RESUMO
Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here, we use RNA-seq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than wild-type and Sp+/- littermates, died soon after birth and had abnormal lung morphogenesis. RNA-seq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development.
Assuntos
Desenvolvimento Embrionário , Fatores de Transcrição , Animais , Camundongos , Divisão Celular , Pulmão , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences.
Assuntos
Macrófagos Alveolares , NF-kappa B , Animais , Cromatina/genética , Cromatina/metabolismo , Pulmão/metabolismo , Camundongos , NF-kappa B/metabolismo , Transcrição GênicaRESUMO
Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common neonatal pathogen. However, the cellular and molecular mechanisms for neonatal susceptibility to GBS pneumonia and sepsis are incompletely understood. Here we optimized a mouse model of GBS pneumonia to test the role of alveolar macrophage (ΑΜΦ) maturation in host vulnerability to disease. Compared with juvenile and adult mice, neonatal mice infected with GBS had increased mortality and persistence of lung injury. In addition, neonatal mice were defective in GBS phagocytosis and killing. ΑΜΦ depletion and disruption of ΑΜΦ differentiation in Csf2-/- mice both impaired GBS clearance. AMΦ engage the heavily sialylated GBS capsule via the cell surface Siglec receptors Sn and Siglec-E. Although both newborn and adult ΑΜΦ expressed Siglec-E, newborn ΑΜΦ expressed significantly lower levels of Sn. We propose that a developmental delay in Sn expression on ΑΜΦ may prevent effective killing and clearing of GBS from the newborn lung.
RESUMO
Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.
Assuntos
Feto/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Citocinas/metabolismo , Feto/citologia , Perfilação da Expressão Gênica , Imunidade Inata , Inflamassomos/metabolismo , Inflamação , Lipopolissacarídeos/farmacologia , Fígado/citologia , Fígado/embriologia , Fígado/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Especificidade de Órgãos , Receptor 4 Toll-Like/metabolismo , Saco Vitelino/citologia , Saco Vitelino/imunologiaRESUMO
Early exposure to inflammatory signals may have a lasting impact on immune function. Present throughout embryogenesis, macrophages are key cells providing innate immune protection to the developing fetus and newborn. Here, we have used an established model of macrophage development to test how early inflammatory signals can impact cellular differentiation and function. Bone marrow-derived macrophages were treated with Escherichia coli lipopolysaccharide (LPS) 2 days after initial isolation and culture. LPS treatment during this early stage of differentiation decreased the expression of CSF1R and increased that of the mature macrophage marker F4/80. These early changes in macrophage differentiation were also measured in cells from mice lacking IKKß, but the change in CSF1R expression after LPS treatment was blocked with MAPK inhibition. LPS-induced changes in macrophage marker expression persisted following LPS removal, suggesting that early inflammatory activation could induce a lasting developmental impact. Early LPS exposure inhibited macrophage phagocytosis of labeled E. coli while LPS had no effect on fully differentiated macrophages. Our data demonstrate that early inflammatory exposure to a microbial stimulus induce lasting phenotypic changes in macrophages.
Assuntos
Diferenciação Celular , Ativação de Macrófagos , Macrófagos , Receptores Toll-Like/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Escherichia coli , Quinase I-kappa B/metabolismo , Imunidade Inata , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagocitose/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de SinaisRESUMO
In the immature lung, inflammation and injury disrupt the epithelial-mesenchymal interactions required for normal development. Innate immune signaling and NF-κB activation disrupt the normal expression of multiple mesenchymal genes that play a key role in airway branching and alveolar formation. To test the role of the NF-κB pathway specifically in lung mesenchyme, we utilized the mesenchymal Twist2-Cre to drive expression of a constitutively active inhibitor of NF-κB kinase subunit ß (IKKßca) mutant in developing mice. Embryonic Twist2-IKKßca mice were generated in expected numbers and appeared grossly normal. Airway branching also appeared normal in Twist2-IKKßca embryos, with airway morphometry, elastin staining, and saccular branching similar to those in control littermates. While Twist2-IKKßca lungs did not contain increased levels of Il1b, we did measure an increased expression of the chemokine-encoding gene Ccl2. Twist2-IKKßca lungs had increased staining for the vascular marker platelet endothelial cell adhesion molecule 1. In addition, type I alveolar epithelial differentiation appeared to be diminished in Twist2-IKKßca lungs. The normal airway branching and lack of Il1b expression may have been due to the inability of the Twist2-IKKßca transgene to induce inflammasome activity. While Twist2-IKKßca lungs had an increased number of macrophages, inflammasome expression remained restricted to macrophages without evidence of spontaneous inflammasome activity. These results emphasize the importance of cellular niche in considering how inflammatory signaling influences fetal lung development.
Assuntos
Quinase I-kappa B/metabolismo , Pulmão/embriologia , Pulmão/enzimologia , Mesoderma/embriologia , Animais , Ativação Enzimática/fisiologia , Pulmão/irrigação sanguínea , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese , NF-kappa B/metabolismoRESUMO
In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2 Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.
Assuntos
Feto/citologia , Imunidade Inata , Pulmão/embriologia , Mesoderma/citologia , Mesoderma/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Lipopolissacarídeos/farmacologia , Mesoderma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Innate immune responses to dsRNA result in signaling through the TLR3 pathway and/or the RIG-I/MDA-5/MAVS pathway which can activate type I IFN, proinflammatory cytokines and apoptosis. It is not clear whether MAVS could play a role in TLR3-dependent responses to extracellular dsRNA. Using a model of epithelial cells that express a functional TLR3 signaling pathway, we found that TLR3-dependent responses to extracellular dsRNA are negatively regulated by MAVS, precisely "miniMAVS", a recently described 50kDa isoform of MAVS. This regulation of TLR3 by a MAVS isoform constitutes an endogenous regulatory mechanism in epithelial cells that could help prevent a potentially damaging excessive inflammatory response.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/fisiologia , Isoformas de Proteínas/metabolismo , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Células HCT116 , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , NF-kappa B/metabolismo , Poli I-C/imunologia , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Receptor 3 Toll-Like/genéticaRESUMO
Inflammation in the developing preterm lung leads to disrupted airway morphogenesis and chronic lung disease in human neonates. However, the molecular mechanisms linking inflammation and the pathways controlling airway morphogenesis remain unclear. In this article, we show that IL-1ß released by activated fetal lung macrophages is the key inflammatory mediator that disrupts airway morphogenesis. In mouse lung explants, blocking IL-1ß expression, posttranslational processing, and signaling protected the formation of new airways from the inhibitory effects ofEscherichia coliLPS. Consistent with a critical role for IL-1ß, mice expressing a gain-of-functionNlrp3allele and subsequent overactive inflammasome activity displayed abnormal saccular-stage lung morphogenesis and died soon after birth. Although the early-stage fetal lung appeared capable of mounting an NF-κB-mediated immune response, airway formation became more sensitive to inflammation later in development. This period of susceptibility coincided with higher expression of multiple inflammasome components that could increase the ability to release bioactive IL-1ß. Macrophages fromNlrp3gain-of-function mice also expressed higher levels of more mature cell surface markers, additionally linking inflammasome activation with macrophage maturation. These data identify developmental expression of the inflammasome and IL-1ß release by fetal lung macrophages as key mechanisms and potential therapeutic targets for neonatal lung disease.
Assuntos
Inflamassomos/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Pulmão/embriologia , Macrófagos/imunologia , Animais , Displasia Broncopulmonar/embriologia , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Interleucina-1beta/biossíntese , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Transdução de Sinais/imunologiaRESUMO
The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4(+), CD8(+) or natural killer T cells, or CD11b(+) or CD11c(+) macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti-programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1-deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1-deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.
Assuntos
Citrobacter rodentium , Infecções por Enterobacteriaceae/imunologia , Mucosa Intestinal/imunologia , Células Matadoras Naturais/imunologia , Receptor de Morte Celular Programada 1/fisiologia , Animais , Colo/imunologia , Feminino , Granzimas/biossíntese , Interferon gama/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perforina/biossíntese , Transdução de SinaisRESUMO
Gut microbiota richness and stability are important parameters in host-microbe symbiosis. Diet modification, notably using dietary fibres, might be a way to restore a high richness and stability in the gut microbiota. In this work, during a 6-week nutritional trial, 19 healthy adults consumed a basal diet supplemented with 10 or 40 g dietary fibre per day for 5 days, followed by 15-day washout periods. Fecal samples were analysed by a combination of 16S rRNA gene pyrosequencing, intestinal cell genotoxicity assay, metatranscriptomics sequencing approach and short-chain fatty analysis. This short-term change in the dietary fibre level did not have the same impact for all individuals but remained significant within each individual gut microbiota at genus level. Higher microbiota richness was associated with higher microbiota stability upon increased dietary fibre intake. Increasing fibre modulated the expression of numerous microbiota metabolic pathways such as glycan metabolism, with genes encoding carbohydrate-active enzymes active on fibre or host glycans. High microbial richness was also associated with high proportions of Prevotella and Coprococcus species and high levels of caproate and valerate. This study provides new insights on the role of gut microbial richness in healthy adults upon dietary changes and host microbes' interaction.
Assuntos
Dieta/métodos , Fibras na Dieta/administração & dosagem , Ácidos Graxos/análise , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Adulto , Clostridiales/genética , Clostridiales/isolamento & purificação , Suplementos Nutricionais , Feminino , Humanos , Masculino , Prevotella/genética , Prevotella/isolamento & purificação , RNA Ribossômico 16S/genética , Simbiose , Adulto JovemRESUMO
GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.
Assuntos
Células da Medula Óssea/imunologia , Proliferação de Células , Colite Ulcerativa/imunologia , Colite Ulcerativa/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos da radiação , Colite Ulcerativa/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Hematopoese/genética , Hematopoese/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Quimera por Radiação , Fatores de Tempo , Cicatrização/genética , Cicatrização/imunologiaRESUMO
Thymic stromal lymphopoietin (TSLP) is constitutively secreted by intestinal epithelial cells. It regulates gut DCs, therefore, contributing to the maintenance of immune tolerance. In the present report, we describe the regulation of TSLP expression in intestinal epithelial cells and characterize the role of several NF-κB binding sites present on the TSLP promoter. TSLP expression can be stimulated by different compounds through activation of p38, protein kinase A, and finally the NF-κB pathway. We describe a new NF-κB binding element located at position -0.37 kb of the promoter that is crucial for the NF-κB-dependent regulation of TSLP. We showed that mutation of this proximal NF-κB site abrogates the IL-1ß-mediated transcriptional activation of human TSLP in several epithelial cell lines. We also demonstrated that both p65 and p50 subunits are able to bind this new NF-κB binding site. The present work provides new insight into epithelial cell-specific TSLP regulation.
Assuntos
Citocinas/genética , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ordem dos Genes , Humanos , Interleucina-1/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurofibromina 1/metabolismo , Neurofibromina 2/metabolismo , Ligação Proteica , Fator de Transcrição AP-1/metabolismo , Linfopoietina do Estroma do TimoRESUMO
TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was independent of TNF signaling. Notably, NF-κB signaling through IκB kinase ß protected crypt IECs but did not protect villus IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium and death. Consistent with direct activation of the TLR3-TRIF-caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3, type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that dsRNA activation of the TLR3-TRIF-caspase 8 signaling pathway in IECs has a significant impact on the structure and function of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral pathogens.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Caspase 8/fisiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Poli I-C/toxicidade , Receptor 3 Toll-Like/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Mucosa Intestinal/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Viral/toxicidade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/deficiênciaRESUMO
BACKGROUND: The intestinal microbiota plays an important role in human health through the modulation of innate immune responses. While selected commensal bacteria are marketed in specific probiotic products to control these responses, relatively little is known about the immune modulation potential of dairy bacteria that have principally been selected for their fermentation properties. The modulation of innate immune responses may reduce chronic inflammation in inflammatory bowel diseases like ulcerative colitis. METHODS: A collection of dairy Lactobacillus delbrueckii strains was screened for immune modulation effects in vitro through the quantification of nuclear factor kappa B (NF-κB) activation in a human intestinal epithelial cell line. Selected bacterial strains were then tested in vivo in a mouse dextran sodium sulfate (DSS) colitis model. RESULTS: All L. delbrueckii strains tested showed anti-inflammatory effects in vitro, to an extent that varied between strains. These effects rely on bacterial surface exposed proteins and affect the central part of the NF-κB activation pathway. One of the selected strains significantly reduced the macroscopic and microscopic symptoms of DSS-induced colitis in the mouse intestinal tract, diminished body weight loss, and improved survival. CONCLUSIONS: The results of this study show that dairy lactobacilli that often are part of a regular diet can modulate innate immune responses, and may thus affect health more than generally thought. One of the strains tested alleviated the symptoms of DSS-induced colitis in mice, a model of human ulcerative colitis.
Assuntos
Anti-Inflamatórios/farmacologia , Colite/microbiologia , Intestinos/microbiologia , Lactobacillus delbrueckii , Animais , Anti-Inflamatórios/imunologia , Colite/induzido quimicamente , Colite/imunologia , Colite/terapia , Sulfato de Dextrana/toxicidade , Células HT29 , Humanos , Intestinos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologiaRESUMO
The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1ß, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations.
Assuntos
Bactérias/química , Bactérias/metabolismo , Intestinos/imunologia , Intestinos/microbiologia , NF-kappa B/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Meios de Cultivo Condicionados , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Expressão Gênica/genética , Células HT29 , Humanos , Interleucina-1beta/química , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Monócitos , NF-kappa B/análise , NF-kappa B/genética , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Streptococcus salivarius exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8).
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , NF-kappa B/antagonistas & inibidores , Streptococcus/imunologia , Linhagem Celular , Citocinas/antagonistas & inibidores , Genes Reporter , Humanos , Fatores Imunológicos/análise , Fatores Imunológicos/isolamento & purificação , Luciferases/metabolismo , Peso Molecular , Streptococcus/químicaRESUMO
BACKGROUND/AIM: The human intestinal microbiota plays an important role in modulation of mucosal immune responses. To study interactions between intestinal epithelial cells (IECs) and commensal bacteria, a functional metagenomic approach was developed. One interest of metagenomics is to provide access to genomes of uncultured microbes. We aimed at identifying bacterial genes involved in regulation of NF-κB signaling in IECs. A high throughput cell-based screening assay allowing rapid detection of NF-κB modulation in IECs was established using the reporter-gene strategy to screen metagenomic libraries issued from the human intestinal microbiota. METHODS: A plasmid containing the secreted alkaline phosphatase (SEAP) gene under the control of NF-κB binding elements was stably transfected in HT-29 cells. The reporter clone HT-29/kb-seap-25 was selected and characterized. Then, a first screening of a metagenomic library from Crohn's disease patients was performed to identify NF-κB modulating clones. Furthermore, genes potentially involved in the effect of one stimulatory metagenomic clone were determined by sequence analysis associated to mutagenesis by transposition. RESULTS: The two proinflammatory cytokines, TNF-α and IL-1ß, were able to activate the reporter system, translating the activation of the NF-κB signaling pathway and NF-κB inhibitors, BAY 11-7082, caffeic acid phenethyl ester and MG132 were efficient. A screening of 2640 metagenomic clones led to the identification of 171 modulating clones. Among them, one stimulatory metagenomic clone, 52B7, was further characterized. Sequence analysis revealed that its metagenomic DNA insert might belong to a new Bacteroides strain and we identified 2 loci encoding an ABC transport system and a putative lipoprotein potentially involved in 52B7 effect on NF-κB. CONCLUSIONS: We have established a robust high throughput screening assay for metagenomic libraries derived from the human intestinal microbiota to study bacteria-driven NF-κB regulation. This opens a strategic path toward the identification of bacterial strains and molecular patterns presenting a potential therapeutic interest.
Assuntos
Infecções Bacterianas/genética , Trato Gastrointestinal/microbiologia , Ensaios de Triagem em Larga Escala/métodos , Metagenômica/métodos , NF-kappa B/genética , Bactérias , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Trato Gastrointestinal/metabolismo , Regulação Bacteriana da Expressão Gênica , Células HT29 , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response. RESULTS: Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-kappaB pathway. CONCLUSIONS: The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients.
Assuntos
Células Epiteliais/imunologia , Interleucina-8/imunologia , Intestinos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas fluorescens/imunologia , Transdução de Sinais , Fator de Transcrição AP-1/imunologia , Aderência Bacteriana , Células CACO-2 , Citotoxicidade Imunológica , Células Epiteliais/microbiologia , Células HT29 , Humanos , Intestinos/microbiologia , NF-kappa B/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas fluorescens/fisiologiaRESUMO
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.