RESUMO
BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (CDI). However, transferring undefined living bacteria entails uncontrollable risks for infectious and metabolic or malignant diseases, particularly in immunocompromised patients. We investigated whether sterile fecal filtrates (containing bacterial debris, proteins, antimicrobial compounds, metabolic products, and oligonucleotides/DNA), rather than intact microorganisms, are effective in patients with CDI. METHODS: We performed a clinical case series to investigate the effects of fecal filtrate transfer (FFT) in 5 patients with symptomatic chronic-relapsing CDI at the Department of Internal Medicine I at the University Hospital Schleswig-Holstein (Kiel, Germany). Patients were followed up for at least 6 months and for up to 33 months. Stool was collected from 5 donors selected by the patients, and fully characterized according to FMT standards. Stool was sterile-filtered to remove small particles and bacteria; the filtrate was transferred to patients in a single administration via nasojejunal tube. Fecal samples were collected from patients before and at 1 week and 6 weeks after FFT. Microbiome, virome, and proteome profiles of donors and patients were compared. RESULTS: In all 5 patients, FFT restored normal stool habits and eliminated symptoms of CDI for a minimum period of 6 months. Proteome analyses of selected FFT filtrates showed no obvious protein candidates associated with therapeutic efficacy. 16S ribosomal RNA gene sequencing detected diverse bacterial DNA signatures in the filtrates. Analysis of virus-like particles from a filtrate found to reduce symptoms of CDI showed a complex signature of bacteriophages. Bacterial phylogeny and virome profile analyses of fecal samples from recipients indicated longitudinal changes in microbial and viral community structures after FFT. CONCLUSIONS: A preliminary investigation of 5 patients with CDI shows that transfer of sterile filtrates from donor stool (FFT), rather than fecal microbiota, can be sufficient to restore normal stool habits and eliminate symptoms. This finding indicates that bacterial components, metabolites, or bacteriophages mediate many of the effects of FMT, and that FFT might be an alternative approach, particularly for immunocompromised patients.
Assuntos
Clostridioides difficile , Enterocolite Pseudomembranosa/terapia , Transplante de Microbiota Fecal/métodos , Esterilização , Idoso , Feminino , Filtração , Microbioma Gastrointestinal , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma , RecidivaRESUMO
PURPOSE: Interleukin-6 (IL-6) production and signalling are increased in the inflamed mucosa in inflammatory bowel diseases (IBD). As published serum levels of IL-6 and its soluble receptors sIL-6R and sgp130 in IBD are from small cohorts and partly contradictory, we systematically evaluated IL-6, sIL-6R and sgp130 levels as markers of disease activity in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Consecutive adult outpatients with confirmed CD or UC were included, and their disease activity and medication were monitored. Serum from 212 CD patients (815 measurements) and 166 UC patients (514 measurements) was analysed, and 100 age-matched healthy blood donors were used as controls. RESULTS: IL-6 serum levels were significantly elevated in active versus inactive CD and UC, also compared with healthy controls. However, only a fraction of IBD patients showed increased serum IL-6. IL-6 levels ranged up to 32.7 ng/mL in active CD (> 5000-fold higher than in controls), but also up to 6.9 ng/mL in inactive CD. Increases in active UC (up to 195 pg/mL) and inactive UC (up to 27 pg/mL) were less pronounced. Associations between IL-6 serum levels and C-reactive protein concentrations as well as leukocyte and thrombocyte counts were observed. Median sIL-6R and sgp130 levels were only increased by up to 15%, which was considered of no diagnostic significance. CONCLUSIONS: Only a minority of IBD patients shows elevated IL-6 serum levels. However, in these patients, IL-6 is strongly associated with disease activity. Its soluble receptors sIL-6R and sgp130 do not appear useful as biomarkers in IBD.
Assuntos
Receptor gp130 de Citocina/sangue , Inflamação/imunologia , Doenças Inflamatórias Intestinais/sangue , Interleucina-6/sangue , Adulto , Bancos de Espécimes Biológicos , Feminino , Alemanha , Humanos , Doenças Inflamatórias Intestinais/imunologia , MasculinoRESUMO
Sarcoidosis is a polygenic immune disorder with predominant manifestation in the lung. Genome-wide linkage analysis previously indicated that the extended major histocompatibility locus on chromosome 6p was linked to susceptibility to sarcoidosis. Here, we carried out a systematic three-stage SNP scan of 16.4 Mb on chromosome 6p21 in as many as 947 independent cases of familial and sporadic sarcoidosis and found that a 15-kb segment of the gene butyrophilin-like 2 (BTNL2) was associated with the disease. The primary disease-associated variant (rs2076530; P(TDT) = 3 x 10(-6), P(case-control) = 1.1 x 10(-8); replication P(TDT) = 0.0018, P(case-control) = 1.8 x 10(-6)) represents a risk factor that is independent of variation in HLA-DRB1. BTNL2 is a member of the immunoglobulin superfamily and has been implicated as a costimulatory molecule involved in T-cell activation on the basis of its homology to B7-1. The G --> A transition constituting rs2076530 leads to the use of a cryptic splice site located 4 bp upstream of the affected wild-type donor site. Transcripts of the risk-associated allele have a premature stop in the spliced mRNA. The resulting protein lacks the C-terminal IgC domain and transmembrane helix, thereby disrupting the membrane localization of the protein, as shown in experiments using green fluorescent protein and V5 fusion proteins.
Assuntos
Glicoproteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Sarcoidose/genética , Lavagem Broncoalveolar , Butirofilinas , Proteínas de Fluorescência Verde/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1 , Células HeLa , Humanos , Monócitos/microbiologia , Monócitos/fisiologia , Mycobacterium tuberculosis/patogenicidade , Conformação Proteica , Proteínas Recombinantes de Fusão , Fatores de Risco , Sarcoidose/imunologia , Sarcoidose/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: Transsignaling of interleukin (IL)-6 is a central pathway in the pathogenesis of disorders associated with chronic inflammation, such as Crohn disease, rheumatoid arthritis, and inflammatory colon cancer. Notably, IL-6 also represents an independent risk factor for coronary artery disease (CAD) in humans and is crucially involved in vascular inflammatory processes. METHODS AND RESULTS: In the present study, we showed that treatment with a fusion protein of the natural IL-6 transsignaling inhibitor soluble glycoprotein 130 (sgp130) and IgG1-Fc (sgp130Fc) dramatically reduced atherosclerosis in hypercholesterolemic Ldlr(-/-) mice without affecting weight gain and serum lipid levels. Moreover, sgp130Fc treatment even led to a significant regression of advanced atherosclerosis. Mechanistically, endothelial activation and intimal smooth muscle cell infiltration were decreased in sgp130Fc-treated mice, resulting in a marked reduction of monocyte recruitment and subsequent atherosclerotic plaque progression. Of note, patients with CAD exhibited significantly lower plasma levels of endogenous sgp130, suggesting that a compromised counterbalancing of IL-6 transsignaling may contribute to atherogenesis in humans. CONCLUSIONS: These data clarify, for the first time, the critical involvement of, in particular, the transsignaling of IL-6 in CAD and warrant further investigation of sgp130Fc as a novel therapeutic for the treatment of CAD and related diseases.
Assuntos
Aterosclerose/fisiopatologia , Doença da Artéria Coronariana/fisiopatologia , Progressão da Doença , Interleucina-6/fisiologia , Transdução de Sinais/fisiologia , Idoso , Animais , Aterosclerose/prevenção & controle , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/prevenção & controle , Receptor gp130 de Citocina/sangue , Receptor gp130 de Citocina/farmacologia , Receptor gp130 de Citocina/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/fisiopatologia , Lipídeos/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de LDL/deficiência , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e.g., Crohn's disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2(wt) or NOD2(L1007fsinsC) to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2(wt) cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-κB (e.g., TNFAIP3 [A20] and IER3). This genotype-dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-κB activation. We show that IER3 impairs the protective effect of NOD2(wt) against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counterregulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation.
Assuntos
Doença de Crohn/imunologia , Regulação para Baixo/imunologia , Mutação da Fase de Leitura/imunologia , Perfilação da Expressão Gênica , Genoma Humano , Proteína Adaptadora de Sinalização NOD2/deficiência , Animais , Linhagem Celular Tumoral , Células Cultivadas , Doença de Crohn/genética , Doença de Crohn/patologia , Regulação para Baixo/genética , Perfilação da Expressão Gênica/métodos , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/imunologia , Monócitos/patologia , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/fisiologiaRESUMO
Crohn disease and ulcerative colitis are two subphenotypes of inflammatory bowel disease (IBD), a complex disorder resulting from gene-environment interaction. We refined our previously defined linkage region for IBD on chromosome 10q23 and used positional cloning to identify genetic variants in DLG5 associated with IBD. DLG5 encodes a scaffolding protein involved in the maintenance of epithelial integrity. We identified two distinct haplotypes with a replicable distortion in transmission (P = 0.000023 and P = 0.004 for association with IBD, P = 0.00012 and P = 0.04 for association with Crohn disease). One of the risk-associated DLG5 haplotypes is distinguished from the common haplotype by a nonsynonymous single-nucleotide polymorphism 113G-->A, resulting in the amino acid substitution R30Q in the DUF622 domain of DLG5. This mutation probably impedes scaffolding of DLG5. We stratified the study sample according to the presence of risk-associated CARD15 variants to study potential gene-gene interaction. We found a significant difference in association of the 113A DLG5 variant with Crohn disease in affected individuals carrying the risk-associated CARD15 alleles versus those carrying non-risk-associated CARD15 alleles. This is suggestive of a complex pattern of gene-gene interaction between DLG5 and CARD15, reflecting the complex nature of polygenic diseases. Further functional studies will evaluate the biological significance of DLG5 variants.
Assuntos
Cromossomos Humanos Par 10/genética , Colite Ulcerativa/genética , Doença de Crohn/genética , Variação Genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas Supressoras de Tumor/genética , Alelos , Substituição de Aminoácidos , Proteínas de Transporte/genética , Estudos de Casos e Controles , Criança , Mapeamento Cromossômico , Clonagem Molecular , Estudos de Coortes , Colite Ulcerativa/epidemiologia , Doença de Crohn/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Genótipo , Haplótipos , Humanos , Masculino , Proteína Adaptadora de Sinalização NOD2 , Linhagem , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
N-Linked glycosylation is an important determinant of protein structure and function. The interleukin-6 signal transducer glycoprotein 130 (gp130) is a common co-receptor for cytokines of the interleukin (IL)-6 family and is N-glycosylated at 9 of 11 potential sites. Whereas N-glycosylation of the extracellular domains D1-D3 of gp130 has been shown to be dispensable for binding of the gp130 ligand IL-6 and its cognate receptor in vitro, the role of the N-linked glycans on domains D4 and D6 is still unclear. We have mutated the asparagines of all nine functional N-glycosylation sites of gp130 to glutamine and systematically analyzed the consequences of deleted N-glycosylation (dNG) in both cellular gp130 and in a soluble gp130-IgG1-Fc fusion protein (sgp130Fc). Our results show that sgp130Fc-dNG is inherently unstable and degrades rapidly under conditions that do not harm wild-type sgp130Fc. Consistently, the bulk of cellular gp130-dNG is not transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of gp130-dNG, which do reach the cell surface, are still able to activate the key gp130 signaling target signal transducer and activator of transcription-3 (STAT3) upon binding of the agonistic complex of IL-6 and soluble IL-6 receptor. In conclusion, N-linked glycosylation is required for the stability but not the signal-transducing function of gp130.
Assuntos
Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Interleucina-6/metabolismo , Nitrogênio/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Cricetinae , Cricetulus , Receptor gp130 de Citocina/genética , Glicoproteínas/genética , Glicosilação , Humanos , Imunoglobulina G/metabolismo , Ligantes , Camundongos , Oxigênio/metabolismo , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT3/metabolismo , Deleção de SequênciaRESUMO
OBJECTIVE: The pleiotropic cytokine interleukin (IL)-6 seems to play a pivotal role in sepsis, but contradictory findings in animal models impede a rationale for therapies directed against IL-6. IL-6 signals by two mechanisms via the ubiquitous transmembrane glycoprotein 130 (gp130): "classic" signaling using membrane-bound IL-6 receptor (IL-6R) and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling is selectively inhibited by soluble gp130 (sgp130). The aim of this study was to systematically compare complete blockade of IL-6 signaling (using a neutralizing anti-IL-6 antibody) and selective blockade of IL-6 trans-signaling (using a fusion protein of sgp130 and the crystallizable fragment of immunoglobulin G1, sgp130Fc) in a standardized cecal ligation and puncture (CLP) sepsis model. DESIGN: Animal study. SETTING: Animal laboratory. SUBJECTS: C57BL/6J mice. INTERVENTIONS: We performed a 96-hr dose-response study and a 24-hr study to investigate short-term mechanisms. In the 96-hr study, CLP was performed in 120 randomized mice (20 mice received vehicle, 10 mice per dose group). Mice were treated with equimolar doses of sgp130Fc (0.01/0.1/1/10 mg/kg) or anti-IL-6 (0.008/0.08/0.8/8 mg/kg) 24 hrs before CLP. Two additional groups received 0.5 mg/kg sgp130Fc 24 hrs before or 1 mg/kg sgp130Fc 24 hrs after CLP. Survival and activity scores were obtained daily until 96 hrs after CLP. In the 24-hr study, mice were randomized into four groups with 10 animals each (sham/vehicle, CLP/vehicle, CLP/anti-IL-6 [0.8 mg/kg], and CLP/sgp130Fc [1 mg/kg]) and killed after 24 hrs. MEASUREMENTS AND MAIN RESULTS: In contrast to anti-IL-6, pretreatment with sgp130Fc significantly and dose-dependently increased survival from 45% to 100%. In addition, 1 mg/kg sgp130Fc administered 24 hrs after CLP increased survival from 45% to 80%. Mechanistically, sgp130Fc efficacy was reflected by complete prevention of epithelial cell apoptosis in the jejunum after CLP, which was not achieved with anti-IL-6. CONCLUSION: Selective inhibition of IL-6 trans-signaling by sgp130Fc has considerable potential for the treatment of sepsis and related disorders.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Sepse/tratamento farmacológico , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sepse/etiologia , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Gut microbiota represent a potential novel target for future prediabetes and type 2 diabetes therapies. In that respect, niacin has been shown to beneficially affect the host-microbiome interaction in rodent models. RESEARCH DESIGN AND METHODS: We characterized more than 500 human subjects with different metabolic phenotypes regarding their niacin (nicotinic acid [NA] and nicotinamide [NAM]) status and their gut microbiome. In addition, NA and NAM delayed-release microcapsules were engineered and examined in vitro and in vivo in two human intervention studies (bioavailability study and proof-of-concept/safety study). RESULTS: We found a reduced α-diversity and Bacteroidetes abundance in the microbiome of obese human subjects associated with a low dietary niacin intake. We therefore developed delayed-release microcapsules targeting the ileocolonic region to deliver increasing amounts of NA and NAM to the microbiome while preventing systemic resorption to avoid negative side effects (e.g., facial flushing). In vitro studies on these delayed-release microcapsules revealed stable conditions at pH 1.4, 4.5, and 6.8, followed by release of the compounds at pH 7.4, simulating the ileocolonic region. In humans in vivo, gut-targeted delayed-release NA but not NAM produced a significant increase in the abundance of Bacteroidetes. In the absence of systemic side effects, these favorable microbiome changes induced by microencapsulated delayed-release NA were associated with an improvement of biomarkers for systemic insulin sensitivity and metabolic inflammation. CONCLUSION: Targeted microbiome intervention by delayed-release NA might represent a future therapeutic option for prediabetes and type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Microbioma Gastrointestinal/efeitos dos fármacos , Niacina/administração & dosagem , Obesidade/sangue , Adulto , Estudos de Casos e Controles , Preparações de Ação Retardada , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/microbiologia , Composição de Medicamentos , Feminino , Humanos , Resistência à Insulina , Masculino , Pessoa de Meia-Idade , Niacina/farmacocinética , Obesidade/tratamento farmacológico , Obesidade/microbiologia , Resultado do TratamentoRESUMO
The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) plays a central role in inflammatory disorders. Transmembrane TNF-alpha and its two receptors are cleaved by the proteinase TNF-alpha converting enzyme (TACE), resulting in appreciable serum levels of soluble TNF-alpha and soluble TNF-alpha receptors (sTNFR1 and -2). The only known functions of sTNFR1 are to antagonize and buffer circulating TNF-alpha. Here, we present evidence that sTNFR1 exerts immunoregulatory functions by induction of apoptosis in monocytes through reverse signaling via transmembrane TNF-alpha. sTNFR1-induced apoptosis is independent of death receptor pathways but depends on autocrine transforming growth factor (TGF)-beta1 signaling through the mitogen-activated protein kinase p38alpha. This novel mechanism has implications for understanding the physiological role of sTNFR1 and for TNF-alpha-blocking therapies of autoimmune diseases.
Assuntos
Apoptose/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas ADAM , Proteína ADAM17 , Adulto , Linhagem Celular , Células Cultivadas , Ativação Enzimática/fisiologia , Feminino , Humanos , Metaloendopeptidases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/enzimologia , Monócitos/metabolismo , Monócitos/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Solubilidade , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Postoperative infectious complications are the leading causes for postoperative sepsis. In severe sepsis, tumor necrosis factor-beta (TNF-beta) NcoI polymorphism was associated with increased mortality. Therefore, the aim of this study was to determine whether the biallelic NcoI polymorphism within the TNF locus is associated with the development of postoperative complications. METHODS: One hundred sixty patients were included in this prospective observation study. Patients undergoing major gastrointestinal surgery, such as esophagectomy, gastrectomy, Whipple operation, major liver resection, or colon resection were included. Patients were monitored during the clinical course, and postoperative complications, divided into severe and minor complications, were documented. The NcoI restriction fragment length polymorphism of the TNF-beta gene was determined by polymerase chain reaction; gene expression as well as complications were correlated. RESULTS: The patients' genotype distribution and demographic characteristics were comparable within the different groups of operations. Patients with the heterozygous genotype TNF-beta1/beta2 had a 1.6-fold higher relative risk for developing complications. If patients with the homozygous genotype TNF-beta2 developed a complication, they had a 1.5-fold higher relative risk for severe complications. Furthermore, the mortality of patients with postoperative sepsis who were homozygous for the genotype TNF-beta2 was significantly elevated. CONCLUSIONS: The TNF-beta NcoI polymorphism influences the development of postoperative complications. While the genotype TNF-beta1/beta2 has a higher risk for developing complications in general, the TNF-beta2/beta2 genotype is associated with more severe complications and mortality from sepsis.
Assuntos
Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Predisposição Genética para Doença/genética , Linfotoxina-alfa/genética , Complicações Pós-Operatórias/etiologia , Sepse/genética , Adulto , Alelos , Feminino , Marcadores Genéticos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Treatment of autoreactive inflammatory diseases such as rheumatoid arthritis with anti-inflammatory drugs is associated with an increased rate of reactivation tuberculosis (TB). Interleukin-6 (IL-6) plays a pivotal role in inflammation and protection against various infectious diseases. IL-6 signals by two mechanisms via the ubiquitous transmembrane protein gp130: 'classic' signaling using the membrane-bound IL-6 receptor (IL-6R), which is expressed mainly on hepatocytes and some leukocytes, and trans-signaling using soluble IL-6R (sIL-6R). Trans-signaling by the IL-6/sIL-6R complex is selectively inhibited by natural soluble gp130 (sgp130) and by sgp130 designer proteins. As specific blockade of IL-6 trans-signaling represents a promising approach for the therapy of inflammatory diseases, we evaluated the potential risk of interfering with this alternative pathway and analyzed the outcome of experimental TB after treatment with an IgG1-Fc fusion protein of soluble gp130 (sgp130Fc) and in sgp130Fc-overexpressing transgenic (sgp130Fc(tg)) mice. In contrast to treatment with anti-tumor necrosis factor (TNF) antibodies, administration of sgp130Fc did not interfere with protective immune responses after infection with Mycobacterium tuberculosis (Mtb). Moreover, Mtb-infected sgp130Fc(tg) mice were capable of controlling mycobacterial growth. Our finding that IL-6 trans-signaling plays no role for protective immune responses against Mtb supports the superior safety of therapeutic targeting of IL-6 trans-signaling compared to anti-TNF treatment.
Assuntos
Interleucina-6/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/administração & dosagem , Resultado do Tratamento , Tuberculose/imunologia , Fatores de Necrose Tumoral/imunologiaRESUMO
Binding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation.
Assuntos
Proteínas de Fase Aguda/imunologia , Receptor gp130 de Citocina/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-6/imunologia , Receptores de Interleucina-6/imunologia , Transdução de Sinais/imunologia , Proteínas de Fase Aguda/genética , Animais , Células CHO , Células COS , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Cricetinae , Cricetulus , Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/farmacologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/imunologia , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/farmacologia , Camundongos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , SolubilidadeRESUMO
The immunoregulatory cytokine interleukin6 (IL6) acts in a pro- and anti-inflammatory fashion. Synthesized by myeloid cells, fibroblasts and endothelial cells, IL6 on target cells, binds to the IL6 receptor (IL6R) and signals via complex formation with the ubiquitously expressed gp130 receptor. Paradoxically, most cells that respond to IL6 during inflammatory states do not express the IL6R and are themselves not directly responsive to the cytokine. A naturally occurring soluble form of the IL6R renders all cells responsive to IL6. This alternative signaling process is called IL6 transsignaling. Here we developed a transgenic strategy based on the overexpression of the soluble form of gp130, which specifically blocks all IL6 responses mediated by the soluble IL6R but does not affect IL6 responses via the membrane bound IL6R. In these mice, inflammatory processes are blocked as in IL6(-/-) mice, strongly arguing for a major role of the soluble IL6R during inflammation in vivo.
Assuntos
Interleucina-6/metabolismo , Doença Aguda , Animais , Linhagem Celular Tumoral , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , DNA Complementar/genética , Modelos Animais de Doenças , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-6/genética , Camundongos , Camundongos Transgênicos , Transdução de Sinais , SolubilidadeRESUMO
NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl dipeptide, have been associated with chronic inflammatory diseases of barrier organs such as Crohn disease, asthma and atopic eczema. In this study we identify CD147 (also known as BSG and EMMPRIN), a membrane-bound regulator of cellular migration, differentiation and inflammatory processes, as a protein interaction partner of NOD2. We demonstrate a complex influence of the CD147-NOD2 interaction on NOD2-dependent signaling responses. We show that CD147 itself acts as an enhancer of the invasion of Listeria monocytogenes, an intracellular bacterial pathogen. We propose that the CD147-NOD2 interaction serves as a molecular guide to regulate NOD2 function at sites of pathogen invasion.
Assuntos
Basigina/metabolismo , Membrana Celular/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Proteína Adaptadora de Sinalização NOD2/metabolismo , Basigina/genética , Western Blotting , Linhagem Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Interleucina-8/metabolismo , Listeria monocytogenes/citologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas do Sistema de Duplo-HíbridoRESUMO
IL-6 plays a pivotal role in immune responses and certain oncologic conditions. The intense investigation of its biological activity and function led to the discovery of two different IL-6-driven signalling pathways. Binding to the membrane-bound IL-6 receptor (mIL-6R, CD126) causes the recruitment of two gp130 co-receptor molecules (CD130) and the activation of intracellular signalling cascades via gp130. Although this classical pathway is mainly limited to hepatocytes, neutrophils, monocytes/macrophages and certain other leukocyte populations, which express IL-6R on their surface, an alternative mechanism has also been described. Proteolytic cleavage of the mIL-6R protein or translation from alternatively spliced mRNA leads to the generation of a soluble form of the IL-6R (sIL-6R), which is likewise able to bind to IL-6. The resulting IL-6/sIL-6R complex is also capable of binding to gp130 and inducing intracellular signalling. Through this so-called 'trans-signalling' mechanism, IL-6 is able to stimulate cells that lack an endogenous mIL-6R. High levels of IL-6 and sIL-6R have been reported in several chronic inflammatory and autoimmune diseases as well as in cancer. Preclinical animal disease models have provided strong evidence that specific blockade of IL-6-regulated signalling pathways represents a promising approach for the therapy of these diseases. An optimised variant of the recently described fusion protein sgp30Fc is now heading towards its clinical evaluation.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Inflamação/tratamento farmacológico , Interleucina-6/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Receptores de Interleucina-6/antagonistas & inibidores , Processamento Alternativo , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Artrite/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Asma/tratamento farmacológico , Doenças Autoimunes/metabolismo , Linhagem Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Colite/tratamento farmacológico , Colite/imunologia , Neoplasias do Colo/tratamento farmacológico , Receptor gp130 de Citocina/fisiologia , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Inflamação/metabolismo , Interleucina-6/fisiologia , Leucócitos/metabolismo , Masculino , Camundongos , Neoplasias/metabolismo , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/fisiologia , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Fator de Crescimento Transformador beta/fisiologiaRESUMO
NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(WT) that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2(WT) and disease-associated NOD2(SNP13) variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteômica/métodos , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Reparo do DNA , Regulação para Baixo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Inativação Gênica , Variação Genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Immunoblotting , Inflamação , Interleucina-8/metabolismo , Luciferases/metabolismo , Modelos Biológicos , Proteína Adaptadora de Sinalização NOD2 , Oxirredução , Peptídeos/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/química , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Transfecção , Tripsina/farmacologia , Regulação para CimaRESUMO
Alterations in splicing patterns of genes contribute to the regulation of gene function by generating endogenous inhibitor or activator molecules. Nucleotide-binding and oligomerization domain (NOD) 2 is an intracellular receptor for bacterial cell wall components and plays an important role in initiating immune responses against cytoinvasive pathogens. NOD2 overexpression sensitizes intestinal epithelial cells toward bacterial cell wall components, activates the proinflammatory transcription factor NF-kappaB, and induces the subsequent release of the chemotactic cytokine IL-8. Here, we have assessed the regulation and function of a transcript isoform of NOD2, NOD2-S, generated by the skipping of the third exon, which encodes for a protein that is truncated within the second caspase recruitment (CARD) domain. NOD2-S is preferentially expressed in the human colon and is up-regulated by the antiinflammatory cytokine IL-10. Overexpression of NOD2-S down-regulates NOD2-induced NF-kappaB activation and IL-8 release. Moreover, NOD2-S also interferes with the maturation and secretion of pro-IL-1beta downstream of NOD2 and its adaptor molecule receptor-interacting protein kinase 2. We provide a molecular basis for these effects, as we show that NOD2-S interacts with both, NOD2 and receptor-interacting protein kinase 2 and inhibits the "nodosome" assembly by interfering with the oligomerization of NOD2. These data unveil another level of complexicity in the regulation of intracellular innate immunity and may have important implications for the molecular understanding of NOD/NALP protein-driven disease pathophysiology.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Linhagem Celular , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2 , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Regulação para CimaRESUMO
Tumor necrosis factor-alpha (TNF-alpha)-induced signaling is pivotally involved in the pathogenesis of chronic inflammatory diseases. A polymorphism in the TNF receptor 2 (TNFR2) gene resulting in a juxtamembrane inversion from methionine (TNFR2(196MET)) to arginine (TNFR2(196ARG)) has been genetically associated with an increased risk for systemic lupus erythematosus and familial rheumatoid arthritis. Albeit the mutation does not affect the TNF binding kinetics of TNFR2, the present study provides evidence that the mutation results in a significantly lower capability to induce TNFR2-mediated NF-kappaB activation. Pretriggering of TNFR2 with a receptor-specific mutein leads to an enhancement of TNFR1-induced apoptosis, which is further increased in cells carrying the TNFR2(196ARG) variant. A diminished induction of NF-kappaB-dependent target genes conveying either anti-apoptotic or pro-inflammatory functions, such as cIAP1, TRAF1, IL-6, or IL-8 is observed. The mutated form TNFR2(196ARG) shows a reduction of inducible TRAF2 recruitment upon TNF-alpha stimulation. The findings suggest a common molecular mechanism for the involvement of the TNFR2(196ARG) variant in the etiopathogenesis of different chronic inflammatory disorders.