscriptional modification profile of prokaryotic when compared with eukaryotic RNA allow innate immune cells to discriminate between "host" and "foreign" RNA. Ribose 2'-O-methylation is of particular importance and has been reported to antagonize TLR7/8 activation. Yet, the exact sequence context in which 2'-O-methylation has to occur to mediate its inhibitory activity remains largely undefined. On the basis of a naturally occurring 2'-O-methylated RNA sequence, we performed a systematic permutation of the methylated nucleotide as well as adjacent bases and hereby identify two minimal trinucleotide motifs within a 9-mer oligoribonucleotide that are necessary and sufficient to antagonize TLR7 and TLR8 activation, respectively. Given the growing interest in the development of inhibitors of nucleic acid-sensing TLRs for therapeutic purposes, these results will facilitate the rational design of such antagonists in the future.


Assuntos
Motivos de Nucleotídeos , RNA/genética , RNA/metabolismo , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Citidina , Humanos , Concentração Inibidora 50 , Leucócitos Mononucleares , Metilação , Mutação , Nucleotídeos/química , Nucleotídeos/metabolismo , RNA/química , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo
7.
J Antimicrob Chemother ; 74(12): 3473-3480, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504593

RESUMO

BACKGROUND: Infection and colonization with multi-resistant Acinetobacter baumannii causes therapeutic and economic problems in the nosocomial setting. Due to the sensitivity issue of screening schemes for A. baumannii, it is difficult to implement adequate transmission prevention measures. The high discriminatory power of WGS for transmission-chain analysis provides us with the necessary tool to study and identify transmission events. We retrospectively sequenced and analysed 39 A. baumannii isolates from 2012-15 to search for possible missed transmission events. METHODS: Molecular typing by WGS was performed for non-repetitive (n=39) carbapenem-resistant A. baumannii. Retrospective assessment of patient records was performed to investigate and confirm possible transmission events. RESULTS: Between July 2012 and September 2015, A. baumannii was isolated from 268 patients, of which 16% (42/268) were carbapenem resistant. Thirty-nine of these isolates were recoverable and sequenced. Fifteen percent (6/39) of these were resistant to all antibiotics tested. Most isolates belong to the circulating IC2 clonal type. SNP analysis revealed four potential outbreak clusters. Two of these clusters showed high concordance with the local spatio-temporal epidemiology, suggesting that transmission events were very likely. CONCLUSIONS: Our data suggest that there were two independent transmission events, which would have been missed by conventional MLST owing to high clonality. The routine implementation of WGS can optimize surveillance and initiation of suitable containment measures. In addition, emerging resistance to salvage therapy is a major therapeutic problem and should be monitored closely.


Assuntos
Infecções por Acinetobacter/transmissão , Acinetobacter baumannii/classificação , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/efeitos dos fármacos , Técnicas de Tipagem Bacteriana , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Surtos de Doenças , Eletroforese em Gel de Campo Pulsado , Feminino , Alemanha , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Estudos Retrospectivos , Sequenciamento Completo do Genoma
8.
Cytokine ; 112: 102-115, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29914791

RESUMO

Osteoclasts are specialised cells that resorb bone and develop from the monocyte/macrophage lineage. While there is a wealth of information on the regulation of macrophage function through metabolic activity, the connection between osteoclast differentiation and metabolism is less well understood. Recent data show that mitochondria participate in switching macrophages from an inflammatory phenotype towards differentiation into osteoclasts. Additionally, it was found that reactive oxygen species (ROS) actively take place in osteoclast differentiation by acting as secondary signalling molecules. Bone resorption is an energy demanding process and differentiating osteoclasts triggers the biogenesis of mitochondria. In addition, the activity of specific OXPHOS components of macrophages and osteoclasts is differentially regulated. This review summarises our knowledge on macrophage-mediated inflammation, its impact on a cell's metabolic activity and its effect on osteoclast differentiation.


Assuntos
Macrófagos/metabolismo , Osteoclastos/metabolismo , Animais , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Humanos , Mitocôndrias/metabolismo , Monócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
9.
J Immunol ; 195(2): 411-8, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26138638

RESUMO

Although DNA of bacterial and viral origin, as well as viral RNA, have been intensively studied as triggers of innate immune responses, the stimulatory properties of bacterial RNA and its role during infections have just begun to be deciphered. Bacterial RNA is a strong inducer of type I IFN and NF-κB-dependent cytokines, and it also can activate the Nlrp3 inflammasome. In this review, we focus on the receptors and signaling pathways involved in innate immune activation by bacterial RNA and analyze the physiological relevance of bacterial RNA recognition during infections. Furthermore, we present the concept that RNA modifications can impair RNA-dependent immune activation. RNA modifications differ between eukaryotes and prokaryotes; thus, they can serve to define the innate pattern that is recognized. In this regard, we discuss the role of ribose 2'-O-methylation as a potential immune-escape mechanism.


Assuntos
Células Dendríticas/imunologia , Imunidade Inata , Inflamassomos/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Ribose/imunologia , Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Células Dendríticas/microbiologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Inflamassomos/genética , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Metilação , Monócitos/microbiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribose/metabolismo , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
10.
J Immunol ; 195(3): 1092-9, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26101323

RESUMO

Microbial nucleic acids constitute an important group of pathogen-associated molecular patterns (PAMPs) that efficiently trigger innate immune activation. In mice, TLR13 has recently been identified to sense a highly conserved region within bacterial 23S rRNA. However, TLR13 is not expressed in humans, and the identity of its human homolog remains elusive. Moreover, the contribution of bacterial RNA to the induction of innate immune responses against entire bacteria is still insufficiently defined. In the current study, we show that human monocytes respond to bacterial RNA with secretion of IL-6, TNF, and IFN-ß, which is critically dependent on lysosomal maturation. Using small interfering RNA and overexpression, we unambiguously identify TLR8 as receptor for bacterial RNA in primary human monocyte-derived macrophages. We further demonstrate that the sequence motif sensed by TLR8 is clearly distinct from that recognized by TLR13. Moreover, TLR8-dependent detection of bacterial RNA was critical for triggering monocyte activation in response to infection with Streptococcus pyogenes. Bacterial RNA within streptococci was also a dominant stimulus for murine immune cells, highlighting the physiological relevance of RNA sensing in defense of infections.


Assuntos
RNA Bacteriano/imunologia , RNA Ribossômico 23S/imunologia , Streptococcus pyogenes/genética , Receptor 8 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Animais , Linhagem Celular , Humanos , Interferon beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Interferência de RNA , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , RNA Interferente Pequeno , Streptococcus pyogenes/imunologia , Receptor 8 Toll-Like/biossíntese , Receptor 8 Toll-Like/genética , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
J Immunol ; 195(11): 5421-31, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26519528

RESUMO

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.


Assuntos
Caspase 8/imunologia , Células Dendríticas/imunologia , Inibidores de Histona Desacetilases/farmacologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Animais , Células da Medula Óssea , Proteínas de Transporte , Caspase 1/genética , Caspase 1/imunologia , Inibidores de Caspase/farmacologia , Caspases/genética , Caspases Iniciadoras , Células Cultivadas , Colite/induzido quimicamente , Sulfato de Dextrana , Histona Desacetilases/imunologia , Inflamassomos/imunologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR
12.
J Neurosci ; 35(2): 583-98, 2015 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-25589753

RESUMO

Acute brain lesions induce profound alterations of the peripheral immune response comprising the opposing phenomena of early immune activation and subsequent immunosuppression. The mechanisms underlying this brain-immune signaling are largely unknown. We used animal models for experimental brain ischemia as a paradigm of acute brain lesions and additionally investigated a large cohort of stroke patients. We analyzed release of HMGB1 isoforms by mass spectrometry and investigated its inflammatory potency and signaling pathways by immunological in vivo and in vitro techniques. Features of the complex behavioral sickness behavior syndrome were characterized by homecage behavior analysis. HMGB1 downstream signaling, particularly with RAGE, was studied in various transgenic animal models and by pharmacological blockade. Our results indicate that the cytokine-inducing, fully reduced isoform of HMGB1 was released from the ischemic brain in the hyperacute phase of stroke in mice and patients. Cytokines secreted in the periphery in response to brain injury induced sickness behavior, which could be abrogated by inhibition of the HMGB1-RAGE pathway or direct cytokine neutralization. Subsequently, HMGB1-release induced bone marrow egress and splenic proliferation of bone marrow-derived suppressor cells, inhibiting the adaptive immune responses in vivo and vitro. Furthermore, HMGB1-RAGE signaling resulted in functional exhaustion of mature monocytes and lymphopenia, the hallmarks of immune suppression after extensive ischemia. This study introduces the HMGB1-RAGE-mediated pathway as a key mechanism explaining the complex postischemic brain-immune interactions.


Assuntos
Proteína HMGB1/metabolismo , Infarto da Artéria Cerebral Média/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Medula Óssea/imunologia , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Proteína HMGB1/genética , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Baço/imunologia , Acidente Vascular Cerebral/metabolismo , Linfócitos T/imunologia
13.
RNA ; 20(9): 1351-5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25051971

RESUMO

RNA can function as a pathogen-associated molecular pattern (PAMP) whose recognition by the innate immune system alerts the body to an impending microbial infection. The recognition of tRNA as either self or nonself RNA by TLR7 depends on its modification patterns. In particular, it is known that the presence of a ribose methylated guanosine at position 18, which is overrepresented in self-RNA, antagonizes an immune response. Here, we report that recognition extends to the next downstream nucleotide and the effectively recognized molecular detail is actually a methylated dinucleotide. The most efficient nucleobases combination of this motif includes two purines, while pyrimidines diminish the effect of ribose methylation. The constraints of this motif stay intact when transposed to other parts of the tRNA. The results argue against a fixed orientation of the tRNA during interaction with TLR7 and, rather, suggest a processive type of inspection.


Assuntos
RNA de Transferência/metabolismo , Receptor 7 Toll-Like/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Células Cultivadas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Transferência/química , Especificidade por Substrato/genética
14.
J Immunol ; 193(8): 4214-4222, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25225670

RESUMO

The nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (Nlrp3) inflammasome plays an important role in inflammation by controlling the maturation and secretion of the cytokines IL-1ß and IL-18 in response to multiple stimuli including pore-forming toxins, particulate matter, and ATP. Although the pathways activated by the latter stimuli lead to a decrease in intracellular K(+) concentration, which is required for inflammasome activation, the mechanism by which microbial RNA activates Nlrp3, remains poorly understood. In this study, we found that cytosolic poly(I:C), but not total RNA from healthy macrophages, macrophages undergoing pyroptosis, or mitochondrial RNA, induces caspase-1 activation and IL-1ß release through the Nlrp3 inflammasome. Experiments with macrophages deficient in Tlr3, Myd88, or Trif, indicate that poly(I:C) induces Nlrp3 activation independently of TLR signaling. Further analyses revealed that the cytosolic sensors Rig-I and melanoma differentiation-associated gene 5 act redundantly via the common adaptor mitochondrial antiviral signaling (Mavs) to induce Nlrp3 activation in response to poly(I:C), but not ATP or nigericin. Mechanistically, Mavs triggered membrane permeabilization and K(+) efflux independently of the inflammasome which were required for poly(I:C)-induced Nlrp3 activation. We conclude that poly (I:C) activates the inflammasome through an Mavs-dependent surveillance pathway that converges into a common K(+) lowering step in the cytosol that is essential for the induction of Nlrp3 activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas de Transporte/imunologia , Potássio/metabolismo , RNA de Cadeia Dupla/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Caspase 1/imunologia , Citosol , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , Inflamação/imunologia , Helicase IFIH1 Induzida por Interferon , Interleucina-18/biossíntese , Interleucina-18/metabolismo , Interleucina-1beta/biossíntese , Interleucina-1beta/metabolismo , Transporte de Íons , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR , Poli I-C/imunologia , RNA Bacteriano/imunologia , RNA Viral/imunologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/genética
15.
J Immunol ; 190(12): 6533-41, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23667107

RESUMO

The significance of bacterial RNA recognition for initiating innate immune responses against invading pathogens has only recently started to be elucidated. Bacterial RNA is an important trigger of inflammasome activation, resulting in caspase-1-dependent cleavage of pro-IL-1ß into the active form. It was reported previously that prolonged treatment with IFN-γ can inhibit IL-1ß production at the level of both transcription and Nlrp3 inflammasome activation in an NO-dependent manner. As a result of the delayed kinetics of NO generation after IFN-γ stimulation, these effects were only observed at later time points. We report that IFN-γ suppressed bacterial RNA and LPS induced IL-1ß transcription in primary murine macrophages and dendritic cells by an additional, very rapid mechanism that was independent of NO. Costimulation with IFN-γ selectively attenuated binding of NF-κB p65 to the IL-1ß promoter, thus representing a novel mechanism of IL-1ß inhibition by IFN-γ. Transcriptional silencing was specific for IL-1ß because expression of other proinflammatory cytokines, such as TNF, IL-6, and IL-12p40, was not affected. Furthermore, by suppressing IL-1ß production, IFN-γ impaired differentiation of Th17 cells and production of neutrophil chemotactic factor CXCL1 in vitro. The findings provide evidence for a rapid immune-modulating effect of IFN-γ independent of NO.


Assuntos
Imunidade Inata/imunologia , Interferon gama/metabolismo , Interleucina-1beta/biossíntese , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Animais , Western Blotting , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Óxido Nítrico/metabolismo , RNA Bacteriano/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th17/citologia , Células Th17/imunologia
16.
J Biol Chem ; 288(51): 36691-702, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24225954

RESUMO

Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1ß, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.


Assuntos
Citocinas/metabolismo , Exossomos/fisiologia , Células Precursoras de Monócitos e Macrófagos/imunologia , Transdução de Sinais , Receptores Toll-Like/metabolismo , Líquido Amniótico/citologia , Líquido Amniótico/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Monócitos e Macrófagos/citologia , Células Precursoras de Monócitos e Macrófagos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
17.
J Immunol ; 189(1): 328-36, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22634614

RESUMO

Recognition of foreign nucleic acids is important for the induction of an innate immune response against invading pathogens. Although the pathways involved in sensing bacterial DNA and viral RNA are now well established, only limited knowledge is available on mechanisms underlying recognition of bacterial RNA. It has been reported that intracellular delivery of Escherichia coli RNA activates the Nlrp3 inflammasome, but whether this is a general property of bacterial RNA remains unclear as are the pathways involved in pro-IL-1ß induction and caspase-1 activation by bacterial RNA. In this study, we report that bacterial RNA from both Gram-positive and Gram-negative bacteria induces activation of caspase-1 and secretion of IL-1ß by murine dendritic cells and bone-marrow derived macrophages. Stimulation was independent of the presence of 5'-triphosphate termini and occurred with whole RNA preparations from bacteria but not from eukaryotes. Induction of pro-IL-1ß as well as the priming for caspase-1 activation by bacterial RNA was dependent on UNC93B, an endoplasmic reticulum protein essential for delivery of TLRs to the endosome, whereas the established nucleic acid sensing endosomal TLRs 3, 7, and 9 were dispensable. Additionally, caspase-1 activation and IL-1ß production by transfected bacterial RNA were absent in MyD88-deficient cells but independent of TRIF. Thus, our data indicate the presence of a yet unidentified intracellular nucleic acid receptor involved in bacterial RNA-induced inflammasome activation and release of IL-1ß.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Caspase 1/metabolismo , Interleucina-1beta/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , RNA Bacteriano/fisiologia , Receptor 3 Toll-Like/fisiologia , Receptor 7 Toll-Like/fisiologia , Receptor Toll-Like 9/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Linhagem Celular , Células Dendríticas/enzimologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Ativação Enzimática/genética , Macrófagos/enzimologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Receptor 3 Toll-Like/deficiência , Receptor 7 Toll-Like/deficiência , Receptor Toll-Like 9/deficiência
19.
Int Immunol ; 23(1): 1-15, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21135031

RESUMO

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 µg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Lactonas/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT5/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Administração Intranasal , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/química , Antiasmáticos/isolamento & purificação , Antiasmáticos/farmacologia , Asma/imunologia , Antígeno CD11c/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interleucina-4/biossíntese , Lactonas/administração & dosagem , Lactonas/química , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Interleucina-3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas com Domínio T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia
20.
Front Immunol ; 13: 828626, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35281009

RESUMO

Staphylococcus aureus is one of the clinically most relevant pathogens causing infections. Humans are often exposed to S. aureus. In approximately one-third of the healthy population it can be found on the skin either for long or short periods as colonizing "commensals", without inducing infections or an inflammatory immune response. While tolerating S. aureus seems to be limited to certain individuals and time periods in most cases, Staphylococcus epidermidis is tolerated permanently on the skin of almost all individuals without activating overwhelming skin inflammation. To investigate this, we co-cultured a keratinocyte cell line (HaCaT) with viable S. aureus or S. epidermidis to study the differences in the immune activation. S. aureus activated keratinocytes depicted by a profound IL-6 and IL-8 response, whereas S. epidermidis did not. Our data indicate that internalization of S. aureus and the subsequent intracellular sensing of bacterial nucleic acid may be essential for initiating inflammatory response in keratinocytes. Internalized dsRNA activates IL-6 and IL-8 release, but not TNF-α or IFNs by human keratinocytes. This is a non-specific effect of dsRNA, which can be induced using Poly(I:C), as well as RNA from S. aureus and S. epidermidis. However, only viable S. aureus were able to induce this response as these bacteria and not S. epidermidis were actively internalized by HaCaT. The stimulatory effect of S. aureus seems to be independent of the TLR3, -7 and -8 pathways.


Assuntos
Ácidos Nucleicos , Infecções Estafilocócicas , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Queratinócitos , Ácidos Nucleicos/metabolismo , Staphylococcus aureus , Staphylococcus epidermidis
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