RESUMO
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Proteoma/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Cromatografia Líquida , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Humanos , Intestinos/citologia , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica , Anotação de Sequência Molecular , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/análise , Plasmídeos , Probióticos/química , Proteólise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
OBJECTIVE: Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. DESIGN: Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1ß. RESULTS: IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. CONCLUSIONS: Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.
Assuntos
Ácidos e Sais Biliares/metabolismo , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/microbiologia , Animais , Área Sob a Curva , Linhagem Celular Tumoral , Distribuição de Qui-Quadrado , Cromatografia Líquida de Alta Pressão , Neoplasias do Colo/patologia , Ensaio de Imunoadsorção Enzimática , Fezes/química , Fezes/microbiologia , Humanos , Metagenoma , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas , Espectrometria de Massas em TandemRESUMO
RATIONALE: Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis. METHODS: Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified. RESULTS: A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA). CONCLUSIONS: Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification.
Assuntos
Quimiocinas/análise , Cromatografia Líquida/métodos , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Espectrometria de Massas em Tandem/métodos , Quimiocina CXCL1/análise , Quimiocina CXCL1/química , Quimiocina CXCL1/metabolismo , Quimiocinas/química , Quimiocinas/metabolismo , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células HT29 , Humanos , Interleucina-8/análise , Interleucina-8/química , Interleucina-8/metabolismo , Mucosa Intestinal/citologia , Lipopolissacarídeos/farmacologia , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Proteoma/efeitos dos fármacosRESUMO
A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-kappaB activity, F. prausnitzii had no effect on IL-1beta-induced NF-kappaB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-gamma production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-kappaB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.
Assuntos
Anti-Inflamatórios/administração & dosagem , Doença de Crohn/terapia , Mucosa Intestinal/microbiologia , Probióticos/uso terapêutico , Ruminococcus/isolamento & purificação , Animais , Células Cultivadas , Colite , Doença de Crohn/microbiologia , Citocinas/biossíntese , Modelos Animais de Doenças , Humanos , Leucócitos/imunologia , Leucócitos/microbiologia , Camundongos , NF-kappa B/metabolismo , Probióticos/administração & dosagem , Probióticos/farmacologia , Resultado do TratamentoRESUMO
Intestinal epithelial cells (IEC) have adapted to the presence of commensal bacteria through a state of tolerance that involves a limited response to lipopolysaccharide (LPS). Low or absent expression of two LPS receptor molecules, the myeloid differentiation (MD)-2 receptor, and toll-like receptor (TLR)4 was suggested to underlie LPS tolerance in IEC. In the present study we performed transfections of TLR4 and MD-2 alone or combined in different IEC lines derived from intestinal cancer (Caco-2, HT-29, and SW837). We found that LPS responsiveness increased more than 100-fold when IEC were transfected with MD-2 alone, but not TLR4. The release of interleukin (IL)-8, but also the expression of cyclooxygenase (Cox-)2 and the related secretion of prostaglandin (PG)E2 were coordinately stimulated by LPS in IEC transfected with MD-2 alone. Supernatants collected from MD-2-transfected IEC supported LPS activation of naïve HT-29, providing additional support to the concept that MD-2 alone endows IEC with LPS responsiveness. LPS responsiveness detected at concentrations as low as 110 pg/ml, and maximal values obtained by 10 ng/ml were clearly beyond those evoked by classical stimuli as IL-1beta. In polarized cells, apical LPS stimulation was markedly more efficient than basolateral. Our data contradict previous opinion that both TLR4 and MD-2 limit IEC response to LPS, and emphasize the prominent role of MD-2 in intestinal immune responses to Gram-negative bacteria.
Assuntos
Células Epiteliais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/fisiologia , Antracenos/farmacologia , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Ciclo-Oxigenase 2/biossíntese , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Citometria de Fluxo , Expressão Gênica , Células HT29 , Humanos , Imidazóis/farmacologia , Interleucina-8/metabolismo , Intestinos/patologia , Antígeno 96 de Linfócito/genética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/fisiologia , TransfecçãoRESUMO
BACKGROUND: Epidemiologic data suggest that smoking increases the risk and the severity of Crohn's disease (CD), although it may protect patients with ulcerative colitis (UC). To investigate this paradox, we evaluated the effect of cigarette smoke in the function of blood mononuclear cells from healthy subjects and patients with CD or UC in flare up. METHODS: The production of mediators associated with inflammation but also with protective functions was evaluated by enzyme-linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), following either in vivo or in vitro exposure to cigarette smoke. RESULTS: We found that mononuclear cells from smokers with CD were functionally impaired. These cells secreted lower levels of chemokines and cytokines as compared with nonsmoker counterparts, whereas healthy smokers or smokers with UC were not affected. Similar findings were noted after in vitro exposure to cigarette smoke extract. In addition, cells from patients with CD who smoke presented a defective sensitivity to antiinflammatory or antioxidant protection, and particularly synthesized lower levels of cytoprotective Hsp70. The effects observed were not due to diminished cell viability. Our experiments suggest that cigarette smoke-related responses were largely dependent on oxidative stress generated, and not on the nicotine component. CONCLUSIONS: Overall, our data point out the presence of biological differences between blood mononuclear cells from patients with CD and UC toward cigarette smoke that might support its opposite role in both diseases.
Assuntos
Células Sanguíneas/efeitos dos fármacos , Colite Ulcerativa/fisiopatologia , Doença de Crohn/fisiopatologia , Leucócitos Mononucleares/efeitos dos fármacos , Fumar/efeitos adversos , Adulto , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , MasculinoRESUMO
Evidence suggests that signalling through lipopolysaccharide (LPS) has a significant role in the development of gastrointestinal malignancies. We previously demonstrated the critical role of myeloid differentiation (MD)-2, the essential co-receptor of LPS, for induction of cyclooxygenase (Cox)-2 in intestinal epithelial cells. Cyclooxigenase-2 was suggested to play a key role in colorectal cancer through the effects of prostaglandin (PG) E(2) generated. We, therefore, addressed the role of MD-2 in several parameters related to malignancy, namely cell proliferation and migration, using colon cancer cells (HT-29). We found that overexpression of MD-2 confers a significantly greater proliferation and migration capacity to these cells. MD-2-dependent proliferation and migration appeared independent of Cox-2 activity but was reduced by endothelial growth factor receptor (EGFR) neutralizing antibodies as well as by pharmacological inhibition of EGFR tyrosine phosphorylation. We propose that MD-2 overexpression contributes to tumour aggressiveness via a Cox-2-independent excessive EGFR signalling. Moreover, MD-2 expression levels were higher in tissue from patients with colorectal cancer as compared with paired control colorectal mucosa. Our data attest to a role of MD-2 activity in colon cancer epithelial cell proliferation and migration, which may be important in the general correlation between innate immune response, chronic inflammation, and cancer.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Antígeno 96 de Linfócito/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Anticorpos Bloqueadores/farmacologia , Carcinoma/imunologia , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Ciclo-Oxigenase 2/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/imunologia , Transgenes/genéticaRESUMO
Myeloid differentiation (MD)-2 is linked to the cell surface as a Toll-like receptor (TLR) 4-bound protein though may also function as a soluble receptor to enable the lipopolysaccharide (LPS)-driven response. We recently demonstrated the importance of MD-2 either as a cell-associated or as a soluble receptor in the control of intestinal epithelial cell response toward LPS. High levels of circulating MD-2 were recently proposed as a risk factor for infectious/ inflammatory diseases as septic shock. We hypothesized that MD-2 might be present in sera from patients with inflammatory bowel disease and have pathogenic consequences. We analysed MD-2 activity in sera from patients with inflammatory bowel disease or from healthy subjects. We measured MD-2 activity as the capacity to mediate LPS-driven stimulation of intestinal epithelial cells (HT29). We found that sera from patients with inflammatory bowel disease, particularly Crohn's disease, endowed HT29 cells with a markedly higher LPS-dependent stimulating capacity as compared to sera from healthy subjects. The effect of sera was specific for LPS activation and was reduced in the presence of anti-MD-2, and anti-TLR4 antibodies. We conclude that sera from patients with inflammatory bowel disease might contain increased MD-2. This might result in higher local availability of the protein leading to a loss of tolerance toward gut microbiota.
Assuntos
Colite Ulcerativa/sangue , Doença de Crohn/sangue , Mucosa Intestinal/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/sangue , Adulto , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Feminino , Células HT29 , Humanos , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Antígeno 96 de Linfócito/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Previous studies demonstrated that the induction of the heat shock protein Hsp70 in response to viral infection is highly specific and differs from one cell to another and for a given virus type. However, no clear consensus exists so far to explain the likely reasons for Hsp70 induction within host cells during viral infection. We show here that upon rotavirus infection of intestinal cells, Hsp70 is indeed rapidly, specifically, and transiently induced. Using small interfering RNA-Hsp70-transfected Caco-2 cells, we observed that Hsp70 silencing was associated with an increased virus protein level and enhanced progeny virus production. Upon Hsp70 silencing, we observed that the ubiquitination of the main rotavirus structural proteins was strongly reduced. In addition, the use of proteasome inhibitors in infected Caco-2 cells was shown to induce an accumulation of structural viral proteins. Together, these results are consistent with a role of Hsp70 in the control of the bioavailability of viral proteins within cells for virus morphogenesis.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/fisiologia , Rotavirus/fisiologia , Proteínas Virais/metabolismo , Células CACO-2/metabolismo , Células CACO-2/virologia , Proteínas de Choque Térmico HSP70/genética , Humanos , Rotavirus/crescimento & desenvolvimento , Proteínas Virais/biossínteseRESUMO
In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Morte Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Hipertermia Induzida , Estresse Oxidativo , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Glutationa/metabolismo , Temperatura Alta , Necrose , Ratos , FumaçaRESUMO
Accumulating evidence suggests that some heat shock proteins (Hsps), in particular the 72-kDa inducible Hsp70, associate to the cell membrane and might be secreted through an unknown mechanism to exert important functions in the immune response and signal transduction. We speculated that specialized structures named lipid rafts, known as important platforms for the delivery of proteins to the cell membrane, might be involved in the unknown mechanism ensuring membrane association and secretion of Hsp70. Lipid rafts are sphingolipid-cholesterol-rich structures that have been mainly characterized in polarized epithelial cells and can be isolated as detergent-resistant microdomains (DRMs). Analysis of soluble and DRM fractions prepared from unstressed Caco-2 epithelial cells revealed that Hsp70, and to a lesser extent calnexin, were present in DRM fractions. Increased expression of Hsps, through heat shock or by using drugs acting on protein trafficking or intracellular calcium level, induced an efficient translocation to DRM. We also found that Hsp70 was released by epithelial Caco-2 cells, and this release dramatically increased after heat shock. Drugs known to block the classical secretory pathway were unable to reduce Hsp70 release. By contrast, release of the protein was affected by the raft-disrupting drug methyl-beta-cyclodextrin. Our data suggest that lipid rafts are part of a mechanism ensuring the correct functions of Hsps and provide a rational explanation for the observed membrane association and release of Hsp70.