RESUMO
BACKGROUND: Soybean (Glycine max) has been bred for thousands of years to produce seeds rich in protein for human and animal consumption, making them an appealing bioreactor for producing valuable recombinant proteins at high levels. However, the effects of expressing recombinant protein at high levels on bean physiology are not well understood. To address this, we investigated whether gene expression within transgenic soybean seed tissue is altered when large amounts of recombinant proteins are being produced and stored exclusively in the seeds. We used RNA-Seq to survey gene expression in three transgenic soybean lines expressing recombinant protein at levels representing up to 1.61 % of total protein in seed tissues. The three lines included: ST77, expressing human thyroglobulin protein (hTG), ST111, expressing human myelin basic protein (hMBP), and 764, expressing a mutant, nontoxic form of a staphylococcal subunit vaccine protein (mSEB). All lines selected for analysis were homozygous and contained a single copy of the transgene. METHODS: Each transgenic soybean seed was screened for transgene presence and recombinant protein expression via PCR and western blotting. Whole seed mRNA was extracted and cDNA libraries constructed for Illumina sequencing. Following alignment to the soybean reference genome, differential gene expression analysis was conducted using edgeR and cufflinks. Functional analysis of differentially expressed genes was carried out using the gene ontology analysis tool AgriGO. RESULTS: The transcriptomes of nine seeds from each transgenic line were sequenced and compared with wild type seeds. Native soybean gene expression was significantly altered in line 764 (mSEB) with more than 3000 genes being upregulated or downregulated. ST77 (hTG) and ST111 (hMBP) had significantly less differences with 52 and 307 differentially expressed genes respectively. Gene ontology enrichment analysis found that the upregulated genes in the 764 line were annotated with functions related to endopeptidase inhibitors and protein synthesis, but suppressed expression of genes annotated to the nuclear pore and to protein transport. No significant gene ontology terms were detected in ST77, and only a few genes involved in photosynthesis and thylakoid functions were downregulated in ST111. Despite these differences, transgenic plants and seeds appeared phenotypically similar to non-transgenic controls. There was no correlation between recombinant protein expression level and the quantity of differentially expressed genes detected. CONCLUSIONS: Measurable unscripted gene expression changes were detected in the seed transcriptomes of all three transgenic soybean lines analyzed, with line 764 being substantially altered. Differences detected at the transcript level may be due to T-DNA insert locations, random mutations following transformation or direct effects of the recombinant protein itself, or a combination of these. The physiological consequences of such changes remain unknown.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Glycine max/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Perfilação da Expressão Gênica , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Sementes/química , Sementes/genética , Glycine max/genéticaRESUMO
There is increasing evidence that the tachykinin substance P (SP) can augment inflammatory immune responses within the CNS. We have recently demonstrated that resident CNS cells express high-affinity receptors for this neuropeptide (neurokinin-1 receptors [NK-1R]), and we have shown that SP can significantly augment glial inflammatory responses to clinically relevant Gram-negative bacteria. Furthermore, we provided evidence that endogenous SP/NK-1R interactions are an essential component in the initiation and/or progression of CNS inflammation following in vivo exposure to these pathogens. In this study, we demonstrate that SP similarly enhances inflammatory glial responses to the major Gram-positive causative agent of bacterial meningitis, Streptococcus pneumoniae, and show that endogenous SP/NK-1R interactions play a critical role in the development of CNS inflammation in an in vivo model of pneumococcal meningitis. Importantly, we provide the first demonstration, to our knowledge, that pharmacological targeting of the NK-1R not only prevents the development of damaging inflammation when administered prophylactically, but can also limit or reverse neuroinflammation associated with an established streptococcal CNS infection when delivered therapeutically. We show that an NK-1R antagonist attenuates increases in CNS inflammatory cytokine levels and decreases in immunosuppressive cytokine production associated with an ongoing S. pneumoniae infection. Furthermore, we demonstrate that such a therapeutic intervention reverses infection-associated gliosis and demyelination in the absence of changes in CNS bacterial burden. Together, these results suggest that targeting SP/NK-1R interactions is a strategy worthy of further study for the treatment of microbially induced neuroinflammation.
Assuntos
Inflamação/tratamento farmacológico , Meningite Pneumocócica/tratamento farmacológico , Antagonistas dos Receptores de Neurocinina-1 , Animais , Sistema Nervoso Central/patologia , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Doenças Desmielinizantes , Bactérias Gram-Negativas/efeitos dos fármacos , Inflamação/microbiologia , CamundongosRESUMO
Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)-a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.
Assuntos
Glycine max/metabolismo , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Tireoglobulina/metabolismo , Transformação Genética , Western Blotting , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Genes Sintéticos , Vetores Genéticos , Humanos , Microscopia Confocal , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Estabilidade Proteica , Rhizobium/genética , Rhizobium/metabolismo , Sementes/genética , Glycine max/genética , Tireoglobulina/genética , TransgenesRESUMO
IL-27 is a heterodimeric cytokine composed of p28 and Epstein Barr virus induced gene 3 (Ebi3) protein subunits. In the present study, we questioned whether murine gammaherpesvirus 68 (HV-68) could induce expression of Ebi3, p28, and IL-27 in this mouse model of an EBV-like infection. Cultured macrophages and dendritic cells exposed to HV-68 upregulated p28 mRNA expression and increased secretion of the p28 and IL-27 (p28+Ebi3) proteins. B220(+) and CD11b(+) cells also upregulated p28 mRNA expression following in vivo infection with this virus. Surprisingly, no significant increases in p28 or IL-27 protein production were observed in vivo during the acute or mononucleosis phases of the disease. The possibility that HV-68-induced upregulation of p28 mRNA expression primed cells for IL-27 secretion was suggested by the ability of a TLR4 agonist to augment cytokine production. When cultured macrophages and dendritic cells were exposed to virus plus a suboptimal dose of LPS, increased levels of p28 protein expression were observed. More importantly, when latently infected mice were challenged with a sublethal dose of LPS, augmented p28 and IL-27 protein production occurred. Using a model of sepsis, mice latently infected with HV-68 had exaggerated p28 protein production when compared to mice that were singularly infected or subjected to cecal ligation and puncture. Taken together, these studies define expression of HV-68 induced IL-27, and suggest that mice latently infected with this gammaherpesvirus will have exaggerated responses when confronted with other stimuli capable of inducing this member of the IL-12 family of cytokines.
Assuntos
Infecções por Herpesviridae/fisiopatologia , Interleucina-17/biossíntese , Receptores de Citocinas/biossíntese , Rhadinovirus , Infecções Tumorais por Vírus/fisiopatologia , Animais , Células Cultivadas , Células Dendríticas/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Subunidades Proteicas/biossíntese , RNA Mensageiro/metabolismo , Rhadinovirus/metabolismo , Receptor 4 Toll-Like/agonistasRESUMO
OBJECTIVE: We questioned whether infection with murine gammaherpesvirus 68 (HV-68) might exacerbate inflammatory bowel disease using mice deficient in IL-10 (IL-10-/-) as a model of developing colitis. METHODS: Groups of C57BL/6 mice and IL-10-/- mice were mock-treated or infected with HV-68. Two months following infection, mice were euthanized and a variety of parameters were measured to quantify the extent of inflammation and the presence of virus. Measurements included survival, body weight, splenomegaly, colonic disease scores, liver histopathology, viable bacteria in the liver, and splenic viral burden. RESULTS: IL-10-/- mice infected with HV-68 displayed reduced survival, lower body weights, increased splenomegaly, exacerbated colonic disease scores, increased numbers of viable bacteria in the liver, and increased leukocyte liver infiltration when compared to mock-treated IL-10-/- mice or HV-68 infected C57BL/6 mice. Surprisingly, levels of infectious or latent virus were not significantly different between the groups of mice exposed to HV-68. CONCLUSIONS: The presence of HV-68 in IL-10-/- mice exacerbates the developing clinical disease in this animal model of colitis.
Assuntos
Infecções por Herpesviridae , Doenças Inflamatórias Intestinais , Interleucina-10 , Camundongos Knockout , Rhadinovirus/imunologia , Animais , Linhagem Celular , Colo/imunologia , Colo/patologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/patologia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/virologia , Interleucina-10/genética , Interleucina-10/metabolismo , Leucócitos/citologia , Leucócitos/imunologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Rhadinovirus/patogenicidade , Baço/patologia , Taxa de Sobrevida , Carga Viral , Latência ViralRESUMO
While Ecstasy (3,4-methylenedioxymethamphetamine, MDMA) has been shown to modulate immune responses, no studies have addressed drug-induced alterations to viral infection. In this study, bone marrow-derived macrophages were exposed to MDMA, then infected with murine gammaherpesvirus-68, and the expression of monokines assessed. MDMA-induced reductions in virus-stimulated monokine mRNA expression were observed in a dose-dependent manner. In particular, IL-6 mRNA expression and secretion was significantly decreased in gammaherpesvirus-infected macrophages exposed to MDMA. Concentrations of MDMA capable of reducing monokine production did not induce significant cell death and allowed normal viral gene expression. These studies represent the first to demonstrate the ability of this drug of abuse to alter a viral-induced macrophage response.
Assuntos
Gammaherpesvirinae/crescimento & desenvolvimento , Macrófagos/efeitos dos fármacos , Monocinas/genética , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Monocinas/biossíntese , Monocinas/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Trace amines such as tyramine, octopamine and beta-phenylethylamine bind with high affinity to the mammalian trace amine-associated receptor 1 (Taar1), potentially activating G-proteins in the synaptic membranes of target neurons. Recently there has been significant interest in Taar1, since this receptor can bind certain psychoactive drugs of abuse such as Ecstasy (3,4-methylenedioxymethamphetamine). Surprisingly, Ecstasy has been shown to alter responses of immune cells, and we questioned whether Taar receptors might be responsible for this effect. Using sensitive and quantitative RT-PCR assays, we found no detectable expression of Taar mRNA in bone marrow, or in primary cultures of mouse macrophages and dendritic cells whether quiescent or activated by exposure to lipopolysaccharide or the mouse gamma herpesvirus-68 (gammaHV-68). Mouse B cells and NK cells isolated from spleen, however, showed expression of several Taar mRNA species. Taar mRNA expression was also upregulated in human peripheral blood lymphocytes following in vitro stimulation with PHA. These studies represent the first to define expression of the mRNAs encoding these trace amine receptors in leukocytes.
Assuntos
Expressão Gênica/fisiologia , Leucócitos/metabolismo , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Células da Medula Óssea , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Contagem de Leucócitos/métodos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rhadinovirus/metabolismo , Fatores de TempoRESUMO
The cytokine storm induced by staphylococcal enterotoxin B (SEB) describes the rapid and dramatic induction of mediators which are likely responsible for the toxin's deleterious effects. However despite the use of numerous animal models for investigating SEB related illness in humans, mechanisms of toxicity and correlates of protection remain unclear. In the present study, we used an LPS-potentiated model of SEB lethality to investigate the toxin-induced cytokine and chemokine responses in untreated and immunized mice. Of 30 separate mediators analyzed, serum levels for 28 or 27 of these cytokines and chemokines were elevated following administration of dosages of 3 or 30 LD50 of native SEB, respectively. Mice immunized with a non-toxic SEB vaccine candidate expressed in either E. coli or transgenic soy expression systems were protected from lethality when challenged with potentiated SEB. The majority of SEB-induced cytokines and chemokines (21 of 28 or 23 of 27 following challenge with dosages of 3 or 30 LD50 of native SEB, respectively) were significantly decreased in mice immunized with an SEB vaccine candidate when compared to control animals. Together, these studies provide the most comprehensive evaluation of the cytokine storm induced in this LPS-potentiated model of SEB lethality to date. As with other animal models, the identification of those mediators which are necessary and sufficient for SEB-induced toxicity remains unclear.
Assuntos
Quimiocinas/sangue , Citocinas/sangue , Enterotoxinas/imunologia , Enterotoxinas/toxicidade , Imunização , Vacinas Antiestafilocócicas/imunologia , Animais , Quimiocinas/imunologia , Quimiocinas/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Escherichia coli/genética , Lipopolissacarídeos/administração & dosagem , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Glycine max/genética , Vacinas Antiestafilocócicas/administração & dosagem , Vacinas Antiestafilocócicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Gender-based differences in the incidence and severity of bacterial sepsis render males more susceptible to septic shock than females. However, the mechanisms that underlie this sexual dimorphism remain unclear. In the present study we confirm that males produce significantly higher levels of the inflammatory cytokine IL-6 and the acute phase protein LPS-binding protein (LBP) than females following in vivo lipopolysaccharide (LPS) exposure. It has also been verified that LPS-challenged male-derived macrophages produce higher levels of IL-1beta and lower levels of PGE(2) than similarly treated female-derived cells. Importantly, we demonstrated that male-derived macrophages produce significantly higher levels of the inflammatory chemokine IP-10 following LPS challenge than their female counterparts. It has been demonstrated further that, although resting macrophage levels of mRNA encoding Toll-like receptor 4 (TLR4) and its co-receptor CD14, are not significantly different between genders, male-derived macrophages constitutively express higher levels of these proteins on their cell surface. Elevated circulating levels of LBP and constitutively higher cell surface expression of TLR4 and CD14 on macrophages in males could result in the observed sexual dimorphism in LPS-induced inflammatory mediator production and the greater susceptibility of males to bacterial sepsis.
Assuntos
Suscetibilidade a Doenças , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Caracteres Sexuais , Choque Séptico/metabolismo , Receptor 4 Toll-Like/metabolismo , Doença Aguda , Proteínas de Fase Aguda/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Escherichia coli/química , Feminino , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Choque Séptico/induzido quimicamente , Choque Séptico/patologiaRESUMO
In vivo responses to bacterially derived superantigen-like toxins have been difficult to define due to the inherent limitations with rodent models and the relevance that the results obtained from such models may, or may not, have for human pathophysiology. Further the use of challenge doses of superantigen toxins that are lethal or supra-lethal complicates analogies to human exposures which are rarely fatal. Here, we utilize the superantigen, staphylococcal enterotoxin B, at doses that are sublethal in a swine model of toxin-induced incapacitation. Relevant dosing using an animal species for which this toxin is a true superantigen distinguishes this model.
Assuntos
Modelos Animais de Doenças , Enterotoxinas/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Animais , Mediadores da Inflamação/sangue , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/mortalidade , SuínosRESUMO
Transgenic crops have been utilized for decades to enhance agriculture and more recently have been applied as bioreactors for manufacturing pharmaceuticals. Recently, we investigated the gene expression profiles of several in-house transgenic soybean events, finding one transformant group to be consistently different from our controls. In the present study, we examined polymorphisms and sequence variations in the exomes of the same transgenic soybean events. We found that the previously dissimilar soybean line also exhibited markedly increased levels of polymorphisms within mRNA transcripts from seed tissue, many of which are classified as gene expression modifiers. The results from this work will direct future investigations to examine novel SNPs controlling traits of great interest for breeding and improving transgenic soybean crops. Further, this study marks the first work to investigate SNP rates in transgenic soybean seed tissues and demonstrates that while transgenesis may induce abundant unanticipated changes in gene expression and nucleotide variation, phenotypes and overall health of the plants examined remained unaltered.
RESUMO
Incidences of osteomyelitis caused by Staphylococcus aureus have increased dramatically in recent years, in part, due to the appearance of community-acquired antibiotic-resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of the pivotal inflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), by osteoblasts during S. aureus-associated bone infection. Quantitative real-time PCR was employed to determine levels of mRNA encoding MCP-1 in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis. Expression of this inflammatory chemokine and osteoblast-specific markers was investigated by confocal laser scanning microscopy in bone tissue from organ cultures of neonatal mouse calvaria and from the in vivo mouse model. Furthermore, the clinical relevancy of these findings was investigated by performing similar studies on infected human bone tissue from patients with S. aureus-associated osteomyelitis. Here, we confirm that expression of mRNA encoding MCP-1 is elevated in bacterially infected murine bone tissue. Importantly, we show that these increases translate into marked elevations in the expression of MCP-1 protein that co-localizes with osteoblast markers in infected bone tissue. Such increases could not be attributed solely to mechanical damage as a similar response was observed in infected but otherwise undamaged organ cultures. Finally, we have demonstrated the in vivo production of MCP-1 by osteoblasts in bone specimens from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases such as staphylococcal osteomyelitis induces cells to produce a key inflammatory chemokine that can direct appropriate host responses or may contribute to progressive inflammatory damage.
Assuntos
Quimiocina CCL2/biossíntese , Osteoblastos/metabolismo , Osteomielite/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/isolamento & purificação , Animais , Animais Recém-Nascidos , Quimiocina CCL2/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Osteomielite/microbiologia , RNA Mensageiro/genética , Infecções Estafilocócicas/microbiologiaRESUMO
Transgenic crops have become a staple in modern agriculture, and are typically characterized using a variety of molecular techniques involving proteomics and metabolomics. Characterization of the transgene insertion site is of great interest, as disruptions, deletions, and genomic location can affect product selection and fitness, and identification of these regions and their integrity is required for regulatory agencies. Here, we present CONTRAILS (Characterization of Transgene Insertion Locations with Sequencing), a straightforward, rapid and reproducible method for the identification of transgene insertion sites in highly complex and repetitive genomes using low coverage paired-end Illumina sequencing and traditional PCR. This pipeline requires little to no troubleshooting and is not restricted to any genome type, allowing use for many molecular applications. Using whole genome sequencing of in-house transgenic Glycine max, a legume with a highly repetitive and complex genome, we used CONTRAILS to successfully identify the location of a single T-DNA insertion to single base resolution.
RESUMO
Ovarian folliculogenesis is characterized, in part, by the formation and expansion of the fluid-filled antrum. Development of this cavity requires water influx, which may occur by transcellular or pericellular transport mechanisms. To assess the contribution of these mechanisms to the water permeability of an antral follicle, the rate of (3)H(2)O and (14)C-inulin (a complex sugar restricted to the extracellular compartment) uptake into isolated follicles was determined. The rate of H(2)O movement was 3.5-fold greater than that of inulin, suggesting that water enters a follicle primarily by transcellular pathways. Preincubation of the follicles with 50 microM HgCl(2) [a nonspecific aquaporin (Aqp) inhibitor] decreased H(2)O movement to levels seen with inulin, indicating that transcellular water movement is mediated through Aqp. To demonstrate the functional presence of Aqp in granulosa cells, we show that swelling in response to a hypotonic insult is attenuated by preincubation with 50 microM HgCl(2). Flow cytometry demonstrated the presence of Aqps-7, -8, and -9, thus identifying candidate Aqp potentially mediating water movement into antral follicles. These results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by Aqps -7, -8, and/or -9 in granulosa cells.
Assuntos
Aquaporinas/fisiologia , Água Corporal/metabolismo , Canais Iônicos/fisiologia , Folículo Ovariano/metabolismo , Animais , Aquaporinas/análise , Feminino , Células da Granulosa/química , Canais Iônicos/análise , Cloreto de Mercúrio/farmacologia , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
A growing body of evidence has shown that bacterially challenged bone-forming osteoblasts are a significant source of an array of cytokines and chemokines that can support immune responses during bone disease. In the present study, Staphylococcus aureus and Salmonella, two common pathogens of bone, were investigated for their ability to induce production of two related inflammatory cytokines, interleukin-1beta (IL-1beta) and IL18, in osteoblasts. Cultured mouse osteoblasts were found to respond rapidly to either bacterial challenge by upregulation in the levels of mRNA encoding both IL-1beta and IL-18. Surprisingly, this mRNA expression did not translate into intracellular accumulation of IL-1beta or IL-18 precursor proteins or secretion of mature cytokines, despite the presence of detectable caspase-1 activity in these cells. These studies demonstrate that although osteoblasts can secrete a number of key proinflammatory mediators in response to bacterial pathogens, IL-1beta and IL-18 are not among this number. We suggest that osteoblasts are an unlikely source of these cytokines during the progression of bacterial infection of bone.
Assuntos
Interleucina-18/biossíntese , Interleucina-1/biossíntese , Osteoblastos/imunologia , Osteoblastos/microbiologia , Salmonella/fisiologia , Staphylococcus aureus/fisiologia , Transcrição Gênica , Animais , Animais Recém-Nascidos , Caspases/metabolismo , Células Cultivadas , Relação Dose-Resposta Imunológica , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Regulação para CimaRESUMO
A growing body of evidence has shown that bacterially challenged osteoblasts can play a significant role in the initiation of inflammatory immune responses at sites of bone disease. We have recently demonstrated the surprising ability of osteoblasts exposed to bacteria to express CD40, a molecule that plays a critical costimulatory role in the activation of T lymphocytes. In the present study, we have extended our investigations into the ability of osteoblasts to interact with CD4+ T lymphocytes by determining the expression of antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human osteoblasts following exposure to two common pathogens of bone, Staphylococcus aureus and Salmonella. Cultured osteoblasts were found to respond rapidly to bacterial challenge by induction of mRNA encoding MHC class II molecules or its transcriptional regulator. Increased mRNA expression translated into expression of MHC class II proteins in murine and human osteoblasts as determined by Western blot analysis and by immunohistochemical and immunofluorescent microscopy. Furthermore, the increased surface expression of these molecules on osteoblasts exposed to bacteria was confirmed by FACS analysis. Finally, we show that bacterial challenge results in the elevated functional expression of MHC class II molecules on osteoblasts by demonstrating the enhanced ability of these cells to interact with T lymphocytes and to initiate antigen-specific T cell activation. Taken together, these data suggest a previously unappreciated role for osteoblasts in the initiation of T lymphocyte activation at sites of bacterial infection in bone tissue.
Assuntos
Infecções Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Genes MHC da Classe II/fisiologia , Osteoblastos/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Salmonella enterica/genética , Salmonella enterica/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Following viral infection, the expression of substance P and its receptor can contribute significantly to the resulting host response. For gammaherpesvirus infection of mice, the presence of this tachykinin and its receptor contributes to the protective host response. It is likely that this augmentation of the immune response is directed toward the developing T helper type 1 response and cytotoxic T lymphocyte activation. However it has also been shown that the presence of substance P and its receptor may contribute to viral diseases by facilitating viral replication or by contributing to a destructive inflammatory response. Specifically, the presence of substance P can augment replication of HIV in cultured macrophages, which is especially significant since levels of this tachykinin are elevated in patients with this viral disease. Furthermore, rodent models of paramyxovirus infection have demonstrated that the presence of neurokinin receptors and their ligands contributes to the destructive inflammatory response in airways. Taken together, these studies demonstrate a surprising role for substance P and the neurokinin-1 receptor in the host response following these viral infections.
Assuntos
Antivirais/farmacologia , Neurotransmissores/farmacologia , Taquicininas/farmacologia , Animais , Gammaherpesvirinae/metabolismo , Humanos , Inflamação , Camundongos , Paramyxovirinae/metabolismo , Receptores da Neurocinina-1/metabolismo , Linfócitos T/virologia , Viroses/metabolismoRESUMO
Tachykinins represent a family of peptides which have significant effects on such diverse physiological responses as gut peristalsis, vascular permeability, and the transmission of pain. Traditionally, neurons have been identified as the major source of tachykinin peptides in peripheral tissues and in the central nervous system, resulting in their classification as neuropeptides or, more specifically, as neurokinins. The fact that a variety of cell types can express one or more members of the family of neurokinin receptors accounts for the remarkable ability of these tachykinin peptides to affect multiple organ systems and numerous biological responses. Perhaps one of the most surprising findings during the last 15 years has been the understanding that expression of neurokinin receptors on leukocyte populations can be constitutive or inducible. This fact establishes a receptor-mediated mechanism by which tachykinin peptides can affect both innate and specific immune responses. Due to the numerous cytokines, chemokines, and other soluble mediators that can modulate leukocyte responses, the importance of tachykinin-mediated modulation of the immune response has met with some skepticism. The following reviews make a strong case for neurokinin receptor expression by leukocytes as a significant participant in host response. From hematopoiesis to antigen-specific T lymphocyte responses, tachykinins are present and can dramatically modulate maturation and responsiveness of these immune cells. Studies which document this surprising role for neurokinin receptor expression by leukocytes are detailed in the following reviews.
Assuntos
Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/patologia , Taquicininas/metabolismo , Animais , Antivirais/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Infecções , Inflamação , Neurocinina A/metabolismo , Neurônios/metabolismo , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Linfócitos T/metabolismoRESUMO
Mammalian tachykinins are traditionally viewed as neuropeptides. This review describes the mammalian tachykinins and evidence for expression of these peptides by non-neuronal cells. Tachykinin expression is defined as evidence for gene transcription, peptide production, or peptide secretion. Since the functions of mammalian tachykinins have been amply reviewed, the biological roles of these peptides will be noted briefly, with emphasis on immune cell action. Of particular interest is the predicted existence and non-neuronal expression of new mammalian tachykinins--hemokinin 1, the endokinins and C14TKL-1. Synthetic forms of these peptides have high affinity for the NK1 receptor, the protein traditionally associated with substance P binding. By acting on the same "substance P" receptor, these tachykinins have the potential for promoting similar post-receptor functions. The structure and action of representative non-mammalian tachykinins acting on mammals are also presented. These peptides, of interest in their own right, also appear to exhibit selectivity for the NK1 receptor. They strengthen the notion that multiple ligands may be capable of binding to one receptor, NK1, effecting similar cellular responses.
Assuntos
Regulação da Expressão Gênica , Taquicininas/biossíntese , Taquicininas/fisiologia , Animais , Ligantes , Neurocinina A/metabolismo , Neurocinina B/metabolismo , Peptídeos/química , Fisalemina/metabolismo , Ligação Proteica , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Taquicininas/metabolismoRESUMO
Hemokinin 1 is encoded by preprotachykinin C (PPT-C) mRNA, and has been proposed as a regulator of B and T cell lymphopoiesis. Here we demonstrate the expression of mouse PPT-C mRNA by CD11b+ macrophages, CD11c+ dendritic cells and in the microglial cell line EOC 13.31. Expression was detected in freshly isolated CD11b+CD11c+ bone marrow cells, as well as in M-CSF expanded bone marrow-derived macrophages and GM-CSF expanded bone marrow-derived dendritic cells. There was preferential expression of PPT-C mRNA in dendritic cell subpopulations that were CD11b+, but not B220+ or GR-1+. These studies are the first to demonstrate PPT-C mRNA expression by cells of the myeloid lineage.