RESUMO
Quantitative PCR (qPCR) is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. Although the methodology is perceived to be relatively simple, there are a number of steps and reagents that require optimization and validation to ensure reproducible data that accurately reflect the biological question(s) being posed. This review article describes and illustrates the critical pitfalls and sources of error in qPCR experiments, along with a rigorous, stepwise process to minimize variability, time, and cost in generating reproducible, publication quality data every time. Finally, an approach to make an informed choice between qPCR and digital PCR technologies is described.
Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Custos e Análise de Custo , Reação em Cadeia da Polimerase em Tempo Real/economia , Reprodutibilidade dos Testes , TempoRESUMO
The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine pathogens and often serves as an entry door for other viral or bacterial pathogens, of which Streptococcus suis is one of the most common. Pre-infection with PRRSV leads to exacerbated disease caused by S. suis infection. Very few studies have assessed the immunological mechanisms underlying this higher susceptibility. Since antigen presenting cells play a major role in the initiation of the immune response, the in vitro transcriptional response of bone marrow-derived dendritic cells (BMDCs) and monocytes in the context of PRRSV and S. suis co-infection was investigated. BMDCs were found to be more permissive than monocytes to PRRSV infection; S. suis phagocytosis by PRRSV-infected BMDCs was found to be impaired, whereas no effect was found on bacterial intracellular survival. Transcription profile analysis, with a major focus on inflammatory genes, following S. suis infection, with and without pre-infection with PRRSV, was then performed. While PRRSV pre-infection had little effect on monocytes response to S. suis infection, a significant expression of several pro-inflammatory molecules was observed in BMDCs pre-infected with PRRSV after a subsequent infection with S. suis. While an additive effect could be observed for CCL4, CCL14, CCL20, and IL-15, a distinct synergistic up-regulatory effect was observed for IL-6, CCL5 and TNF-α after co-infection. This increased pro-inflammatory response by DCs could participate in the exacerbation of the disease observed during PRRSV and S. suis co-infection.
Assuntos
Coinfecção/genética , Células Dendríticas/metabolismo , Inflamação/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Infecções Estreptocócicas/genética , Doenças dos Suínos/genética , Suínos/genética , Animais , Células Cultivadas , Coinfecção/imunologia , Suscetibilidade a Doenças/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Celular/genética , Inflamação/microbiologia , Inflamação/virologia , Mediadores da Inflamação/metabolismo , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Infecções Estreptocócicas/imunologia , Streptococcus suis/patogenicidade , Suínos/imunologia , Suínos/microbiologia , Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Regulação para Cima/genéticaRESUMO
BACKGROUND: Swine influenza is a highly contagious viral infection in pigs affecting the respiratory tract that can have significant economic impacts. Streptococcus suis serotype 2 is one of the most important post-weaning bacterial pathogens in swine causing different infections, including pneumonia. Both pathogens are important contributors to the porcine respiratory disease complex. Outbreaks of swine influenza virus with a significant level of co-infections due to S. suis have lately been reported. In order to analyze, for the first time, the transcriptional host response of swine tracheal epithelial (NPTr) cells to H1N1 swine influenza virus (swH1N1) infection, S. suis serotype 2 infection and a dual infection, we carried out a comprehensive gene expression profiling using a microarray approach. RESULTS: Gene clustering showed that the swH1N1 and swH1N1/S. suis infections modified the expression of genes in a similar manner. Additionally, infection of NPTr cells by S. suis alone resulted in fewer differentially expressed genes compared to mock-infected cells. However, some important genes coding for inflammatory mediators such as chemokines, interleukins, cell adhesion molecules, and eicosanoids were significantly upregulated in the presence of both pathogens compared to infection with each pathogen individually. This synergy may be the consequence, at least in part, of an increased bacterial adhesion/invasion of epithelial cells previously infected by swH1N1, as recently reported. CONCLUSION: Influenza virus would replicate in the respiratory epithelium and induce an inflammatory infiltrate comprised of mononuclear cells and neutrophils. In a co-infection situation, although these cells would be unable to phagocyte and kill S. suis, they are highly activated by this pathogen. S. suis is not considered a primary pulmonary pathogen, but an exacerbated production of proinflammatory mediators during a co-infection with influenza virus may be important in the pathogenesis and clinical outcome of S. suis-induced respiratory diseases.
Assuntos
Células Epiteliais/microbiologia , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Vírus da Influenza A Subtipo H1N1/fisiologia , Streptococcus suis/fisiologia , Suínos , Traqueia/citologia , Animais , Linhagem Celular , Coinfecção , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Inflamação/metabolismo , Transcrição GênicaRESUMO
Streptococcus suis is an important swine pathogen and an emergent zoonotic pathogen. Excessive inflammation caused by S. suis is responsible for early high mortality in septic shock-like syndrome cases. Polyunsaturated fatty acids (PUFAs) may contribute to regulating inflammatory processes. This study shows that mouse infection by S. suis is accompanied by an increase of arachidonic acid, a proinflammatory omega-6 (ω-6) PUFA, and by a decrease of docosahexaenoic acid, an anti-inflammatory ω-3 PUFA. Macrophages infected with S. suis showed activation of mitogen-activated protein kinase pathways and cyclooxygenase-2 upregulation. Fenretinide, a synthetic vitamin A analog, reduced in vitro expression of inflammatory mediators. Pretreatment of mice with fenretinide significantly improved their survival by reducing systemic proinflammatory cytokines during the acute phase of an S. suis infection. These findings indicate a beneficial effect of fenretinide in diminishing the expression of inflammation and improving survival during an acute infection by a virulent S. suis strain.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus suis/fisiologia , Animais , Anticarcinógenos/farmacologia , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-6/sangue , Fenretinida/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estreptocócicas/sangue , ZoonosesRESUMO
We developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains.
Assuntos
Cápsulas Bacterianas/genética , Genes Bacterianos/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Polissacarídeos/genética , Infecções Estreptocócicas/diagnóstico , Streptococcus suis/genética , Animais , Humanos , Sorotipagem/métodos , Infecções Estreptocócicas/microbiologia , Suínos/microbiologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/microbiologiaRESUMO
Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.
Assuntos
Células Epiteliais , Vírus da Influenza A Subtipo H1N1/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus suis/metabolismo , Animais , Aderência Bacteriana , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Células Epiteliais/virologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/classificação , Suínos , Traqueia/imunologiaRESUMO
We previously identified Fragile X-related protein 1 (FXR1) as an RNA-binding protein involved in the post-transcriptional control of TNF and other cytokines in macrophages. Macrophages derived from FXR1-KO mice overexpress several inflammatory cytokines including TNF. Recently, we showed that fenretinide (4HPR) is able to inhibit several inflammatory cytokines in the lungs of cystic fibrosis mice, which also have abnormal immune responses. Therefore, we hypothesized that 4HPR might also be able to downregulate excessive inflammation even in macrophages with ablated FXR1. Indeed, our results demonstrate that 4HPR inhibited the excessive production of inflammatory mediators, including TNF, IL-6, CCL2 and CCL-5 in LPS-stimulated FXR1-KO macrophages, by selectively inhibiting phosphorylation of ERK1/2, which is naturally more phosphorylated in FXR1-KO cells. We also found that LPS stimulation of FXR1-KO macrophages led to significantly higher ratio of arachidonic acid/docosahexaenoic acid than observed in FXR1-WT macrophages. Interestingly, treatment with 4HPR was associated with the normalization of arachidonic acid/docosahexaenoic acid ratio in macrophages, which we found to impact phosphorylation of ERK1/2. Overall, this study shows for the first time that 4HPR modulates inflammatory cytokine expression in macrophages by correcting a phospholipid-bound fatty acid imbalance that impacts the phosphorylation of ERK1/2.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Fenretinida/farmacologia , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular , Ácidos Docosa-Hexaenoicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Fatores de Necrose Tumoral/genética , Fatores de Necrose Tumoral/metabolismoRESUMO
Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1ß (IL-1ß), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.
Assuntos
Células Dendríticas/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Polissacarídeos Bacterianos/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/imunologia , Streptococcus suis/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Fator Ativador de Células B/imunologia , Fator Ativador de Células B/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Células Dendríticas/metabolismo , Imunidade Humoral/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , Polissacarídeos Bacterianos/metabolismo , Transdução de Sinais/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/metabolismo , Streptococcus suis/metabolismo , Receptor 2 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Streptococcus suis is an emerging zoonotic agent causing meningitis and septicemia. Outbreaks in humans in China with atypical cases of streptococcal toxic shock-like syndrome have been described to be caused by a clonal epidemic S. suis strain characterized as sequence type (ST) 7 by multilocus sequence typing, different from the classical ST1 usually isolated in Europe. Previous in vitro studies showed that Toll-like receptor (TLR) 2 plays a major role in S. suis ST1 interactions with host cells. In the present study, the in vivo role of TLR2 in systemic infections caused by S. suis ST1 or ST7 strains using TLR2 deficient (TLR2(-/-)) mice was evaluated. TLR2-mediated recognition significantly contributes to the acute disease caused by the highly virulent S. suis ST1 strain, since the TLR2(-/-) mice remained unaffected when compared to wild type (WT) mice. The lack of mortality could not be associated with a lower bacterial burden; however, a significant decrease in the induction of pro-inflammatory mediators, as evaluated by microarray, real-time PCR and protein assays, was observed. On the other hand, TLR2(-/-) mice infected with the epidemic ST7 strain presented no significant differences regarding survival and expression of pro-inflammatory mediators when compared to the WT mice. Together, these results show a TLR2-independent host innate immune response to S. suis that depends on the strain.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Choque Séptico/microbiologia , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/imunologia , Streptococcus suis/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Bacteriemia/microbiologia , Quimiocinas/genética , Quimiocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Choque Séptico/epidemiologia , Choque Séptico/imunologia , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus suis/patogenicidade , Análise de Sobrevida , VirulênciaRESUMO
Streptococcus suis, a major porcine pathogen, can be transmitted to humans and cause severe symptoms. A large human outbreak associated with an unusual streptococcal toxic shock-like syndrome (STSLS) was described in China. Albeit an early burst of proinflammatory cytokines following Chinese S. suis infection was suggested to be responsible for STSLS case severity, the mechanisms involved are still poorly understood. Using a mouse model, the host response to S. suis infection with a North American intermediately pathogenic strain, a European highly pathogenic strain, and the Chinese epidemic strain was investigated by a whole-genome microarray approach. Proinflammatory genes were expressed at higher levels in mice infected with the Chinese strain than those infected with the European strain. The Chinese strain induced a fast and strong gamma interferon (IFN-γ) response by natural killer (NK) cells. In fact, IFN-γ-knockout mice infected with the Chinese strain showed significantly better survival than wild-type mice. Conversely, infection with the less virulent North American strain resulted in an IFN-ß-subjugated, low inflammatory response that might be beneficial for the host to clear the infection. Overall, our data suggest that a highly virulent epidemic strain has evolved to massively activate IFN-γ production, mainly by NK cells, leading to a rapid and lethal STSLS.
Assuntos
Doenças Transmissíveis Emergentes/microbiologia , Interferon gama/metabolismo , Choque Séptico/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/patogenicidade , Animais , China/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Regulação Bacteriana da Expressão Gênica/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , América do Norte/epidemiologia , Análise Serial de Proteínas , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Choque Séptico/epidemiologia , Choque Séptico/patologia , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/patologia , Streptococcus suis/classificação , Suínos , Doenças dos Suínos/microbiologia , VirulênciaRESUMO
The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.
Assuntos
Espinhas Dendríticas/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Hipocampo/metabolismo , Ribossomos/metabolismo , Animais , Análise por Conglomerados , Proteína 4 Homóloga a Disks-Large , Feminino , Proteínas de Fluorescência Verde/metabolismo , Guanilato Quinases/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Polirribossomos/metabolismo , Transporte Proteico , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Extratos de TecidosRESUMO
Streptococcus suis is a major swine pathogen and important zoonotic agent causing mainly septicemia and meningitis. However, the mechanisms involved in host innate and adaptive immune responses toward S. suis as well as the mechanisms used by S. suis to subvert these responses are unknown. Here, and for the first time, the ability of S. suis to interact with bone marrow-derived swine dendritic cells (DCs) was evaluated. In addition, the role of S. suis capsular polysaccharide in modulation of DC functions was also assessed. Well encapsulated S. suis was relatively resistant to phagocytosis, but it increased the relative expression of Toll-like receptors 2 and 6 and triggered the release of several cytokines by DCs, including IL-1ß, IL-6, IL-8, IL-12p40 and TNF-α. The capsular polysaccharide was shown to interfere with DC phagocytosis; however, once internalized, S. suis was readily destroyed by DCs independently of the presence of the capsular polysaccharide. Cell wall components were mainly responsible for DC activation, since the capsular polysaccharide-negative mutant induced higher cytokine levels than the wild-type strain. The capsular polysaccharide also interfered with the expression of the co-stimulatory molecules CD80/86 and MHC-II on DCs. To conclude, our results show for the first time that S. suis interacts with swine origin DCs and suggest that these cells might play a role in the development of host innate and adaptive immunity during an infection with S. suis serotype 2.
Assuntos
Cápsulas Bacterianas/metabolismo , Células Dendríticas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/fisiologia , Doenças dos Suínos/imunologia , Animais , Medula Óssea , Sobrevivência Celular , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/fisiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Citometria de Fluxo/veterinária , Microscopia Confocal/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Fagocitose , Reação em Cadeia da Polimerase/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
The murine astrocyte response to virulent Streptococcus suis, a swine and an emerging human meningitis-causing pathogen, is reported. Albeit astrocytes do not internalize S. suis, all S. suis strains studied enhanced Toll-like receptor (TLR)2 expression and the production of pro-inflammatory cytokines and inducible nitric oxide synthase. Cell wall components and hemolysin (suilysin) are shown to be mainly responsible for cell activation. Astrocytes from TLR2 knockout mice presented a partial but significant reduction of S. suis-induced production of pro-inflammatory cytokines. These results contribute to increase the knowledge on mechanisms underlying S. suis inflammation in the brain.
Assuntos
Astrócitos/metabolismo , Astrócitos/microbiologia , Regulação da Expressão Gênica/fisiologia , Streptococcus suis , Receptor 2 Toll-Like/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Sobrevivência Celular , Células Cultivadas , Córtex Cerebral/citologia , Cricetinae , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Mutação/genética , Óxido Nítrico Sintase Tipo II , Fagocitose/fisiologia , RNA Mensageiro/metabolismo , Streptococcus suis/patogenicidade , Fatores de Tempo , Receptor 2 Toll-Like/deficiênciaRESUMO
The inflammatory response contributes importantly to secondary tissue damage and functional deficits after spinal cord injury (SCI). In this work, we identified mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of p38 MAPK, as a potential target using microarray analysis of contused spinal cord tissue taken at the peak of the inflammatory response. There was increased expression and phosphorylation of MK2 after SCI, with phospho-MK2 expressed in microglia/macrophages, neurons and astrocytes. We examined the role of MK2 in spinal cord contusion injury using MK2(-/-) mice. These results show that locomotor recovery was significantly improved in MK2(-/-) mice, compared with wild-type controls. MK2(-/-) mice showed reduced neuron and myelin loss, and increased sparing of serotonergic fibers in the ventral horn caudal to the injury site. We also found differential expression of matrix metalloproteinase-2 and 9 in MK2(-/-) and wild-type mice after SCI. Significant reduction was also seen in the expression of proinflammatory cytokines and protein nitrosylation in the injured spinal cord of MK2(-/-) mice. Our previous work has shown that macrophages lacking MK2 have an anti-inflammatory phenotype. We now show that there is no difference in the number of macrophages in the injured spinal cord between the two mouse strains and little if any difference in their phagocytic capacity, suggesting that macrophages lacking MK2 have a beneficial phenotype. These findings suggest that a lack of MK2 can reduce tissue damage after SCI and improve locomotor recovery. MK2 may therefore be a useful target to treat acute SCI.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microglia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Análise de Variância , Animais , Western Blotting , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Atividade Motora/genética , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Traumatismos da Medula Espinal/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Streptococcus suis is an important swine and human pathogen responsible for septicemia and meningitis. In vivo research in mice suggested that in the brain, microglia might be involved in activating the inflammatory response against S. suis. The aim of this study was to better understand the interactions between S. suis and microglia. Murine microglial cells were infected with a virulent wild-type strain of S. suis. Two isogenic mutants deficient at either capsular polysaccharide (CPS) or hemolysin production were also included. CPS contributed to S. suis resistance to phagocytosis and regulated the inflammatory response by hiding proinflammatory components from the bacterial cell wall, while the absence of hemolysin, a potential cytotoxic factor, did not have a major impact on S. suis interactions with microglia. Wild-type S. suis induced enhanced expression of Toll-like receptor 2 by microglial cells, as well as phosphotyrosine, protein kinase C, and different mitogen-activated protein kinase signaling events. However, cells infected with the CPS-deficient mutant showed overall stronger and more sustained phosphorylation profiles. CPS also modulated inducible nitric oxide synthase expression and further nitric oxide production from S. suis-infected microglia. Finally, S. suis-induced NF-κB translocation was faster for cells stimulated with the CPS-deficient mutant, suggesting that bacterial cell wall components are potent inducers of NF-κB. These results contribute to increase the knowledge of mechanisms underlying S. suis inflammation in the brain and will be useful in designing more efficient anti-inflammatory strategies for meningitis.
Assuntos
Doenças Transmissíveis Emergentes/microbiologia , Encefalite/microbiologia , Meningites Bacterianas/microbiologia , Microglia/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/fisiologia , Zoonoses/microbiologia , Animais , Linhagem Celular , Quimiocinas/fisiologia , Citocinas/fisiologia , Encefalite/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Regulação Bacteriana da Expressão Gênica/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Meningites Bacterianas/fisiopatologia , Camundongos , Microglia/fisiologia , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Fagocitose/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry. Our results demonstrate that patients with CF display significantly lower levels of several ceramide sphingolipid species, specifically C14:0, C20:1, C22:0, C22:1, and C24:0 ceramides, and dihydroxy ceramide (DHC16:0). We report that Cftr-knockout mice display diminished ceramide levels in CF-related organs (lung, pancreas, ileum, and plasma) compared with their littermate controls. Since it has been previously reported that in vitro treatment with fenretinide induced ceramide in neuroblastoma cell lines, we decided to test this drug in vivo using our Cftr-knockout mice in an attempt to correct this newly identified defect in ceramide levels. We demonstrate that treatment with fenretinide is able to increase ceramide concentrations in CF-related organs. We further assessed the biological effect of fenretinide on the ability of Cftr-knockout mice to combat lung infection with P. aeruginosa. Our data show dramatic improvement in the ability of Cftr-knockout mice to control P. aeruginosa infection. Overall, these findings not only document a novel deficiency in several ceramide species in patients with CF, but also demonstrate a pharmacologic means to correct this defect in Cftr-knockout mice. Our data provide a strong rationale for clinical intervention that may benefit patients with CF suffering from CF lung disease.
Assuntos
Anticarcinógenos/farmacologia , Ceramidas/deficiência , Fibrose Cística/sangue , Fenretinida/farmacologia , Infecções por Pseudomonas/sangue , Pseudomonas aeruginosa , Esfingolipídeos/deficiência , Adulto , Animais , Anticarcinógenos/uso terapêutico , Linhagem Celular Tumoral , Ceramidas/sangue , Cromatografia Líquida de Alta Pressão , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Fenretinida/uso terapêutico , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Pessoa de Meia-Idade , Especificidade de Órgãos/genética , Infecções por Pseudomonas/genética , Esfingolipídeos/sangueRESUMO
An RNA-binding protein (RBP) was recently identified, FXR1P, which regulates tumour necrosis factor (TNF) gene expression at the posttranscriptional level in response to lipopolysaccharide, was recently identified resulting in higher TNF production in macrophages from FXR1 knockout (KO) mice compared with wild-type (WT) macrophages. In this study, the importance of FXR1P in the induction of TNF by toll-like receptor 7 (TLR7) ligand S28463 and TLR9 ligand CpG is evaluated. The results clearly reveal a much higher level of TNF protein expression in FXR1-KO than in WT macrophages following stimulation with CpG but not with S28463. To better understand the molecular mechanism, both the steady-state levels and the stability of TNF mRNA were assessed. It was found that the TNF mRNA steady-state level was more elevated in CpG-stimulated FXR1-KO macrophages, while the stability of TNF mRNA was not affected in CpG-stimulated FXR1-KO macrophages. It was also established that FXR1P is involved in regulating the expression of several other inflammatory cytokines and chemokines. Together, the data clearly demonstrate the importance of FXR1P RBP in the regulation of a wide spectrum of inflammatory genes and suggest an important role of MAP signalling in the response of macrophages to selected TLR ligands, including CpG.
Assuntos
Regulação da Expressão Gênica , Macrófagos/imunologia , Oligodesoxirribonucleotídeos/imunologia , Proteínas de Ligação a RNA/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Interleucina-12/biossíntese , Interleucina-12/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Macrófagos/química , Camundongos , Camundongos Knockout , Estabilidade de RNA , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
Toll-like receptor ligands (TLRLs) produced by various pathogens activate mitogen-activated protein kinases (MAPKs). While the dependence on p38 MAPK activation for the induction of inflammatory genes by the TLR4L, lipopolysaccharide (LPS), has been well documented, the importance of the p38 pathway in gene regulation by other TLRLs is less well understood. We have focused our analysis on two TLRLs with therapeutic potential, imidazoquinoline S28463 (TLR7L) and CpG DNA (TLR9L), to explore in detail their effects on the regulation of gene expression in macrophages. Here we report that activation of the p38 MAPK/MK2 pathway is crucial for both S28463- and CpG-induced cytokine and chemokine production. We show that the stability of TNF mRNA induced by CpG DNA and S28463 is not dependent on the p38 MAPK/MK2 pathway, in contrast to LPS-induced TNF mRNA. Using a GFP reporter construct under the control of the 3' untranslated region of the TNF gene, we demonstrate that S28463 and CpG DNA-induced MK2 signalling regulates TNF mRNA primarily at the translational level, whereas LPS-induced MK2 signalling regulates both the stability and translational efficiency of TNF mRNA. Overall, these data provide insight into distinct molecular mechanisms of gene expression regulation by different Toll-like receptor ligands.
Assuntos
Aminoquinolinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Quimiocinas/genética , Quimiocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3'-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation.
Assuntos
Regulação da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/genética , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Sequência Rica em At/genética , Animais , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Síndrome do Cromossomo X Frágil , Genes Reporter/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transcrição Gênica/genéticaRESUMO
Cystic fibrosis females have a worse prognosis compared to male patients. Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and lower levels of IL-10 are found in the bronchoalveolar lavage fluid compared to healthy controls. The present study was aimed at investigating the importance of gender and IL-10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa. We found that wildtype females were more susceptible than males to infection, as we observed greater weight loss, higher bacterial load, and inflammatory mediators in their lungs. IL-10 knockout mice, both females and males, had higher levels of TNF-alpha in the lungs compared to wildtype mice and maintained higher levels of polymorphonuclear cells and lower levels of macrophages for a longer period of time. Our results demonstrate that the number of bacteria recovered from the lungs of IL-10 knockout male mice was significantly higher than that observed in their wildtype male counterparts and we show that neutralization of IL-10 in infected female mice for a prolonged period of time leads to increased susceptibility to infection. Results reported in this study clearly demonstrate that females, both wildtype and IL-10 knockout mice are more susceptible to Pseudomonas aeruginosa infection than males, and that they mount a stronger inflammatory response in the lungs.