Lactic acid bacteria secrete metabolites retaining anti-inflammatory properties after intestinal transport.

BACKGROUND: Probiotic bacteria have a beneficial effect on intestinal inflammation. In this study, we have examined the effect of lactic acid and commensal Gram positive (+) bacteria conditioned media (CM) on tumour necrosis factor alpha (TNF-alpha) release and the mechanisms involved. METHODS: Lipopolysaccharide (LPS) induced TNF-alpha secretion by peripheral blood mononuclear cells or the THP-1 cell line was monitored in the presence or absence of bacteria CM obtained from two probiotic strains, Bifidobacterium breve (Bb) and Streptococcus thermophilus (St), and three commensal bacterial strains (Bifidobacterium bifidum, Ruminococcus gnavus, and unidentified Streptococcus). Bb and St bacteria CM were allowed to cross filter grown intestinal epithelial cell monolayers (HT29-19A) to assess intestinal transport of active bacterial products. These products were characterised and their effect on LPS binding to THP-1 cells and nuclear factor kappa B (NF kappa B) activation assessed. RESULTS: Dose dependent inhibition of LPS induced TNF-alpha secretion was noted for both probiotic bacteria CM (64% and 71% inhibition for Bb and St, respectively) and to a lesser extent commensal bacteria CM (21-32% inhibition). Active products from Bb and St were resistant to digestive enzymes and had a molecular mass <3000 Da. Their inhibitory effect was preserved after transepithelial transport across intestinal cell monolayers, mainly in inflammatory conditions. LPS-FITC binding to THP-1 cells and NF kappa B activation were significantly inhibited by Bb and St CM. CONCLUSION: B breve and S thermophilus release metabolites exerting an anti-TNF-alpha effect capable of crossing the intestinal barrier. Commensal bacteria also display a TNF-alpha inhibitory capacity but to a lesser extent. These results underline the beneficial effect of commensal bacteria in intestinal homeostasis and may explain the role of some probiotic bacteria in alleviating digestive inflammation.

Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice.

BACKGROUND: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. METHODS: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon gamma (IFN-gamma) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. RESULTS: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-gamma) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-gamma (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. CONCLUSIONS: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.

Goats' milk of defective alpha(s1)-casein genotype decreases intestinal and systemic sensitization to beta-lactoglobulin in guinea pigs.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) alpha(s1)-casein content. Parenteral sensitization to GM1 and GM2 proteins as also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (delta Isc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-beta-lg IgG1, with an important cross reactivity between goat and cow beta-lg. However, in guinea pigs fed on GM2, anti-goat beta-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3.5+/-4.5 microA/cm2) compared with that observed in GM1 guinea pigs (8.3+/-7.6 microA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

Intestinal epithelial cells secrete exosome-like vesicles.

BACKGROUND & AIMS: Given the observations that intestinal epithelial cells (IECs) can present antigens to CD4(+) T lymphocytes and that professional antigen-presenting cells secrete exosomes (antigen-presenting vesicles), we hypothesized that IECs may secrete exosomes carrying molecules implicated in antigen presentation, which may be able to cross the basement membrane and convey immune information to noncontiguous immune cells. METHODS: Human IEC lines HT29-19A and T84-DRB1*0401/CIITA were grown on microporous filters. Release of exosomes under basal or inflammatory conditions was evaluated in conditioned apical and basolateral media after differential ultracentrifugations. Morphologic and biochemical characterization of exosomes was performed using immunoelectron microscopy, Western blotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: The intestinal cell lines released 30-90-nm-diameter vesicles from the apical and basolateral sides, and this release was significantly increased in the presence of interferon gamma. MHC class I, MHC class II, CD63, CD26/dipeptidyl-peptidase IV, and A33 antigen were present in epithelial-derived exosomes. CONCLUSIONS; Human IEC lines secrete exosomes bearing accessory molecules that may be involved in antigen presentation. These data are consistent with a model in which IECs may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.

The time-course of milk antigen-induced TNF-alpha secretion differs according to the clinical symptoms in children with cow's milk allergy.

BACKGROUND: TNF-alpha secretion by blood mononuclear cells stimulated with cow's milk proteins is significantly higher in infants with active cow's milk allergy (CMA) manifested by digestive symptoms than in children who have recovered from CMA. OBJECTIVE: The current study was undertaken to analyze the kinetics of TNF-alpha secretion and to evaluate the usefulness of the measurement of TNF-alpha release in whole blood cultures in the prediction of clinical outcome after milk challenge. METHODS: Blood samples were obtained from 83 children maintained on a cow's milk-free diet and examined just before a cow's milk provocation. Children were divided into 4 groups according to clinical outcome: group I (active CMA with cutaneous symptoms), group II (active CMA with predominantly digestive symptoms), group III (children recovered from CMA), and group IV (control). The kinetics of TNF-alpha secretion was measured in blood cultured for 1 to 5 days at different cow's milk protein concentrations. RESULTS: On day 1 TNF-alpha secretion was significantly higher in group I (485 [453] pg/mL, mean [SD], P <.005) and in group II (269 [102] pg/mL, P <. 005) than that observed in groups III and IV (149 [95] and 87 [71] pg/mL, respectively). Then TNF-alpha was rapidly degraded and a second peak of secretion was observed on day 5 but only in group II (278 [221] pg/mL), whereas in groups I, III, and IV a low secretion was observed (70 [61], 45 [40], and 11 [12] pg/mL, respectively, P <. 02). CONCLUSION: These results show that the pattern of TNF-alpha secretion in response to cow's milk proteins is different in CMA infants with cutaneous or digestive symptoms and suggest that TNF-alpha release might predict clinical relapse on challenge.

Intestinal barrier function and cow's milk sensitization in guinea pigs fed milk or fermented milk.

BACKGROUND: The respective effect of milk and fermented milks on intestinal barrier capacity and on sensitization to beta-lactoglobulin was studied using a guinea pig model of cow's milk allergy. METHODS: Guinea pigs were fed a control diet or the same diet supplemented with milk, fermented milk (Streptococcus thermophilus and Bifidobacterium breve), or dehydrated fermented milk. Intestinal barrier capacity to macromolecules was assessed in an Ussing chamber, and sensitization to cow's milk proteins was measured by systemic anti-beta-lactoglobulin immunoglobulin G1 titers and by intestinal anaphylaxis, the latter assessed by the beta-lactoglobulin-induced increase in short-circuit current of jejunal fragments (deltaIsc(beta-LG)). RESULTS: The electrical resistance of jejunum was similar in the four groups (approximately 80 omega/cm2) suggesting the same paracellular permeability. The transport of 14C-beta-lactoglobulin from mucosa to serosa was significantly decreased in the animals fed dehydrated fermented milk (403+/-131 ng / hr x cm2) compared with that in control animals or animals fed milk (767+/-250 ng / hr x cm2 and 749+/-475 ng / hr x cm2, respectively; p < 0.05). Milk fermentation did not modify native beta-lactoglobulin concentration but anti-beta-lactoglobulin immunoglobulin G1 titers were higher in fermented milk and dehydrated fermented milk (log10 titer = 2.86 and 2.79, respectively) than in guinea pigs fed milk (log10 titer = 2.5; p < 0.007). However, beta-lactoglobulin-induced intestinal anaphylaxis remained the same in the three groups (deltaIsc(beta-LG), 9.6+/-4.1 microA/cm2, 8.5+/-4.3 microA/cm2, and 8.5+/-3.4 microA/cm2 in milk-fed, fermented milk-fed, and dehydrated fermented milk-fed guinea pigs, respectively). CONCLUSIONS: The intestinal barrier capacity to milk proteins seems to be reinforced by dehydrated fermented milk, but milk and fermented milks are equally efficient in inducing cow's milk allergy in guinea pigs.

Effect of terfenadine on TNF alpha release from peripheral blood mononuclear cells during cow's milk allergy.

BACKGROUND: In infants with cow's milk allergy and intestinal symptoms, peripheral blood mononuclear cells stimulated in vitro with cow's milk proteins, secrete large amounts of the proinflammatory cytokine TNF alpha thus altering intestinal barrier capacity. Terfenadine, an antihistaminic drug, inhibits the release of several inflammatory mediators, including histamine, prostaglandins and leukotrienes. OBJECTIVES: To test the potential ability of terfenadine to inhibit TNF alpha secretion by mononuclear cells from infants with cow's milk allergy. METHODS: Mononuclear cells from infants allergic to cow's milk proteins were stimulated in vitro for 6 days by a mixture of milk proteins (beta-lactoglobulin, alpha-lactalbumin and casein) with or without terfenadine (0.1-1 microM) and culture supernatants were assayed for TNF alpha by enzyme immunoassay. The effect of culture supernatants on intestinal barrier capacity was evaluated by measuring the electrical resistance (index of integrity) of filter-grown HT29-19 A intestinal cells in Ussing chambers. RESULTS: During active cow's milk allergy, mononuclear cells stimulated with cow's milk proteins secreted large amounts of TNF alpha which significantly reduced the electrical resistance of HT29-19 A intestinal cells. There was a dose-dependent decrease in TNF alpha secretion in the presence of terfenadine, with a maximal inhibition of 62% of this secretion at 1 microM. Accordingly, terfenadine-treated mononuclear cells supernatants did not alter the electrical resistance of intestinal HT29.19 A cells. CONCLUSION: These results indicate that in infants with intestinal dysfunction due to cow's milk allergy, terfenadine is a potent inhibitor of the TNF alpha secretion induced by sensitizing milk protein antigens. This inhibition prevents the degradation of intestinal function as measured in an intestinal cell line, in vitro.

Zinc and intestinal anaphylaxis to cow's milk proteins in malnourished guinea pigs.

Zinc supplementation could favor recovery from diarrhea in malnourished children. As the recent experimental evidence suggests that oxidative stress and intestinal anaphylaxis may contribute to the intestinal dysfunction associated with malnutrition, we postulated that zinc could act through antioxidant or antianaphylactic properties. Control (C), malnourished (M), and malnourished zinc-treated (MZ) guinea pigs were, respectively, fed a normal 30% protein diet, a low 4% protein diet, and a low 4% protein diet plus 1800 ppm of zinc. Milk proteins were included in the diets to trigger intestinal anaphylaxis. Milk sensitization was assessed by passive cutaneous anaphylaxis (PCA) against beta-lactoglobulin and by intestinal anaphylaxis measured in Ussing chambers by the increase in short circuit-current after addition of beta-lactoglobulin (deltaIsc(betaLg)). Oxidative stress was assessed by intestinal lipid peroxidation. The intestinal secretion was assessed by deltaIsc induced by inflammatory mediators. Malnutrition increased the level of anti-betaLg reaginic antibodies [PCA = 1.19 +/- 0.79 and 0.69 +/- 0.67 log(l/titer) in M versus C guinea pigs, p = 0.07] and enhanced intestinal anaphylaxis (deltaIsc(betaLg)) = 16.4 +/- 9.9 and 9.1 +/- 5.8 microA/cm2 in M versus C guinea pigs, p = 0.07), without inducing intestinal lipid peroxidation. Moreover, malnutrition enhanced significantly the intestinal secretory response to histamine and 5-hydroxytryptamine. Administration of pharmacologic doses of zinc during malnutrition inhibited the increase in milk sensitization induced by malnutrition, both at the systemic [PCA = 0.35 +/- 0.55 log(l/titer) in MZ guinea pigs, p = 0.03 versus M] and intestinal (deltaIsc(betaLg)) = 2.8 +/- 2.5 microA/cm2 in MZ guinea pigs; p = 0.001 versus M) level, and prevented the hypersecretion in response to histamine and 5-hydroxytryptamine. These data suggest that zinc has antianaphylactic and antisecretory properties that may contribute to its capacity to prevent intestinal dysfunction during malnutrition.

The threshold for immune cell reactivity to milk antigens decreases in cow's milk allergy with intestinal symptoms.

BACKGROUND: In cow's milk allergy (CMA) with intestinal symptoms, peripheral blood mononuclear cells (PBMCs) secrete tumor necrosis factor-alpha (TNF-alpha), altering intestinal function. However, the type of cow's milk protein (CMP) that triggers symptoms (intact or intestinally processed) is not known, and neither is the minimal amount required. METHODS: PBMCs were isolated from infants with active CMA or cured infants just before a new challenge and stimulated with intact or intestinally processed CMP. Supernatants were tested for cytokine content and for their ability to perturb intestinal barrier capacity, measured in Using chambers in HT29-19A intestinal cells. RESULTS: PBMCs from infants with active CMA secreted more TNF-alpha, when they were stimulated with intact rather than intestinally processed CMPs, and more TNF-alpha than PBMCs from cured infants. Accordingly, supernatants from PBMCs stimulated with intact but not intestinally processed CMPs significantly increased intestinal permeability. The CMP concentration required to trigger TNF-alpha secretion capable of altering intestinal function was very small in infants with active CMA (approximately 2 micrograms/ml), but about 300 times higher in cured infants. CONCLUSION: Intact rather than intestinally processed proteins stimulate PBMCs to release TNF-alpha and alter intestinal barrier capacity. The threshold for PBMC reactivity to milk antigens drops considerably during active CMA with intestinal symptoms.

Intestinal paracellular permeability during malnutrition in guinea pigs: effect of high dietary zinc.

BACKGROUND: Zinc has been shown to have beneficial effects in vitro on epithelial barrier function, and in vivo to reduce intestinal permeability in malnourished children with diarrhoea. AIMS: To determine whether malnutrition alters intestinal paracellular permeability, and whether zinc prevents such alterations. METHODS: Guinea pigs were fed a normal protein diet (NP group), a low protein diet (LP group), or a low protein diet enriched with 1800 ppm zinc (LPZn group) for three weeks. Intestinal permeability was measured on jejunal segments mounted in Ussing chambers by measuring ionic conductance and mucosal to serosal fluxes of 14C-mannitol, 22Na, and horseradish peroxidase. Tight junction morphology was assessed on cryofracture replicas. RESULTS: Mannitol and Na fluxes and ionic conductance increased in the LP group compared with the NP group but remained normal in the LPZn group. Accordingly, jejunal epithelia from the LP group, but not from the LPZn group, showed a small decrease in number of tight junctional strands compared with epithelia from the NP group. Neither malnutrition nor zinc treatment modified horseradish peroxidase fluxes. CONCLUSIONS: Malnutrition is associated with increased intestinal paracellular permeability to small molecules, and pharmacological doses of zinc prevent such functional abnormality.

Anaphylactic intestinal response to milk proteins during malnutrition in guinea pigs.

We investigated whether sensitization to cow's milk occurs during malnutrition and alters intestinal ion and macromolecular transport. Malnourished guinea pigs received a low-protein diet containing either 4% soy or 4% milk proteins, and well-nourished sensitized controls received 26% soy plus 4% milk proteins. To assess milk sensitization, we measured immunoglobulin (Ig) G and passive cutaneous anaphylactic (PCA) responses to beta-lactoglobulin (beta-Lg) and the intestinal anaphylaxis, reflected by the rise in short-circuit current (delta Isc) induced by beta-Lg in tissues mounted in Ussing chambers. To assess intestinal function, we measured ionic conductance and unidirectional fluxes of -14C-mannitol and -3H-horseradish peroxidase (HRP). In malnourished animals fed milk proteins, IgG, PCA, and delta Isc (beta-Lg) increased more than in well-nourished animals. Ionic conductance and mannitol permeability rose in both malnourished groups. Malnourished animals fed milk proteins also displayed enhanced permeability to HRP. These data suggest that increased paracellular permeability is due to malnutrition per se, whereas increased macromolecular transport seems to require both malnutrition and sensitization. They indicate that intestinal anaphylaxis in response to milk proteins is persistent and even enhanced during experimental malnutrition.

Tumour necrosis factor-alpha induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers.

TNF-alpha is a widely distributed proinflammatory cytokine, involved in many disease states. Although it has widely distributed effects, a precise mechanism of action has never been described, in particular at the epithelial level. Morpho-functional changes of the intestinal epithelial monolayer HT29 cl.19A exposed to TNF-alpha were therefore assessed, using electron microscopy (including freeze-fracture replica analysis), as well as measurement of mannitol, Na+ and horseradish peroxidase fluxes across intestinal HT29 cl.19A cell monolayers using Ussing chambers. TNF-alpha receptors were induced on HT29 cl.19A cells by a small non-toxic dose of IFN-gamma (5 U/ml). After 4 h of the combined presence of TNF-alpha (10 ng/ml) and IFN-gamma (5 U/ml), the tight junction structure was altered as shown by a significant decrease in the average strand number measured in the apico-basal direction (5.50 +/- 2.70 vs 3.73 +/- 1.39 in control and treated cells respectively, P < 0.0001) and by a significant decrease in junctional depth (0.27 +/- 0.14 and 0.17 +/- 0.10 microns in control and treated cells respectively, P < 0.0001). These results are in agreement with a decrease in number of 'kiss' sites between contiguous membranes of TNF-alpha treated cells observed in ultrathin sections.(ABSTRACT TRUNCATED AT 250 WORDS)