RESUMEN
BACKGROUND: The effects of Arula-7 powder (ASP) on diarrhea and intestinal barrier function associated with its regulation of intestinal microflora in calves infected with pathogenic Escherichia coli O1 (E. coli O1) were studied. METHOD: Twenty Holstein calves were randomly divided into four treatment groups: normal control (NC), model control (MC), 0.5 mg/kg ciprofloxacin (CIP) and 2.50 g/kg ASP groups. RESULTS: ASP inhibited the relative abundance of Proteobacteria, Selenomonadales, and Enterobacteriales, and increased the relative abundance of Lactobacillus, Faecalibacterium, and Alloprevotella. Moreover, we demonstrated for the first time that the ASP and CIP promoted weight gain, reduced the diarrhea rate (P < 0.05), and enhanced antioxidant capacity (P < 0.05) due to the increase in average daily gain (ADG), total protein (TP), and albumin (ALB). In addition, ASP and CIP increased the expression of Zunola occludens-1 (ZO-1), Occludin, and Claudin-1 in the ileum (P < 0.05), and improved immunity due to increase levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), immunoglobulin A (IgA), and immunoglobulin G (IgG) in the serum, strengthened CD4+T levels in the ileal mucosa and reducing CD8+T and CD11c+T (P < 0.05). CONCLUSION: Hence, The intestinal microbiota environment formed by early intervention of ASP powder has a protective effect on the intestinal mucosal function of calves infected with pathogenic E. coli. Video Abstract.
Asunto(s)
Infecciones por Escherichia coli , Microbioma Gastrointestinal , Animales , Bovinos , Polvos/metabolismo , Polvos/farmacología , Escherichia coli/metabolismo , Uniones Estrechas/microbiología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/veterinaria , Diarrea/tratamiento farmacológico , Diarrea/microbiología , Mucosa Intestinal/microbiologíaRESUMEN
Iron supplementation is necessary for the treatment of anemia, one of the most frequent complications in inflammatory bowel disease (IBD). However, oral iron supplementation leads to an exacerbation of intestinal inflammation. Gut barrier plays a key role in the pathogenesis of IBD. The aim of this study was to characterize the interrelationship between systemic iron, intestinal barrier and the development of intestinal inflammation in a dextran sulfate sodium (DSS) induced experimental colitis mice model. We found that DSS-treated mice developed severe inflammation of colon, but became much healthy when intraperitoneal injection with iron. Iron supplementation alleviated colonic and systemic inflammation by lower histological scores, restorative morphology of colonic villi, and reduced expression of pro-inflammatory cytokines. Moreover, intraperitoneal supplementation of iron enhanced intestinal barrier function by upregulating the colonic expressions of tight junction proteins, restoring intestinal immune homeostasis by regulating immune cell infiltration and T lymphocyte subsets, and increasing mucous secretion of goblet cells in the colon. High-throughput sequencing of fecal 16 S rRNA showed that iron injection significantly increased the relative abundance of Bacteroidetes, which was suppressed in the gut microbiota of DSS-induced colitis mice. These results provided evidences supporting the protective effects of systemic iron repletion by intraperitoneal injection of iron on intestinal barrier functions. The finding highlights a novel approach for the treatment of IBD with iron injection therapy.
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Colitis/tratamiento farmacológico , Colon/efectos de los fármacos , Suplementos Dietéticos , Células Caliciformes/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Complejo Hierro-Dextran/administración & dosificación , Proteínas de Uniones Estrechas/metabolismo , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Sulfato de Dextran , Modelos Animales de Enfermedad , Disbiosis , Microbioma Gastrointestinal/efectos de los fármacos , Células Caliciformes/metabolismo , Células Caliciformes/microbiología , Inyecciones Intraperitoneales , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Ratones Endogámicos C57BL , Permeabilidad , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Regulación hacia ArribaRESUMEN
As the first line of defense against intestinal bacteria and toxins, intestinal epithelial cells are always exposed to bacteria or lipopolysaccharide (LPS), whereas pathogenic bacteria or LPS can cause intestinal epithelial cell damage. Previous studies have shown that konjac mannan oligosaccharides (KMOS) have a positive effect on maintaining intestinal integrity, and Bacillus subtilis (BS) can promote the barrier effect of the intestine. However, it is still unknown whether KMOS and BS have a synergistic protective effect on the intestines. In this study, we used the LPS-induced Caco-2 cell injury model and mouse intestinal injury model to study the synergistic effects of KMOS and BS. Compared with KMOS or BS alone, co-treatment with KMOS and BS significantly enhanced the activity and antioxidant capacity of Caco-2 cell, protected mouse liver and ileum from LPS-induced oxidative damage, and repaired tight junction and mucus barrier damage by up-regulating the expression of Claudin-1, ZO-1 and MUC-2. Our results demonstrate that the combination of KMOS and BS has a synergistic repair effect on inflammatory and oxidative damage of Caco-2 cells and aIIeviates LPS-induced acute intestinal injury in mice.
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Bacillus subtilis/fisiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Enfermedades del Íleon/prevención & control , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mananos/farmacología , Probióticos , Animales , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/microbiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Enfermedades del Íleon/metabolismo , Enfermedades del Íleon/microbiología , Enfermedades del Íleon/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Lipopolisacáridos , Ratones , Estrés Oxidativo/efectos de los fármacos , Permeabilidad , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
Salmonella pathogenicity island (SPI) 2 type three secretion system (T3SS)-mediated effector molecules facilitate bacterial survival in phagocytes but their role in the intestinal epithelium in vivo remains ill-defined. Using our neonatal murine infection model in combination with SPI2 reporter technology and RNA-Seq of sorted primary enterocytes, we demonstrate expression of SPI2 effector molecules by intraepithelial Salmonella Typhimurium (S. Typhimurium). Contrary to expectation, immunostaining revealed that infection with SPI2 T3SS-mutants resulted in significantly enlarged intraepithelial Salmonella-containing vacuoles (SCV) with altered cellular positioning, suggesting impaired apical to basolateral transmigration. Also, infection with isogenic tagged S. Typhimurium strains revealed a reduced spread of intraepithelial SPI2 T3SS mutant S. Typhimurium to systemic body sites. These results suggest that SPI2 T3SS effector molecules contribute to enterocyte apical to basolateral transmigration of the SCV during the early stage of the infection.
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Proteínas Bacterianas/metabolismo , Mucosa Intestinal/microbiología , Proteínas de la Membrana/metabolismo , Salmonella typhimurium/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Vacuolas/microbiología , Animales , Proteínas Bacterianas/genética , Células Cultivadas , Enterocitos/metabolismo , Enterocitos/microbiología , Mucosa Intestinal/citología , Macrófagos/inmunología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , RNA-Seq , Infecciones por Salmonella/patología , Uniones Estrechas/microbiología , Sistemas de Secreción Tipo III/genética , Vacuolas/metabolismoRESUMEN
The present study evaluated the protection of Lactiplantibacillus plantarum CCFM8661, a candidate probiotic with excellent benzopyrene (B[a]P)-binding capacity in vitro, against B[a]P-induced toxicity in the colon and brain of mice. Mice that received B[a]P alone served as the model group. Each mouse in the L. plantarum treatment groups were administered 2×109 colony forming unit (CFU) of L. plantarum strains once daily, followed by an oral dose of B[a]P at 50 mg/kg body weight. Behavior, biochemical indicators in the colon and brain tissue, and the gut microbiota composition and short-chain fatty acid (SCFA) levels in the gut were investigated. Compared to the treatment in the model group, CCFM8661 treatment effectively reduced oxidative stress in the brain, improved behavioral performance, increased intestinal barrier integrity, and alleviated histopathological changes in mice. Moreover, CCFM8661 increased the gut microbiota diversity and abundance of Ruminococcus and Lachnospiraceae and reduced the abundance of pro-inflammatory Turicibacter spp. Additionally, the production of SCFAs was significantly increased by L. plantarum CCFM8661. Our results suggest that CCFM8661 is effective against acute B[a]P-induced toxicity in mice and that it can be considered as an effective and easy dietary intervention against B[a]P toxicity.
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Benzo(a)pireno/toxicidad , Encéfalo/efectos de los fármacos , Colon/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Lactobacillus plantarum/crecimiento & desarrollo , Probióticos , Animales , Conducta Animal/efectos de los fármacos , Benzo(a)pireno/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiología , Encéfalo/patología , Colon/metabolismo , Colon/microbiología , Colon/patología , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Disbiosis , Ácidos Grasos Volátiles/metabolismo , Lactobacillus plantarum/metabolismo , Masculino , Ratones Endogámicos BALB C , Prueba de Campo Abierto , Estrés Oxidativo/efectos de los fármacos , Permeabilidad , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
Glaesserella parasuis (G. parasuis) causes inflammation and damage to piglets. Whether polyserositis caused by G. parasuis is due to tight junctions damage and the protective effect of baicalin on it have not been examined. Therefore, this study aims to investigate the effects of baicalin on peritoneal tight junctions of piglets challenged with G. parasuis and its underlying molecular mechanisms. Piglets were challenged with G. parasuis and treated with or without baicalin. RT-PCR was performed to examine the expression of peritoneal tight junctions genes. Immunofluorescence was carried out to detect the distribution patterns of tight junctions proteins. Western blot assays were carried out to determine the involved signaling pathways. Our data showed that G. parasuis infection can down-regulate the tight junctions expression and disrupt the distribution of tight junctions proteins. Baicalin can alleviate the down-regulation of tight junctions mRNA in peritoneum, prevent the abnormalities and maintain the continuous organization of tight junctions. Our results provide novel evidence to support that baicalin has the capacity to protect peritoneal tight junctions from G. parasuis-induced inflammation. The protective mechanisms of baicalin could be associated with inhibition of the activation of PKC and MLCK/MLC signaling pathway. Taken together, these data demonstrated that baicalin is a promising natural agent for the prevention and treatment of G. parasuis infection.
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Flavonoides/farmacología , Infecciones por Pasteurellaceae/tratamiento farmacológico , Pasteurellaceae/efectos de los fármacos , Enfermedades de los Porcinos/tratamiento farmacológico , Animales , Pasteurellaceae/genética , Pasteurellaceae/patogenicidad , Infecciones por Pasteurellaceae/genética , Infecciones por Pasteurellaceae/microbiología , Infecciones por Pasteurellaceae/veterinaria , Peritoneo/efectos de los fármacos , Peritoneo/microbiología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Porcinos , Enfermedades de los Porcinos/microbiología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genética , Uniones Estrechas/microbiologíaRESUMEN
The role of dietary components in immune function has acquired considerable attention in recent years. An important focus area is to unravel the role of bioactive dietary compounds in relation to enteric disease and their impact on gut mucosal immunity. Proanthocyanidins (PAC) are among the most common and most consumed dietary polyphenols, and are characterised by their variable molecular structures and diverse bioactivities. In particular, their anti-oxidative effects and ability to modulate gut microbiota have been widely described. However, there is limited evidence on the mechanism of action of PAC on the immune system, nor is it clearly established how PAC may influence susceptibility to enteric infections. Establishing the sites of action of PAC and their metabolites within the gut environment is fundamental to determine the applicability of PAC against enteric pathogens. Some mechanistic studies have shown that PAC have direct modulatory effects on immune cell signalling, isolated pathogens, and gut mucosal barrier integrity. Boosting the recruitment of immune cells and suppressing the amount of pro-inflammatory cytokines are modulating factors regulated by PAC, and can either be beneficial or detrimental in the course of re-establishing gut homeostasis. Herein, we review how PAC may alter distinct immune responses towards enteric bacterial, viral and parasitic infections, and how the modulation of gut microbiota may act as a mediating factor. Furthermore, we discuss how future studies could help unravel the role of PAC in preventing and/or alleviating intestinal inflammation and dysbiosis caused by enteric disease.
Asunto(s)
Antioxidantes/farmacología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Proantocianidinas/farmacología , Uniones Estrechas/fisiología , Antioxidantes/administración & dosificación , Dieta , Disbiosis/inmunología , Microbioma Gastrointestinal/inmunología , Humanos , Inmunidad Mucosa/inmunología , Proantocianidinas/administración & dosificación , Uniones Estrechas/inmunología , Uniones Estrechas/microbiologíaRESUMEN
MicroRNA-155 (miR-155) is implicated in the pathological processes of sepsis. However, the function and regulatory mechanism of miR-155 in sepsis-induced inflammation and intestinal barrier dysfunction remain unknown. In this study, mouse models of sepsis were established by caecal ligation and puncture (CLP). To reduce miR-155 expression, the mice were injected for three consecutive days with an miR-155 inhibitor (80 mg/kg) before CLP. The serum DAO concentration was measured by ELISA, and histological changes in the intestine were identified by H&E staining 24 h after CLP. FITC-dextran assays were used to evaluate intestinal permeability. MiR-155 gene expression was evaluated with RT-PCR, and relative protein expression was assessed by Western blotting. NCM460 cells were transfected with an miR-155 mimic/miR-155 inhibitor or pretreated with an NF-κB inhibitor before LPS treatment, and the cytokines levels, miR-155 gene expression and relative protein expression were measured. Sepsis increased miR-155, DAO and FITC-dextran levels and reduced Occludin and ZO-1 expression. Mice injected with the miR-155 inhibitor recovered from the damages. Transfection of NCM460 cells with the miR-155 mimic elevated the NF-κB (P65) and p-NF-κB (p-P65) localization and expression in the nucleus, which was reversed by the miR-155 inhibitor. Pretreatment with an NF-κB inhibitor suppressed inflammation, improved cell permeability to FITC-dextran and increased Occludin and ZO-1 levels. Transfection with the miR-155 inhibitor decreased TNF-α and IL-6 levels, reduced cell permeability to FITC-dextran and increased ZO-1 and Occludin expression. The effects induced by transfection with the miR-155 mimic, including elevated TNF-α and IL-6 levels, hyperpermeability to FITC-dextran and reduced ZO-1 and Occludin expression, were partly rescued by pretreatment with the NF-κB inhibitor. These findings reveal that the miR-155 inhibitor alleviates inflammation and intestinal barrier dysfunction by inactivating NF-κB signaling during sepsis.
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Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , MicroARNs/antagonistas & inhibidores , FN-kappa B/metabolismo , Sepsis/tratamiento farmacológico , Animales , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/microbiología , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Permeabilidad , Sepsis/genética , Sepsis/metabolismo , Sepsis/microbiología , Transducción de Señal , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiologíaRESUMEN
The beneficial effects of prebiotic, such as fructo-oligosaccharides (FOS), in intestinal inflammation have been demonstrated in several studies. Herein, we evaluate whether joint treatment with FOS, both before and during mucositis, had additional beneficial effects and investigated the mechanisms underlying in the action of FOS on the intestinal barrier. BALB/c mice were randomly divided into five groups: CTR (without mucositisâ¯+â¯saline solution), FOS (without mucositisâ¯+â¯6 % FOS), MUC (mucositisâ¯+â¯saline solution), PT (mucositisâ¯+â¯6 % FOS supplementation before disease induction), and TT (mucositisâ¯+â¯6 % FOS supplementation before and during disease induction). Mucositis was induced by intraperitoneal injection (300â¯mg/kg) of 5-fluorouracil (5-FU). After 72â¯h, the animals were euthanized and intestinal permeability (IP), tight junction, bacterial translocation (BT), histology and morphometry, and immunoglobulin A secretory (sIgA), inflammatory infiltrate, and production of short-chain fatty acids (acetate, butyrate and propionate) were evaluated. The MUC group showed an increase in the IP, BT, and inflammatory infiltrate but a decrease in the tight junction expression and butyrate and propionate levels (Pâ¯<â¯0.05). In the PT and TT groups, FOS supplementation maintained the IP, tight junction expression, and propionate concentration within physiologic levels, increased butyrate levels, and reduced BT and inflammatory infiltrate (Pâ¯<â¯0.05). Total treatment with FOS (TT group) was more effective in maintaining histological score, morphometric parameters, and sIgA production. Thus, total treatment (prophylactic and therapeutic supplementation) with FOS was more effective than pretreatment alone, in reducing 5-FU-induced damage to the intestinal barrier.
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Bacterias/efectos de los fármacos , Ácidos Grasos Volátiles/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Íleon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucositis/inducido químicamente , Oligosacáridos/farmacología , Prebióticos , Uniones Estrechas/efectos de los fármacos , Acetatos/metabolismo , Animales , Bacterias/metabolismo , Traslocación Bacteriana/efectos de los fármacos , Butiratos/metabolismo , Modelos Animales de Enfermedad , Fluorouracilo , Íleon/metabolismo , Íleon/microbiología , Íleon/patología , Inmunoglobulina A Secretora/metabolismo , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos BALB C , Mucositis/metabolismo , Mucositis/microbiología , Mucositis/patología , Permeabilidad , Propionatos/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
Obesity is associated with impaired intestinal barrier function and dysbiosis of the gut microbiota. Spermidine, a polyamine that acts as an autophagy inducer, has important benefits in patients with aging-associated diseases and metabolic dysfunction. However, the mechanism of spermidine on obesity remains unclear. Here, we show that spermidine intake is negatively correlated with obesity in both humans and mice. Spermidine supplementation causes a significant loss of weight and improves insulin resistance in diet-induced obese (DIO) mice. These effects are associated with the alleviation of metabolic endotoxemia and enhancement of intestinal barrier function, which might be mediated through autophagy pathway and TLR4-mediated microbial signaling transduction. Moreover, spermidine causes the significant alteration of microbiota composition and function. Microbiota depletion compromises function, while transplantation of spermidine-altered microbiota confers protection against obesity. These changes might partly be driven by an SCFA-producing bacterium, Lachnospiraceae NK4A136 group, which was decreased in obese subjects and subsequently increased by spermidine. Notably, the change of Lachnospiraceae NK4A136 group is significantly correlated with enhanced gut barrier function induced by spermidine. Our results indicate that spermidine supplementation may serve as a viable therapy for obesity.
Asunto(s)
Disbiosis/tratamiento farmacológico , Microbioma Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Obesidad/tratamiento farmacológico , Espermidina/farmacología , Uniones Estrechas/efectos de los fármacos , Animales , Autofagia/fisiología , Peso Corporal , Células CACO-2 , Línea Celular Tumoral , Clostridiales/metabolismo , Disbiosis/microbiología , Endotoxemia/tratamiento farmacológico , Humanos , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/microbiología , Transducción de Señal , Uniones Estrechas/microbiología , Receptor Toll-Like 4/metabolismoRESUMEN
Our skin provides a physical barrier to separate the internal part of our body from the environment. Maintenance of complex barrier functions is achieved through anatomical structures in the skin, the stratified squamous epithelium specialized junctional organelles, called tight junctions (TJs). Several members of our microbial communities are known to affect the differentiation state and function of the colonized organ. Whether and how interactions between skin cells and cutaneous microbes, including Cutibacterium acnes (C. acnes), modify the structure and/or function of our skin is currently only partly understood. Thus, in our studies, we investigated whether C. acnes may affect the epidermal barrier using in vitro model systems. Real-time cellular analysis showed that depending on the keratinocyte differentiation state, the applied C. acnes strains and their dose, the measured impedance values change, together with the expression of selected TJ proteins. These may reflect barrier alterations, which can be partially restored upon antibiotic-antimycotic treatment. Our findings suggest that C. acnes can actively modify the barrier properties of cultured keratinocytes, possibly through alteration of tight cell-to-cell contacts. Similar events may play important roles in our skin, in the maintenance of cutaneous homeostasis.
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Acné Vulgar/microbiología , Acné Vulgar/patología , Epidermis/metabolismo , Queratinocitos/metabolismo , Queratinocitos/microbiología , Propionibacteriaceae/patogenicidad , Acné Vulgar/metabolismo , Diferenciación Celular , Células Cultivadas , Humanos , Queratinocitos/patología , Fenómenos Fisiológicos de la Piel , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.
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Colitis/tratamiento farmacológico , Colon/efectos de los fármacos , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Células Epiteliales/efectos de los fármacos , Fármacos Gastrointestinales/farmacología , Ensayos Analíticos de Alto Rendimiento , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Animales , Células CACO-2 , Citrobacter rodentium/patogenicidad , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/microbiología , Colon/metabolismo , Colon/microbiología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/metabolismo , Infecciones por Enterobacteriaceae/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/patología , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Permeabilidad , Putrescina/farmacología , Taurina/farmacología , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.
Asunto(s)
Bronquios/metabolismo , Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Infecciones por Bacterias Gramnegativas/metabolismo , Proteoma , Vías Secretoras , Stenotrophomonas maltophilia/patogenicidad , Uniones Estrechas/microbiología , Bronquios/patología , Línea Celular , Cromatografía Liquida , Fibrosis Quística/patología , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Mapas de Interacción de Proteínas , Proteómica/métodos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Serina Proteasas/metabolismo , Stenotrophomonas maltophilia/enzimología , Espectrometría de Masas en Tándem , Uniones Estrechas/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (µFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa, ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli, MRSA, and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1ß production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence, which may have broader implications governing the course of treatment compared to traditional methods alone.
Asunto(s)
Bacterias/metabolismo , Fenómenos Fisiológicos Celulares/fisiología , Impedancia Eléctrica , Células Epiteliales/microbiología , Células A549 , Técnicas Biosensibles/métodos , Línea Celular , Citocinas/metabolismo , Enterococcus/aislamiento & purificación , Enterococcus/metabolismo , Enterococcus/patogenicidad , Escherichia coli/aislamiento & purificación , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Humanos , Microelectrodos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidad , Uniones Estrechas/microbiologíaRESUMEN
A clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Although broad-spectrum antibiotics have been shown to disrupt the structure of the gut microbiota, some antibiotics appear to increase CDAD risk without being highly active against intestinal anaerobes, suggesting direct nonantimicrobial effects. We examined cell biological effects of antibiotic exposure that may be involved in bacterial pathogenesis using an in vitro germfree human colon epithelial culture model. We found a marked loss of mucosal barrier and immune function with exposure to the CDAD-associated antibiotics clindamycin and ciprofloxacin, distinct from the results of pretreatment with an antibiotic unassociated with CDAD, tigecycline, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent gut mucosal barrier changes in the increased risk of CDAD with specific antibiotics and warrant further studies in in vivo systems. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD.
Asunto(s)
Antibacterianos/efectos adversos , Clostridioides difficile/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Membrana Mucosa/fisiología , Uniones Estrechas/efectos de los fármacos , Antibacterianos/farmacología , Células CACO-2 , Línea Celular Tumoral , Ciprofloxacina/efectos adversos , Ciprofloxacina/farmacología , Clindamicina/efectos adversos , Clindamicina/farmacología , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterocolitis Seudomembranosa/microbiología , Células HT29 , Humanos , Membrana Mucosa/microbiología , Factores de Riesgo , Tigeciclina/efectos adversos , Tigeciclina/farmacología , Uniones Estrechas/microbiologíaRESUMEN
Enhanced gut permeability due to dysregulated epithelial tight junction is often associated with inflammatory bowel diseases (IBD), which have a greater risk for developing colorectal cancer. STAT6 activation was detected in inflamed colonic epithelium of active IBD patients, suggesting a role of epithelial STAT6 in colitis development. Here, we demonstrated that non-hematopoietic STAT6, but not hematopoietic STAT6, triggered DSS-induced colitis and subsequent tumorigenesis. This could be due to the enhancing-effect of STAT6 on gut permeability and microbiota translocation via interruption of epithelial tight junction integrity. Mechanistically, long-myosin light-chain kinase (MLCK1) was identified as a target of STAT6, leading to epithelial tight junction dysfunction and microbiota-driven colitis. Furthermore, neutralization of IL-13, which was primarily derived from type 2 innate lymphoid cells (ILC2) in a microbiota-dependent way, inhibited epithelial STAT6 activation and improved gut permeability and DSS-induced colitis. Importantly, pharmacological inhibition of STAT6 reduces murine intestinal tumor formation, and tumoral p-STAT6 levels positively correlated to the clinical stage and poor prognosis of human colorectal cancer. Thus, our study reveals a direct role of STAT6 in the disruption of epithelial tight junction integrity and colitis development, and suggests STAT6 as a potential therapeutic and prophylactic target for IBD and colitis-associated cancer.
Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Mucosa Intestinal/metabolismo , Factor de Transcripción STAT6/metabolismo , Uniones Estrechas/metabolismo , Animales , Traslocación Bacteriana , Células CACO-2 , Colitis/genética , Colitis/microbiología , Colitis/patología , Colon/efectos de los fármacos , Colon/microbiología , Colon/patología , Neoplasias del Colon/microbiología , Neoplasias del Colon/patología , Neoplasias del Colon/prevención & control , Modelos Animales de Enfermedad , Impedancia Eléctrica , Microbioma Gastrointestinal , Genes APC , Humanos , Interleucina-13/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Linfocitos/metabolismo , Linfocitos/patología , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Permeabilidad , Fosforilación , Pirimidinas/farmacología , Factor de Transcripción STAT6/antagonistas & inhibidores , Factor de Transcripción STAT6/deficiencia , Factor de Transcripción STAT6/genética , Transducción de Señal , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/microbiología , Uniones Estrechas/patología , Técnicas de Cultivo de TejidosRESUMEN
Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.
Asunto(s)
Inflamación/genética , Ocludina/genética , Nacimiento Prematuro/genética , Progesterona/genética , Adulto , Células CACO-2 , Femenino , Microbioma Gastrointestinal , Regulación de la Expresión Génica/genética , Humanos , Inflamación/sangre , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/microbiología , Lipopolisacáridos/sangre , Permeabilidad , Embarazo , Nacimiento Prematuro/sangre , Nacimiento Prematuro/microbiología , Nacimiento Prematuro/patología , Progesterona/metabolismo , Uniones Estrechas/genética , Uniones Estrechas/microbiología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Tight junction barrier is critical to intestinal homeostasis. Applying antibiotics to treat infections is common in clinical practice, which may affect intestinal microbiota. Intestinal microbiota dysbiosis is involved in the occurrence of some gastrointestinal diseases. Therefore, this study was aimed to investigate the influence of antibiotics on intestinal tight junction barrier and the possible underlying mechanisms. Healthy adult female C57BL/6 mice were treated with a broad-spectrum antibiotic cocktail for 14 days. 16S rDNA Illumina sequencing and headspace gas chromatography-mass spectrometry (HS-GC/MS) were respectively used to analyze microbial community and to detect short-chain fatty acids (SCFAs) contents. In vivo intestinal paracellular permeability to fluorescein isothiocyanate-dextran (FITC-dextran) was measured. Protein expression was determined by immunoblotting. Immunofluoresence was applied to observe the distributions of ZO-1, LC3B and ASC. Antibiotics remarkably altered intestinal microbiota composition in healthy mice, accompanying reduced SCFAs' concentrations. In addition, the intestinal tight junction barrier was disrupted by antibiotic treatment, as evidenced by increased intestinal paracellular permeability to FITC-dextran, decreased tight junction protein expressions, and disrupted ZO-1 morphology. Furthermore, NLRP3 inflammasome and autophagy were activated by antibiotic treatment. In conclusion, intestinal epithelial tight junction barrier dysfunction induced by antibiotics is associated with intestinal microbiota dysbiosis, activated NLRP3 inflammasome and autophagy in mice.
Asunto(s)
Antibacterianos/efectos adversos , Intestinos/efectos de los fármacos , Microbiota/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Uniones Estrechas/efectos de los fármacos , Animales , Antibacterianos/farmacología , Autofagia/efectos de los fármacos , ADN Ribosómico/genética , Dextranos/química , Modelos Animales de Enfermedad , Disbiosis/inducido químicamente , Disbiosis/microbiología , Disbiosis/patología , Ácidos Grasos Volátiles/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Intestinos/microbiología , Intestinos/patología , Ratones , Uniones Estrechas/microbiología , Uniones Estrechas/patologíaRESUMEN
BACKGROUND: Blood-brain barrier (BBB) disruption and neuroinflammation are considered key mechanisms of pathogenic Escherichia coli invasion of the brain. However, the specific molecules involved in meningitic E. coli-induced BBB breakdown and neuroinflammatory response remain unclear. Our previous RNA-sequencing data from human brain microvascular endothelial cells (hBMECs) revealed two important host factors: platelet-derived growth factor-B (PDGF-B) and intercellular adhesion molecule-1 (ICAM-1), which were significantly upregulated in hBMECs after meningitic E. coli infection. Whether and how PDGF-B and ICAM-1 contribute to the development of E. coli meningitis are still unclear. METHODS: The western blot, real-time PCR, enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence were applied to verify the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in vivo and in vitro. Evan's blue assay and electric cell-substrate impedance sensing assay were combined to identify the effects of PDGF-B on BBB permeability. The CRISPR/Cas9 technology, cell-cell adhesion assay, and electrochemiluminescence assay were used to investigate the role of ICAM-1 in neuroinflammation subversion. RESULTS: We verified the significant induction of PDGF-B and ICAM-1 by meningitic E. coli in mouse as well as monolayer hBMECs models. Functionally, we showed that the increase of PDGF-B may directly enhance the BBB permeability by decreasing the expression of tight junction proteins, and the upregulation of ICAM-1 contributed to neutrophils or monocytes recruitment as well as neuroinflammation subversion in response to meningitic E. coli infection. CONCLUSIONS: Our findings demonstrated the roles of PDGF-B and ICAM-1 in mediating bacterial-induced BBB damage as well as neuroinflammation, providing new concepts and potential targets for future prevention and treatment of bacterial meningitis.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Infecciones por Escherichia coli/metabolismo , Mediadores de Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Linfocinas/biosíntesis , Meningitis Bacterianas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Animales , Barrera Hematoencefálica/microbiología , Barrera Hematoencefálica/patología , Células Cultivadas , Escherichia coli , Infecciones por Escherichia coli/patología , Femenino , Meningitis Bacterianas/patología , Ratones , Uniones Estrechas/metabolismo , Uniones Estrechas/microbiología , Regulación hacia Arriba/fisiologíaRESUMEN
Escherichia coli (E. coli) is one of the common pathogenic bacteria in veterinary clinical infection. As an opportunistic microorganism, E. coli normally does not cause diseases. However, it causes infections under certain circumstance to domesticated animal and poultry, resulting in severe diarrhea, septicemia, and respiratory infections. Although there are increasing reports regarding the infections of E. coli to domestic animals and poultry, the infection of E. coli in dogs is relatively less reported, especially on septicemia and meningoencephalitis. Here, we reported the isolation and identification of an E. coli isolate named CEC-GZL17 from dogs characterized by septicemia and sudden death, and found that CEC-GZL17 is able to cause meningoencephalitis. Exploration on the potential mechanism underlying meningoencephalitis demonstrated that CEC-GZL17 infection significantly increases TNF-α expression and inhibits ZO-1 and occludin expressions in brain tissue, indicating that the E coli likely use the mechanism to penetrate the blood-brain barrier via disrupting tight junction architecture, thus leading to the invasion to brain tissue.