RESUMEN
BACKGROUND AND AIMS: Pathogenic bacteria and host cells counteract or neutralize each other's effect in two fundamental ways: Direct invasion and secretion of various substances. Among these, lipases secreted by pathogenic bacteria and host cell lysozyme are key actors. Secreted lipases from pathogenic bacterial are suggested as a key player in the pathogen-host interaction. Among the gut microbial energy sources, glucose and fats have been referred to as one of the best inducers and substrates for bacterial lipases. Enrichment of bacterial growth medium with extra glucose or oil has been shown to induce lipase production in pathogenic bacteria. More recently, research has focused on the role of human gut phage alterations in the onset of dysbiosis because the bacteria-phage interactions can be dramatically affected by the nutrient milieu of the gut. However, the reciprocal role of bacterial lipases and phages in this context has not been well studied and there is no data available about how high glucose or fat availability might modulate the cellular milieu of the pathogenic bacteria-phageeukaryotic host cell interface. The purpose of this study was to evaluate the immunologic outcome of pathogenic bacteria-phage interaction under normal, high glucose, and high butter oil conditions to understand how nutrient availability affects lipase activity in pathogenic bacteria and, ultimately, the eukaryotic host cell responses to pathogenic bacteria-phage interaction. MATERIALS AND METHODS: 10 groups of co-cultured T84 and HepG2 cells were treated with Pseudomonas aeruginosa strain PAO1 (P.a PAO1) in the presence and absence of its KPP22 phage and incubated in three different growth media (DMEM, DMEM + glucose and DMEM + butter oil). Structural and physiological (barrier function and cell viability), inflammatory (IL-6 and IL-8), metabolic (glucose and triglycerides), and enzymatic (lipases and lysozyme) parameters were determined. RESULTS: Excess glucose or butter oil enhanced additively extracellular lipase activity of P.a PAO1. Excess glucose or butter oil treatments also magnified P. a PAO1- induced secretion of inflammatory signal molecules (IL-1ß, IL-6) from co-cultured cells, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells, these effects being abolished by phage KPP22. CONCLUSION: The results of the present study imply that KPP22 phage influences the interplay between food substances, gut bacterial lipases, and the gut cellular milieu. This can be applied in two-way interaction: by affecting the microbial uptake of excess free simple sugars and fats from the gut milieu leading to decreased bacterial lipases and by modulating the immune system of the intestinal -liver axis cells. Further studies are needed to see if the biological consequences of these effects also occur in vivo.
RESUMEN
BACKGROUND AND AIMS: The host micronutrient milieu is a compilation of factors of both endogenous and exogenous origin. This milieu shapes the host's immune responses and can control the inflammatory response of the host when infected. Among vitamins, B12 plays a key role in the defense process because there is intense competition for it between pathogenic invaders and infected host cells. Alcoholic beverages and antibiotics can cause biological (in vivo) interferences that affect pathogenhost crosstalk. Ethanol is known to interfere with the absorption, distribution, and excretion of vitamin B12 in men and animals. However, the molecular mechanisms underlying this backdrop are not fully understood. Here, we explored how Gram-positive ethanol-producing and Gram-negative vitamin B12- producing microbes of the infected milieu interact to influence biomarkers of host cell defense responses in absorbing, digesting, and defensive cells. MATERIAL AND METHODS: We investigated two different cell types of colon and liver origin, hepatic-like Huh7 cells and HT- 29/B6 colon cells. To assess the ability of secreted factors from bacteria to exert influence on co-cultured cell's secretion of host-defense markers in response to invading pathogens, cocultured human colonic HT-29/B6 and human hepatic Huh-7 (hereafter Huh7) cells were stimulated or not with Klebsiella pneumoniae 52145 for 24 h in the presence or absence of either Weissella confusa strain NRRL-B-14171 (as a Gram-positive producer of ethanol), Limosilactobacillus reuteri 20016 (as a Gram-positive producer of vitamin B12), or Pseudomonas nitroreducens 1650 (as a Gram-negative producer of vitamin B12). After stimulation, molecular functional biomarkers of host cell defense responses including total MMP-1, lysozyme activity, ALP, and IL-25 were measured. RESULTS: While simultaneously reducing IL-25 secretion, Kp52145 alone significantly elicited MMP-1, lysozyme, and ALP secretion from co-cultured cells, as compared to no treatment. When compared with Kp 52145 stimulation alone, Pn1650 significantly potentiated MMP-1 and lysozyme secretions from Kp 52145-stimulated co-cultured cells by 29.7% and 67.4%, respectively. Simultaneously, a potentiated suppression (an overall decrease of 77.3%) in IL-25 secretion occurred 24 hours after Kn52145 plus Pn1650 administration. Compared to Kp52145-stimulation alone, treatment with W. confusa NRRL-B-14171 and Kp52145-stimulated co-cultured cells was associated with significant additive induction of MMP-1 and lysozyme secretions. However, compared to Kp52145-stimulation alone, W. confusa NRRL-B-14171 treatment significantly potentiated Kp52145-induced suppression of IL-25. Using the same condition as mentioned above and compared to Kp52145-stimulation alone, L. reuteri 20016 treatment altered the secretion pattern in response to Kp52145: L. reuteri 20016-treated cells displayed less aversive responses towards Kp52145, suggesting that L. reuteri 20016 modulation may act differently on Kp52145 - induced signaling. CONCLUSION: Gram-negative and Gram-positive vitamin B12- producing bacteria differently affect the secretion of key immune biomarkers in co-cultured HT-29/B6 and Huh7 cells following exposure to Kp52145. Ethanol-producing bacteria additively potentiate pathogenicity and inflammatory responses upon infection. To confirm the biological consequences of these effects on human gut microbiota and health, further studies are warranted, incorporating ex vivo studies of human colon samples and host biomarkers such as cytohistological, molecular, or biochemical measurements.
Asunto(s)
Etanol , Metaloproteinasa 1 de la Matriz , Masculino , Animales , Humanos , Muramidasa , Colon , Vitamina B 12RESUMEN
BACKGROUND AND AIMS: Dietary habits, food, and nutrition-associated oral dysbiosis lead to the formation of microbial biofilm, which affects the overall health of an individual by promoting systemic diseases like cardiovascular disease, immunological disorders, and diabetes. Today's diets contain a variety of fermentable carbohydrates, including highly processed starch and novel synthetic carbohydrates such as oligofructose, sucralose, and glucose polymers. These constitute risk factors in the initiation and progression of oral dysbiosis. Oral, lung and gut microbiomes are interlinked with each other via direct and indirect ways. It is unknown whether or not lactobacilli and Lactobacillus phages are able to rescue dysbiotic effects by decreasing the uptake into the cells of excess simple sugars and their derivatives present within the digestive tract. MATERIALS AND METHODS: Using transwell cell culture plate inserts, six groups of in vitro co-cultured TR146 and HepG2 cells, grown in DMEM medium either with or without sucrose (10 % v/v), were treated with 1) PBS, 2) Fructilactobacillus sanfranciscensis (F.s) H2A, 3) F.s H2A and sucrose, 4) F.s H2A plus sucrose plus phage EV3 lysate, 5) F.s H2A plus sucrose plus phage EV3 supernatant, and 6) F.s H2A plus sucrose plus phage EV3 particles. The pH of the culture medium (indicating lactic acid production) and key oral biomarkers, including cytokines (IL-1ß and IL-6), inflammatory chemokines (e.g., CXCL8 and CCL2), and homeostatic chemokines (e.g., CXCL4 and CCL18) were measured. RESULTS: Excess sucrose significantly enhanced inflammatory signal molecules (e.g., IL-1ß, IL-6, and CCL2) secretion, concomitant with the enhancement of intracellular triglycerides in co-cultured HepG2 cells. Co-culture with F.s H2A decreased the sucrose-induced release of inflammatory signal molecules from co-cultured cells, these effects being abolished by F.s phage EV3. CONCLUSION: This study shows that Lactobacillus phages apparently influence the interplay between food components, oral microbiota, and the oral cellular milieu, at least in part by affecting the microbial uptake of excess free simple sugars from the oral milieu. To confirm the biological consequences of these effects on human oral microbiota and health, further studies are warranted, incorporating ex vivo studies of human dental plaque biofilms and host biomarkers, such as cytohistological, molecular, or biochemical measurements.
Asunto(s)
Bacteriófagos , Microbiota , Humanos , Biomarcadores , Técnicas de Cultivo de Célula , Quimiocinas , Disbiosis , Interleucina-6 , Monosacáridos/metabolismo , SacarosaRESUMEN
Lactobacilli are the most common probiotic bacteria found in the human gut microbiota, and the presence of acquired antibiotic resistance determinants carried on mobile genetic elements must be screened due to safety concerns. Unnecessary and inappropriate antibiotic therapy, as well as ingested antibiotic resistance bacteria (originating from food or food products), influence the abundance of antibiotic resistance genes in human guts, with serious clinical consequences. The current study looked into the antibiotic resistance of lactobacilli isolated from the guts of sepsis patients on long-term antibiotic therapy. The broth microdilution method was used to investigate the minimum inhibitory concentrations (MICs) of antibiotics such as imipenem, meropenem, erythromycin, tetracycline, cefepime, ciprofloxacin, and gentamycin, and the molecular genetic basis of resistance was studied based on the MIC values. The isolates were phenotypically resistant to tetracycline (20%), fluoroquinolone (20%), and macrolide (5%). Following that, resistance genes for tetracycline [tet(L), tet(O), tet(K), and tet(M)], macrolide [erm(B) and erm(C)], and beta-lactams [bla(CMY)] were investigated. Tetracycline or macrolide resistance genes were not found in the isolates, and only one isolate possessed the bla(CMY) resistance gene. The findings suggested that tetracycline and macrolide resistance may be linked to other resistance genes that were not investigated in this study. Because tetracyclines, fluoroquinolones, and macrolides are commonly used in clinics and animals, there has been concern about the spread of resistance in humans. If acquired antibiotic resistance is passed down through mobile genetic elements, it may serve as a reservoir of resistance for gut pathogens and other microbiome environments.
Asunto(s)
Antibacterianos , Sepsis , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias , Farmacorresistencia Bacteriana/genética , Humanos , Lactobacillus/genética , Macrólidos/farmacología , Prevalencia , Sepsis/tratamiento farmacológico , Tetraciclina/farmacologíaRESUMEN
BACKGROUND: Asparagus contains different bioactive and volatile components including pyrazines, sulphur-containing compounds, and polyphenols. Asparagus juice is a new low-calorie LAB-containing natural juice product, the usage of which is expanding. Pyrazines and sulphur-containing compounds are degraded by bacteria on one hand, but on the other hand, dietary polyphenols prevent human colorectal diseases as modulators of the composition and/or activity of gut microbiota. However, the utility of these asparagus compounds for reversal of age-associated microbial dysbiosis and the immunometabolic disorders that dysbiosis incites body inflammatory reactions was not much explored so far. Hence, using middle-aged mice, we conducted the current study to verify the effect of freshly squeezed domestic white asparagus juice on the biomarkers reflecting immuno-metabolic pathways linking age-related dysbiosis and metabolic events. MATERIALS AND METHODS: Thirty-two conventional Harlan Laboratories C57BL/6 mice aged between 11-12 months were randomly divided into two groups (n=16). Mice in control group 1 received sterile tap water. Animals in group 2 had 60 days ad libitum free-choice access to sterile tap water supplemented with 5% (v/v) freshly squeezed domestic white asparagus juice. Clinical signs of general health, hydration, and inflammation were monitored daily. Caecal content samples were analysed by qPCR for microbial composition. Histology of relevant organs was carried out on day 60 after sacrificing the mice. Universal markers of metabolic- and liver function were determined in serum samples. Caecal SCFAs contents were measured using HPLC. RESULTS: Overall, no significant differences in general health or clinical signs of inflammation between the two groups were observed. The liver to body weight ratio in asparagus juice-drank mice was lowered. The qPCR quantification showed that asparagus juice significantly decreased the caecal Clostridium coccoides group while causing an enhancement in Clostridium leptum, Firmicutes, and bifidobacterial groups as well as total caecal bacterial count. Asparagus juice significantly elevated the caecal contents of SCFAs. Enhanced SCFAs (acetate, butyrate, and propionate) in mice receiving asparagus juice, however, did coincide with altered lipid levels in plasma or changes in the abundance of relevant bacteria for acetate-, butyrate-, and propionate production. DISCUSSION: To the best of our knowledge, this is the first study aiming at evaluating the effect of freshly squeezed German domestic white asparagus juice on universal markers of metabolic- and liver function in middle- aged mice and the role of gut microbiota in this regard. The effectiveness of asparagus juice to improve metabolism in middle-aged mice was associated with alterations in intestinal microbiota but maybe also due to uptake of higher amounts of SCFAs. CONCLUSION: Hence, the key signal pathways corresponding to improved immune-metabolic homeostasis will be an important research scheme in the future.
Asunto(s)
Microbioma Gastrointestinal , Animales , Bacterias , Biomarcadores/metabolismo , Butiratos/metabolismo , Disbiosis , Ácidos Grasos Volátiles/metabolismo , Femenino , Homeostasis , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Polifenoles/metabolismo , Propionatos/metabolismo , Pirazinas/metabolismo , Azufre/metabolismo , AguaRESUMEN
BACKGROUND AND AIMS: Following a fat-rich diet, alterations in gut microbiota contribute to enhanced gut permeability, metabolic endotoxemia, and low grade inflammation-associated metabolic disorders. To better understand whether commensal bifidobacteria influence the expression of key metaflammation-related biomarkers (chemerin, MCP-1, PEDF) and modulate the pro-inflammatory bacteria- and lipid-coupled intracellular signaling pathways, we aimed at i) investigating the influence of the establishment of microbial signaling molecules-based cell-cell contacts on the involved intercellular communication between enterocytes, immune cells, and adipocytes, and ii) assessing their inflammatory mediators' expression profiles within an inflamed adipose tissue model. MATERIAL AND METHODS: Bifidobacterium animalis R101-8 and Escherichia coli TG1, respectively, were added to the apical side of a triple co-culture model consisting of intestinal epithelial HT-29/B6 cell line, human monocyte-derived macrophage cells, and adipose-derived stem cell line in the absence or presence of LPS or palmitic acid. mRNA expression levels of key lipid metabolism genes HILPDA, MCP-1/CCL2, RARRES2, SCD, SFRP2 and TLR4 were determined using TaqMan qRT-PCR. Protein expression levels of cytokines (IL-1ß, IL-6, and TNF-α), key metaflammation-related biomarkers including adipokines (chemerin and PEDF), chemokine (MCP- 1) as well as cellular triglycerides were assessed by cell-based ELISA, while those of p-ERK, p-JNK, p-p38, NF-κB, p-IκBα, pc-Fos, pc-Jun, and TLR4 were assessed by Western blotting. RESULTS: B. animalis R101-8 inhibited LPS- and palmitic acid-induced protein expression of inflammatory cytokines IL-1ß, IL-6, TNF-α concomitant with decreases in chemerin, MCP-1, PEDF, and cellular triglycerides, and blocked NF-kB and AP-1 activation pathway through inhibition of p- IκBα, pc-Jun, and pc-Fos phosphorylation. B. animalis R101-8 downregulated mRNA and protein levels of HILPDA, MCP-1/CCL2, RARRES2, SCD and SFRP2 and TLR4 following exposure to LPS and palmitic acid. CONCLUSION: B. animalis R101-8 improves biomarkers of metaflammation through at least two molecular/signaling mechanisms triggered by pro-inflammatory bacteria/lipids. First, B. animalis R101-8 modulates the coupled intracellular signaling pathways via metabolizing saturated fatty acids and reducing available bioactive palmitic acid. Second, it inhibits NF-kB's and AP-1's transcriptional activities, resulting in the reduction of pro-inflammatory markers. Thus, the molecular basis may be formed by which commensal bifidobacteria improve intrinsic cellular tolerance against excess pro-inflammatory lipids and participate in homeostatic regulation of metabolic processes in vivo.
Asunto(s)
Bifidobacterium animalis/metabolismo , Mediadores de Inflamación/metabolismo , Inflamación/fisiopatología , Metabolismo de los Lípidos/fisiología , Transducción de Señal/fisiología , Biomarcadores , Línea Celular , Técnicas de Cocultivo , Citocinas/biosíntesis , Humanos , Metabolismo de los Lípidos/genética , Lipopolisacáridos/farmacologíaRESUMEN
BACKGROUND AND AIMS: Pglyrp3 is a bactericidal innate immunity protein known to sustain the habitual gut microbiome and protect against experimental colitis. Intestinal inflammation and metaflammation are commonly associated with a marked reduction of commensal bifidobacteria. Whether Pglyrp3 and bifidobacteria interact synergistically or additively to alleviate metaflammation is unknown. We investigated the extent to which Pglyrp3 and bifidobacteria regulate metaflammation and gut bacterial dysbiosis in DSS-induced mouse models of intestinal inflammation. MATERIAL & METHODS: 8-10 weeks old male mice were used. In both WT and Pglyrp3 -/- experiments, the mice were randomly divided into three groups of 16 mice per group: (1) a control group receiving sterile tap water, (2) an experimental group receiving sterile tap water supplemented with only 5% DSS, and (3) an experimental group receiving sterile tap water supplemented with 5% DSS and 1 × 109 CFU/ml of Bifidobacterium adolescentis (B.a.) for 7 days. Wild-type (WT) littermates of the respective gene (i.e. Pglyrp3) were used as controls throughout the study. Clinical signs of general health and inflammation were monitored daily. Faecal pellet samples were analysed by qRT-PCR for microbial composition. Histology of relevant organs was carried out on day 8. Metabolic parameters and liver inflammation were determined in serum samples. RESULTS: Intestinal inflammation in mice of group 2 were significantly increased compared to those of control group 1. There was a significant difference in mean scores for inflammation severity between DSS-treated WT and DSS-treated Pglyrp3 -/- mice. Buildup of key serum metabolic markers (cholesterol, triglyceride and glucose) was set off by colonic inflammation. qRT-PCR quantification showed that DSS significantly decreased the Clostridium coccoides and Bifidobacterium cell counts while increasing those of Bacteroides group in both WT and Pglyrp3 -/- mice. These manifestations of DSS-induced dysbiosis were significantly attenuated by feeding B.a. Both the local and systemic ill-being of the mice alleviated when they received B.a. DISCUSSION: This study shows that Pglyrp3 facilitates recognition of bifidobacterial cell wall-derived peptidoglycan, thus leading additively to a reduction of metaflammation through an increase in the number of bifidobacteria, which were able to mitigate intestinal immunopathology in the context of Pglyrp3 blockade.
Asunto(s)
Bifidobacterium adolescentis/fisiología , Proteínas Portadoras/metabolismo , Colitis/metabolismo , Disbiosis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Terapia Biológica , Proteínas Portadoras/genética , Células Cultivadas , Colitis/terapia , Sulfato de Dextran , Modelos Animales de Enfermedad , Disbiosis/terapia , Microbioma Gastrointestinal , Humanos , Enfermedades Inflamatorias del Intestino/terapia , Ratones , Ratones Endogámicos BALB C , Ratones NoqueadosRESUMEN
BACKGROUND AND PURPOSE: In previous investigations, Weissella confusa was shown to lack the metabolic pathway from fructose to mannitol and to produce ethanol when cultivated in the presence of fructose. Hence, we assessed the effect of oral administration of W. confusa (strain NRRL-B-14171) on blood and fecal ethanol concentrations, glucose and lipid metabolism and traits of the metabolic syndrome in Wistar rats (n=27) fed diets with two different fat and fructose levels and with or without the addition of W. confusa during a total intervention time of 15 weeks (105 days). MATERIALS AND METHODS: From week 1 to 6, rats were given a medium fructose and fat (MFru-MF) diet containing 28% fructose and 10% fat without the addition of W. confusa (control group, n=13) or mixed with 30 g per kg diet of lyophilized W. confusa (10.56 ± 0.20 log CFU/g; W. confusa group, n=14). From week 7 to 15, the percentage of dietary fructose and fat in the control and W. confusa group was increased to 56% and 16%, respectively (high fructose-high fat (HFru-HF) diet). RESULTS: In HFru-HF-fed rats, W. confusa was detected in feces, regardless of whether W. confusa was added to the diet or not, but not in rats receiving the MFru-MF diet without added W. confusa or in an additional control group (n=10) fed standard rat food without fructose, increased fat content and W. confusa. This indicates that fecal W. confusa may be derived from orally administered W. confusa as well as - in the case of high fructose and fat intake and obesity of rats - from the intestinal microbiota. As shown by multifactorial ANOVA, blood ethanol, the relative liver weight, serum triglycerides, and serum cholesterol as well as fecal ethanol, ADH, acetate, propionate and butyrate, but not lactate, were significantly higher in the W. confusa - compared to the control group. DISCUSSION: This is the first in vivo trial demonstrating that heterofermentative lactic acid bacteria lacking the mannitol pathway (like W. confusa) can increase fecal and blood ethanol concentrations in mammals on a high fructose-high fat diet. This may explain why W. confusa resulted in hyperlipidemia and may promote development of NAFLD in the host.
RESUMEN
Increased concentration of ferrous iron in the gastrointestinal tract increases the number of various pathogens and induces inflammation. LPS and/or high-fat diet-associated metaflammation is mediated through a quaternary receptor signaling complex containing iron-regulated pathway, IL-6/STAT inflammatory signaling pathway, hepcidin regulatory pathway, and common TLR4/NF-κB signaling pathway. We, therefore, investigated whether bifidobacteria directly or indirectly ameliorate LPS- and/or high-fat diet-associated metaflammation by reduction of intestinal iron concentration and/or the above-mentioned pathways. MATERIAL & METHODS: We used a triple co-culture model of HT-29/B6, HMDM and HepG2 cells with apically added Bifidobacterium pseudolongum (DSMZ 20099), in the absence or presence of iron, LPS or oleate. Expressions of the biomarkers of interest were determined after 24 h incubation by TaqMan qRT-PCR, cell-based ELISA or Western blot. RESULTS: Bifidobacteria inhibited LPS- and oleate-induced protein expression of inflammatory cytokines (IL-6, TNF-α) concomitantly with decreases in cellular TG and iron concentration. Exposure of co-cultured cells to bifidobacteria blocked NF-kB activity through inhibition of IκBα, p38 MAPK, and phosphorylation of NF-kB 65 subunit. TaqMan qRT-PCR and Western blot analysis revealed that bifidobacteria downregulated mRNA and protein expression of BMP6, DMT1, hepcidin, l-ferritin, ferroportin, IL-6, TfR1, Stat3, and TLR4 following exposure to excessive extracellular LPS, oleate and iron. However, the patterns of TLR2 mRNA and protein expression were quite the opposite of those of TLR4. CONCLUSION: Commensal bifidobacteria ameliorate metaflammation/inflammatory responses to excessive extracellular LPS, oleate and iron through at least two molecular/signaling mechanisms: i. modulation of interactions of the hepcidin- and iron-signaling pathways via reduction of excess iron; ii. reduction of pro-inflammatory cytokines and hepcidin production through inhibition of the TLR4/NF-kB pathway. This may be a molecular basis by which commensal bifidobacteria enhance intrinsic cellular tolerance against excess consumption of energy-yielding substrates and/or free iron.
Asunto(s)
Bifidobacterium/fisiología , Hepcidinas/metabolismo , Inflamación/metabolismo , Mucosa Intestinal/inmunología , Hierro/metabolismo , Obesidad/inmunología , Biomarcadores/metabolismo , Técnicas de Cocultivo , Dieta Alta en Grasa , Células HT29 , Células Hep G2 , Humanos , Mucosa Intestinal/microbiología , Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Ácido Oléico/metabolismo , Transducción de Señal , Simbiosis , Receptor Toll-Like 4/metabolismoRESUMEN
We hypothesised that probiotic feeding would alter the fatty acid (FA) profile of sheep's milk. Sixteen lactating ewes, kept under the same feeding and management practices, were randomly allocated to receive either a control diet or the same diet supplemented with a commercial multi-strain bacterial probiotic. Milk fat FA contents were monitored fortnightly for eight consecutive weeks from 14 d after lambing. Probiotic supplementation increased the contents of butyric and caproic acids in milk fat and had no negative effects on other relevant FA from the human's health point of view (i.e., no differences in branched chain, vaccenic, rumenic and n-3 FA were observed). Under the conditions assayed in the present work, the contents of milk FA originated from rumen microbial metabolism were scantly altered, which suggests that the rumen conversion pathways of FA were not substantially modified by the probiotics.
Asunto(s)
Ácidos Grasos/análisis , Leche/química , Probióticos/administración & dosificación , Ovinos/metabolismo , Animales , Ácido Butírico/análisis , Caproatos/análisis , Dieta/veterinaria , Suplementos Dietéticos , Ácidos Grasos/metabolismo , Femenino , Lactancia , Rumen/metabolismo , Rumen/microbiologíaRESUMEN
BACKGROUND AND OBJECTIVES: We characterized the immunomodulating potential of a number of lactobacilli isolated from an African fermented food by co-incubation with peripheral blood mononuclear cells (PBMCs). Two strains with different immune modulating properties were genetically compared by suppression subtractive hybridization (SSH). METHODS: From 48 Lactobacillus strains isolated from Kimere, African fermented pearl millet dough, 10 were selected based on their bile salt tolerance. Their effects on secretion by PBMCs of the T-helper cells Th1- and Th2-cytokines IFN-γ and IL-4, respectively, in the presence or absence of staphylococcal enterotoxin A (SEA) were assessed. To study the genetic basis of different immune-modulating properties, a subtracted cDNA library for L. fermentum strains K1-Lb1 (Th1 inducer) and K8-Lb1 (Th1 and Th2 suppressor) was constructed using SSH. Finally, adhesion of these strains to hydrocarbons (relative hydrophobicity) and to human HT-29 colonic epithelial cell line was assessed. RESULTS: Two strains, K1-Lb1 and K4-Lb6, induced basal IFN-γ secretion. Four strains, K1-Lb6, K6-Lb2, K7-Lb1, and K8-Lb1 diminished INF-γ secretion by SEA-stimulated PBMCs. All strains, except K1-Lb1, K2-Lb4, and K9-Lb3, inhibited SEA-stimulated IL-4 secretion. Comparing the genomes of K1-Lb1 and K8-Lb1 by SSH indicated that K1-Lb1 is able to synthetize polysaccharides, for the synthesis of which K1-Lb8 appears to lack enzymes. A difference in the hydrophobicity properties of the surfaces of both strains indicated that this has impact on their surface. CONCLUSION: The K1-Lb1-specific sequences encoding putative glycosyltransferases and enzymes for polysaccharides synthesis may account for the observed differences in immunomodulation and surface properties between the two strains and for mediating potential probiotic effects.
Asunto(s)
Conductos Biliares/microbiología , Lactobacillaceae/inmunología , Leucocitos Mononucleares/inmunología , Células TH1/inmunología , Células Th2/inmunología , Células Cultivadas , Sondas de ADN , Enterotoxinas/inmunología , Biblioteca de Genes , Genes Bacterianos/genética , Glicosiltransferasas/genética , Humanos , Inmunomodulación , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Lactobacillaceae/genética , Lactobacillaceae/aislamiento & purificación , Leucocitos Mononucleares/microbiología , Polisacáridos Bacterianos/biosíntesis , Probióticos , Balance Th1 - Th2RESUMEN
The pathophysiology of IBD is characterized by a complex interaction between genes and the environment. Genetic and environmental differences are attributed to the heterogeneity of the disease pathway and to the epigenetic modifications that lead to altered gene expression in the diseased tissues. The epigenetic machinery consists of short interfering RNA, histone modifications, and DNA methylation. We evaluated the effects of Bifidobacterium breve (DSMZ 20213) and LGG (ATCC 53103), as representatives of commensal probiotics on the expression of IL-17 and IL-23, which play an important role in IBD, and on the epigenetic machinery in a 3D coculture model composed of human intestinal HT-29/B6 or T84 cells and PBMCs. The cells were treated with LPS in the presence or absence of bacteria for 48 h, and the expression of IL-17, IL-23, and CD40 at the mRNA and protein levels was assessed using TaqMan qRT-PCR and ELISA, respectively. Western blotting was used to assess the expression of the MyD88, the degradation of IRAK-1 and IκBα, the expression of the NF-κB p50/p65 subunits, the p-p38 MAPK and p-MEK1, as well as histone modifications. NF-κB activity was assessed by NF-κB-dependent luciferase reporter gene assays. The accumulation of Ac-H4 and DNA methylation was quantitatively assessed using colorimetric assays. B. breve and LGG diminished the LPS-induced expression of IL-17, IL-23, CD40, and histone acetylation, while slightly enhancing DNA methylation. These effects were paralleled by a decrease in the nuclear translocation of NF-κB, as demonstrated by a decrease in the expression of MyD88, degradation of IRAK-1 and IκBα expression of the nuclear NF-κB p50/p65 subunits, p-p38 MAPK and p-MEK1, and NF-κB-dependent luciferase reporter gene activity in LPS-stimulated cells. B. breve and LGG may exert their anti-inflammatory effects in the gut by down-regulating the expression of the IBD-causing factors (IL-23/IL-17/CD40) associated with epigenetic processes involving the inhibition of histone acetylation and the optimal enhancement of DNA methylation, reflected in the limited access of NF-κB to gene promoters and reduced NF-κB-mediated transcriptional activation. We describe a new regulatory mechanism in which commensal probiotics inhibit the NF-κB-mediated transcriptional activation of IBD-causing factors (IL-23/IL-17/CD40), thereby simultaneously reducing histone acetylation and enhancing DNA methylation.
Asunto(s)
Epigénesis Genética , Interleucina-17/antagonistas & inhibidores , Interleucina-23/antagonistas & inhibidores , Mucosa Intestinal/inmunología , Probióticos/farmacología , Bifidobacterium , Proliferación Celular , Técnicas de Cocultivo , Metilación de ADN , Células HT29 , Histonas/metabolismo , Humanos , Inmunidad Mucosa , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Mucosa Intestinal/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/fisiologíaRESUMEN
SCOPE: Human ß-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. It has been suggested that probiotic bacteria sustain gut barrier function via induction of defensins. The goals of this study were (i) to evaluate the potential immunomodulatory effects of 11 different Lactobacillus fermentum strains isolated from Kimere, an African fermented pearl millet (Pennisetum glaucum) dough, on the hBD-2 secretion by human intestinal CaCo-2 cell line and (ii) to examine genetic differences between two strains of L. fermentum (K2-Lb4 and K11-Lb3) which differed in their effect on the production of hBD-2 in this study. METHODS AND RESULTS: Totally, 46 strains of L. fermentum from Kimere were isolated and characterized using molecular biology methods including pulsed-field gel electrophoresis patterns. After performing time- and dose-experiments, CaCo-2 cells were incubated with or without bacteria for 12 h. L. fermentum PZ1162 was included as the positive control. Cell-free supernatants were analyzed for hBD-2 protein by enzyme-linked immunosorbent assay (ELISA). To identify potential bacterial genes associated with hBD-2 regulation, suppression subtractive hybridization (SSH) was used. Among the 11 strains tested, only two strains of bacteria, K11-Lb3 and K2-Lb6, significantly induced the production of hBD-2 by CaCo-2 cells. This effect was strain-specific, dose-dependent and particularly seems to be bacterial genomic-dependent as manifested by SSH. L. fermentum strains with and without hBD-2 inducing effect differed in genes encoding proteins involved in glycosylation of cell-wall proteins e.g. glycosyltransferase, UDP-N-acetylglucosamine 2-epimerase, rod shape-determining protein MreC, lipoprotein precursors, sugar ABC transporters, and glutamine ABC transporter ATP-binding protein. CONCLUSION: This study implies that certain strains of L. fermentum isolated from Kimere may stimulate the intestinal innate defense through the induction of hBD-2. The molecular basis of hBD-2 induction by L. fermentum strain K11-Lb3 may be based on glycosylated cell-surface structures synthesized with the aid of glycosyltransferase, UDP-N-acetylglucosamine 2-epimerase, and rod shape-determining protein MreC.
Asunto(s)
Enterocitos/metabolismo , Enterocitos/microbiología , Limosilactobacillus fermentum/genética , beta-Defensinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Células CACO-2 , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Genoma Bacteriano , Glicosilación , Humanos , Hibridación in Situ/métodos , Datos de Secuencia Molecular , Pennisetum/microbiología , Especificidad de la Especie , beta-Defensinas/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Control of the intracellular Mycobacterium tuberculosis (Mtb), mainly requires an appropriate ratio of Th1/Th2 cytokines to induce autophagy, a physiologically, and immunologically regulated process that has recently been highlighted as an innate defense mechanism against intracellular pathogens. Current vaccines/adjuvants induce both protective Th1 autophagy-promoting cytokines, such as IFN-gamma, and immunosuppressive Th2 autophagy-restraining cytokines, such as IL-4 and IL-13. TB infection itself is also characterized by relatively high levels of Th2 cytokines, which down-regulate Th1 responses and subsequently subvert adequate protective immunity, and a low ratio of IFN-gamma/IL-4. Therefore, there is a need for a safe and non-toxic vaccine/adjuvant that will induce Th1 autophagy-promoting cytokine (IFN-gamma) secretion and suppress the pre-existing subversive Th2 autophagy-restraining cytokines (IL-4 and IL-13). As lactic acid bacteria (LAB) belonging to the natural intestinal microflora and their components have been shown to shift immune responses against other antigens from Th2-type cytokines toward Th1-type cytokines like IFN-gamma, we investigated whether LAB can improve the polarization of Th1/Th2 cytokines and autophagic ability of mononuclear phagocytes in response to Mtb antigen. METHODS: Peripheral blood mononuclear cells (PBMCs), which are a part of the mononuclear phagocyte system and source of crucial macrophage activators in the in vivo situation, and human monocyte-derived macrophages (HMDMs) were treated with Mtb antigen in the presence or absence of two strains of LAB, L. rhammosus GG (LGG) and Bifidobacterium bifidum MF 20/5 (B.b). PBMCs cell culture supernatants were analyzed for the production of the autophagy-promoting factors IFN-gamma, and nitric oxide (NO) and the autophagy-restraining cytokines IL-4 and IL-13, using ELISA and Griess assays to detect the production of cytokines and NO, respectively. In HMDMs, expression of microtubule-associated protein 1 light chain 3 (LC3-I), membrane-associated (LC3-II) forms of LC3 protein and Beclin-1, as hallmarks of autophagy, were assessed using Western blot to detect the autophagy markers. The secreted interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin (IL)-12 and transformig growth factor-beta (TGF-beta), and chemokine (C-C motif) ligand 18 (CCL18) from HMDMs were determined by ELISA. Also, reverse transcription polymerase chain reaction (RT-PCR) analysis was used to assess the mRNA expressions of CCL18 in HMDMs. RESULTS: Treatment of PBMCs with either Mtb antigen or with LAB significantly increased the IFN-gamma and NO production. Combination of Mtb antigen and LAB led to synergistic increase in IFN-gamma, and an additive increase in NO. Treatment with Mtb antigen alone significantly increased the IL-4 and IL-13 production. LAB significantly decreased IL-4 and IL-13 secretion in both unstimulated and Mtb antigen-stimulated PBMCs. The IFN-gamma/IL-4+IL-13 ratio was enhanced, indicating Th1/Th2 polarization. Treatment of macrophages with combined use of Mtb antigen and LAB led to an additive increase in Beclin-1, LC3-II expression, as well as in synergistic increase in IL-12 production. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in IL-6, IL-10, and CCL18 secretion. LAB inhibited the secretion of TGF-beta by Mtb-stimulated macrophages, however not significantly. Treatment of macrophages with combined use of Mtb antigen and LAB led to a decrease in CCL18 mRNA expression. CONCLUSION: Our study implies that LAB may reinforce the response of the mononuclear phagocytes to Mtb antigen by inducing production of the autophagy-promoting factors IFN-gamma and NO, while decreasing the Th2 autophagy-restraining cytokines IL-4 and IL-13. Hence, combination of Mtb antigen and LAB may perhaps be safer in more efficacious TB vaccine formulation.
Asunto(s)
Antígenos Bacterianos/inmunología , Autofagia/inmunología , Bifidobacterium/inmunología , Interferón gamma/inmunología , Lacticaseibacillus rhamnosus/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Adulto , Bifidobacterium/metabolismo , Femenino , Humanos , Interleucina-13/análisis , Interleucina-13/metabolismo , Interleucina-4/análisis , Interleucina-4/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Macrófagos/microbiología , Masculino , Proteínas Asociadas a Microtúbulos/inmunología , Monocitos/inmunología , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Células TH1/inmunología , Células Th2/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND AND AIM: The intestinal epithelium is constantly exposed to high levels of genetic material like bacterial DNA. Under normal physiological conditions, the intestinal epithelial monolayer as a formidable dynamic barrier with a high-polarity structure facilitates only a controlled and selective flux on components between the lumen and the underlining mucosa and even is able to facilitate structure-based macromolecules movement. The aim of this study was to test the effect of natural commensal-origin DNA on the TLR9 signaling cascade and the barrier integrity of polarized intestinal epithelial cells (IECs). METHODS: : Polarized HT-29 and T84 cells were treated with TNF-alpha in the presence or absence of DNA from Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum. TLR9 and interleukin-8 (IL-8) mRNA expression was assessed by semiquantitative and TaqMan real-time reverse-transcription polymerase chain reaction. Expression of TLR9 protein, degradation of inhibitor of kappa B alpha (IkappaBalpha), and p38 mitogen-activated protein kinase (p38 MAP) phosphorylation were assessed by Western blotting. To further reveal the role of TLR9 signaling, the TLR9 gene was silenced by siRNA. IL-8 secretion was measured by an enzyme-linked immunosorbent assay. Nuclear factor-kappa B (NF-kappaB) activity was assessed by the electrophoretic mobility shift assay (EMSA) and NF-kappaB-dependent luciferase reporter gene assays. As an indicator of tight junction formation and monolayer integrity of epithelial cell monolayers, transepithelial electrical resistance (TER) was repetitively monitored. Transmonolayer movement of natural commensal-origin DNA across monolayers was monitored using qRT-PCR and nested PCR based on bacterial 16S rRNA genes. RESULTS: In response to apically applied natural commensal-origin DNA, polarized HT-29 and T84 cells enhanced expression of TLR9 in a specific manner, which was subsequently associated with attenuation of TNF-alpha-induced NF-kappaB activation and NF-kappaB-mediated IL-8 expression. TLR9 silencing abolished this inhibitory effect. Apically applied LGG DNA attenuated TNF-alpha-enhanced NF-kappaB activity by reducing IkappaBalpha degradation and p38 phosphorylation. LGG DNA did not decrease the TER but rather diminished the TNF-alpha-induced TER reduction. Translocation of natural commensal-origin DNA into basolateral compartments did not occur under tested conditions. CONCLUSIONS: Our study indicates that TLR9 signaling mediates, at least in part, the anti-inflammatory effects of natural commensal-origin DNA on the gut because TLR9 silencing abolished the inhibitory effect of natural commensal-origin DNA on TNF-alpha-induced IL-8 secretion in polarized IECs. The nature of the TLR9 agonist, the polarity of cells, and the tight junction integrity of IECs has to be taken into account in order to predict the outcome of TLR9 signaling. (Inflamm Bowel Dis 2010).
Asunto(s)
Productos Biológicos/genética , Interleucina-8/inmunología , Mucosa Intestinal/inmunología , Lacticaseibacillus rhamnosus/genética , Transducción de Señal/inmunología , Receptor Toll-Like 9/inmunología , Productos Biológicos/inmunología , Polaridad Celular/fisiología , Supervivencia Celular/inmunología , ADN Bacteriano/farmacología , Impedancia Eléctrica , Expresión Génica/inmunología , Células HT29 , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/inmunología , Lipopolisacáridos/farmacología , Luciferasas/genética , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación/inmunología , Probióticos , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Among the factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal flora or lack of microbial exposure during childhood has been proposed. T(H)2-cytokines increase the production of IgE and stimulate mast cells and eosinophils, whereas T(H)1-cytokines, such as IFN-gamma, may suppress IgE synthesis and stimulate the expression of the secretory piece of IgA. Thus, a dysregulation in the expression of T(H)1- and T(H)2-cytokines may contribute to the initiation and maintenance of allergic diseases. Lactobacilli belonging to the natural intestinal microflora were reported to reduce the incidence of atopic dermatitis and the severity of allergic manifestations and to modulate T(H)1/T(H)2 responses. The mechanisms still remain to be elucidated. We sought to assess the effect of different probiotics, Lactobacillus rhamnosus GG, Lactobacillus gasseri (PA16/8), Bifidobacterium bifidum (MP20/5), and Bifidobacterium longum (SP07/3), on the T(H)1 and T(H)2 responses of peripheral blood mononuclear cells (PBMCs) from healthy subjects and from patients with allergy against house dust mite to Staphylococcus enterotoxin A (SEA) and Dermatophagoides pteronyssinus (Dpt). To elucidate the molecular basis of these effects, the effects of bacterial genomic DNA were compared with the effects of viable bacteria. PBMCs from allergic patients and from healthy donors were incubated for 24 or 48 h, respectively, with or without SEA and Dpt allergens. The effects of preincubation with live probiotic bacteria and the effect of their genomic DNA, added simultaneously to cultures and incubated for 24h, were assessed by measuring T(H)1/T(H)2-cytokine production. The tested live Gram-positive probiotic bacteria and their genomic DNA inhibited SEA- and Dpt-stimulated secretion of T(H)2-cytokines (IL-4 and IL-5) and enhanced the stimulation of IFN-gamma. This effect was dose-dependent with a dosage-optimum, which was identical for all lactic acid producing bacteria (LAB) tested (10 bacteria per PBMC) and their DNA (75 ng/ml). Based on the maximal effects achieved with LAB and their DNA, more than 50% of the effects seem to be contributed by DNA. No significant effect was induced by the control, Gram-negative Escherichia coli TG1. Lactobacilli and bifidobacteria reduced SEA-stimulated IL-4 and IL-5 production more effectively in PBMCs from healthy subjects than from allergic patients. In contrast to this, inhibition of Dpt-stimulated IL-4- and IL-5-secretion was more pronounced in cells from allergic subjects. Compared with living LAB, bacterial DNA inhibited IL-4- and IL-5-secretion in a similar manner. SEA- and even more so Dpt-stimulated IFN-gamma stimulation by living LAB was less pronounced in allergic than in healthy subjects, whereas IFN-gamma stimulation by their DNA was more pronounced in allergic subjects. The tested probiotic bacteria as well as their genomic DNA modulated the T(H)1/T(H)2 response to some allergens dose-dependently. DNA seems to contribute to 50% of the effect exerted by living bacteria in this in vitro model. The magnitude of the probiotic effects differed between healthy and allergic subjects. Whether the modulation found for the tested strains might be useful for the prevention and treatment of allergic diseases has to be assessed in clinical trials.
Asunto(s)
Bifidobacterium , ADN Bacteriano/inmunología , Hipersensibilidad/inmunología , Lacticaseibacillus rhamnosus , Probióticos , Células TH1/inmunología , Células Th2/inmunología , Adulto , Antígenos Dermatofagoides/inmunología , Citocinas/metabolismo , Relación Dosis-Respuesta Inmunológica , Enterotoxinas/inmunología , Femenino , Humanos , Hipersensibilidad/sangre , Activación de Linfocitos , Masculino , Células TH1/metabolismo , Células TH1/microbiología , Células Th2/metabolismo , Células Th2/microbiologíaRESUMEN
Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. In particular the intestinal mucosal surfaces are in continuous contact with a heterogeneous population of microorganisms of the endogenous flora and are exposed to food and microbes. As a result, the immune system of the host has to discriminate between pathogenic and commensal microorganisms. This article reviews the types of sentinel cells that continuously sense the environment and coordinate immune defenses as well as the mechanisms of the innate and adaptive immune systems that are activated by bacterial and viral molecular patterns leading to inflammatory, allergic, or regulatory immune response with special emphasis on probiotic bacteria.