Hanne Frøkiær

Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells

Dendritic cells (DC) play a pivotal immunoregulatory role in the Th1, Th2, and Th3 cell balance and are present throughout the gastrointestinal tract. Thus, DC may be targets for modulation by gut microbes, including ingested probiotics. In the present study, we tested the hypothesis that species of Lactobacillus, important members of the gut flora, differentially activate DC. Bone marrow-derived murine DC were exposed to various lethally irradiated Lactobacillus spp. and resultant culture supernatants were analyzed for IL-6, IL-10, IL-12, and TNF-α. Substantial differences were found among strains in the capacity to induce IL-12 and TNF-α production in the DC. Similar but less pronounced differences were observed among lactobacilli in the induction of IL-6 and IL-10. Although all strains up-regulated surface MHC class II and B7-2 (CD86), which is indicative of DC maturation, those lactobacilli with greatest capacity to induce IL-12 were most effective. Remarkably, Lactobacillus reuteri DSM12246, a poor IL-12 inducer, inhibited IL-12, IL-6, and TNF-α induction by the otherwise strong cytokine inducer L. casei CHCC3139, while IL-10 production remained unaltered. In analogous fashion, L. reuteri reduced L. casei-induced up-regulation of B7-2. These results suggest that different species of Lactobacillus exert very different DC activation patterns and, furthermore, at least one species may be capable of inhibiting activities of other species in the genus. Thus, the potential exists for Th1/Th2/Th3-driving capacities of the gut DC to be modulated according to composition of gut microflora, including ingested probiotics.

Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-β

Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte‐derived DC in a transwell model where Gram‐positive (G+) commensals prevent Toll‐like receptor‐4 (TLR4)‐dependent Escherichia coli‐induced semimaturation in a TLR2‐dependent fashion. These findings add to our understanding of the hypo‐responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)‐12p70, and tumour necrosis factor‐α when matured with G+ and Gram‐negative (G–) commensals, while IL‐10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G– bacteria. The Caco2 SM‐induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor‐β (TGF‐β) and markedly reduced levels of bacteria‐induced interferon‐γ production. Caco2 cell production of IL‐8, thymic stromal lymphopoietin (TSLP) and TGF‐β increases upon microbial stimulation in a strain dependent manner. TSLP and TGF‐β co‐operate in inducing the tolerogenic DC phenotype but other mediators might be involved.

Dietary soya saponins increase gut permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.)

Saponins are naturally occurring amphiphilic molecules and have been associated with many biological activities. The aim of the present study was to investigate whether soya saponins trigger the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.), and to examine if dietary soya saponins increase the epithelial permeability of the distal intestine in Atlantic salmon. Seven experimental diets containing different levels of soya saponins were fed to seawater-adapted Atlantic salmon for 53 d. The diets included a fishmeal-based control diet, two fishmeal-based diets with different levels of added soya saponins, one diet containing 25% lupin kernel meal, two diets based on 25% lupin kernel meal with different levels of added soya saponins, and one diet containing 25% defatted soyabean meal. The effect on intestinal morphology, intestinal epithelial permeability and faecal DM content was examined. Fish fed 25% defatted soyabean meal displayed severe enteritis, whereas fish fed 25% lupin kernel meal had normal intestinal morphology. The combination of soya saponins and fishmeal did not induce morphological changes but fish fed soya saponins in combination with lupin kernel meal displayed significant enteritis. Increased epithelial permeability was observed in fish fed 25% defatted soyabean meal and in fish fed soya saponin concentrate independent of the protein source in the feed. The study demonstrates that soya saponins, in combination with one or several unidentified components present in legumes, induce an inflammatory reaction in the distal intestine of Atlantic salmon. Soya saponins increase the intestinal epithelial permeability but do not, per se, induce enteritis.

Lactic acid bacteria inducing a weak interleukin-12 and tumor necrosis factor alpha response in human dendritic cells inhibit strongly stimulating lactic acid bacteria but act synergistically with gram-negative bacteria.

ABSTRACT The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G−) commensals, such as members of the family Enterobacteriaceae, may, however, be immunomodulators that are as important as G+ organisms but tend to be overlooked. Dendritic cells (DCs) are crucial immune regulators, and therefore, the present study aimed at investigating differences among human gut flora-derived LAB and G− bacteria in their patterns of DC polarization. Human monocyte-derived DCs were exposed to UV-killed bacteria, and cytokine secretion and surface marker expression were analyzed. Profound differences in the DC polarization patterns were found among the strains. While strains of LAB varied greatly in their capacity to induce interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α), G− strains were consistently weak IL-12 and TNF-α inducers. All strains induced significant amounts of IL-10, but G− bacteria were far more potent IL-10 inducers than LAB. Interestingly, we found that when weakly IL-12- and TNF-α-inducing LAB and strong IL-12- and TNF-α-inducing LAB were mixed, the weakly IL-12- and TNF-α-inducing LAB efficiently inhibited otherwise strong IL-12- and TNF-α-inducing LAB, yet when weakly IL-12- and TNF-α-inducing LAB were mixed with G− bacteria, they synergistically induced IL-12 and TNF-α. Furthermore, strong IL-12- and TNF-α-inducing LAB efficiently up-regulated surface markers (CD40, CD83, CD86, and HLA-DR), which were inhibited by weakly IL-12- and TNF-α-inducing LAB. All G− bacteria potently up-regulated surface markers; however, these markers were not inhibited by weakly IL-12- and TNF-α-inducing LAB. These much divergent DC stimulation patterns among intestinal bacteria, which encompass both antagonistic and synergistic relationships, support the growing evidence that the composition of the gut flora affects immune regulation and that compositional imbalances may be involved in disease etiology.

Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll‐like receptor‐2‐dependent mechanism

Lactobacilli are probiotics that, among other health‐promoting effects, have been ascribed immunostimulating and virus‐preventive properties. Certain Lactobacillus spp. have been shown to possess strong interleukin‐12 (IL‐12) ‐inducing properties. As IL‐12 production depends on the up‐regulation of type I interferons (IFNs), we hypothesized that the strong IL‐12‐inducing capacity of Lactobacillus acidophilus NCFM in murine bone‐marrow‐derived dendritic cells (DCs) is caused by an up‐regulation of IFN‐β, which subsequently induces IL‐12 and the double‐stranded RNA binding Toll‐like receptor‐3 (TLR‐3). The expression of the genes encoding IFN‐β, TLR‐3, IL‐12 and IL‐10 in DCs upon stimulation with L. acidophilus NCFM was determined. Lactobacillus acidophilus NCFM induced a much stronger expression of Ifn‐β, Il‐12 and Il‐10 compared with the synthetic double‐stranded RNA ligand Poly I:C, whereas the levels of expressed Tlr‐3 were similar. Whole genome microarray gene expression analysis revealed that other genes related to viral defence were significantly up‐regulated and among the strongest induced genes in DCs stimulated with L. acidophilus NCFM. The ability to induce IFN‐β was also detected in another L. acidophilus strain (X37), but was not a property of other probiotic strains tested, i.e. Bifidobacterium bifidum Z9 and Escherichia coli Nissle 1917. The IFN‐β expression was markedly reduced in TLR‐2−/− DCs, dependent on endocytosis, and the major cause of the induction of Il‐12 and Tlr‐3 in DCs stimulated with L. acidophilus NCFM. Collectively, our results reveal that certain lactobacilli trigger the expression of viral defence genes in DCs in a TLR‐2 manner dependent on IFN‐β.

Fish oil supplementation of lactating mothers affects cytokine production in 2 1/2-year-old children

Abstractn−3 PUFA influence immune functioning and may affect the cytokine phenotype during development. To examine whether maternal fish oil supplementation during lactation could modify later immune responses in children, 122 lactating Danish mothers with a fish intake below the population median were randomized to groups supplemented for the first 4 mon of lactation with 4.5 g/d of fish oil (equivalent to 1.5 g/d of n−3 long-chain PUFA) or olive oil. Fifty-three mothers with a fish intake in the highest quartile of the population were also included. The FA composition of erythrocyte membranes was measured at 4 mon and at 2 1/2 yr. Plasma immunoglobulin E (IgE) levels and cytokine production in lipopolysaccharide-stimulated whole-blood cultures were determined at 2 1/2 yr. Erythrocyte n−3 PUFA at 4 mon were higher in infants from the fish oil group compared with the olive oil group (P<0.001) but were no longer different at 2 1/2 yr. The median production of lipopolysaccharide-induced interferon γ(IFN-γ) in the fish oil group was fourfold higher than that in the olive oil group (P=0.034), whereas interleukin-10 (IL-10) production was similar. The IFN-γ/IL-10 ratio was twofold higher in the fish oil group (P=0.019) and was positively correlated with 20∶5n−3/20∶4n−6 in erythrocytes at 4 mon (P=0.050). The percentages of atopic children and plasma IgE were not different in the two groups, but the study was not designed to look at atopy. Cytokine responses and erythrocyte FA composition in children of mothers with a high fish intake were intermediate in comparison with those in the randomized groups. Fish oil supplementation during lactation resulted in increased in vitro IFN-γ production in the children 2 yr after the supplementation was given, which may reflect a faster maturation of the immune system.

Whole-blood culture is a valid low-cost method to measure monocytic cytokines - A comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes

Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19-40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L. acidophilus. Cytokines produced from whole-blood was found to be more strongly correlated with monocytic cytokines than cytokines from PBMC, particularly after LPS-stimulation: r=0.57, P<0.001 versus r=0.33, P=0.01 for IL-6 and r=0.43, P<0.001 versus r=0.30, P=0.02 for TNF-alpha. Adjustment for a preceding 8-week dietary fatty acid-intervention did not change any of the associations. Based on measurements at three time-points 8 weeks apart the intra-individual variation was > or = 50% smaller than the inter-individual variation (P<0.05) in most whole-blood cytokine responses and LPS-stimulated IL-6 from PBMC. We conclude that whole-blood cultures are well-suited low-cost proxy-measures of monocytic cytokine production. Moreover, large inter-individual variation in cytokine production was demonstrated whereas the individual responses in whole-blood were reproducible even over long time-periods.

Reduction of immunoreactivity of bovine β-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

Solonamide B Inhibits Quorum Sensing and Reduces Staphylococcus aureus Mediated Killing of Human Neutrophils

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be a serious human pathogen, and particularly the spread of community associated (CA)-MRSA strains such as USA300 is a concern, as these strains can cause severe infections in otherwise healthy adults. Recently, we reported that a cyclodepsipeptide termed Solonamide B isolated from the marine bacterium, Photobacterium halotolerans strongly reduces expression of RNAIII, the effector molecule of the agr quorum sensing system. Here we show that Solonamide B interferes with the binding of S. aureus autoinducing peptides (AIPs) to sensor histidine kinase, AgrC, of the agr two-component system. The hypervirulence of USA300 has been linked to increased expression of central virulence factors like α-hemolysin and the phenol soluble modulins (PSMs). Importantly, in strain USA300 Solonamide B dramatically reduced the activity of α-hemolysin and the transcription of psma encoding PSMs with an 80% reduction in toxicity of supernatants towards human neutrophils and rabbit erythrocytes. To our knowledge this is the first report of a compound produced naturally by a Gram-negative marine bacterium that interferes with agr and affects both RNAIII and AgrA controlled virulence gene expression in S. aureus.

Lactobacilli and bifidobacteria induce differential interferon-β profiles in dendritic cells.

The health promoting effects of probiotics are well-documented; however, current knowledge on immunostimulatory effects is based on data from a single strain or a limited selection of strains or species. Here, we compared the capacity of 27 lactobacilli and 16 bifidobacteria strains to stimulate bone marrow-derived dendritic cells (DC). Most lactobacilli strains, including Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus casei and Lactobacillus plantarum, induced strong IL-12 and TNF-α production and up-regulation of maturation markers. In contrast, all bifidobacteria and certain lactobacilli strains were low IL-12 and TNF-α inducers. IL-10 and IL-6 levels showed less variation and no correlation with IL-12 and TNF-α. DC matured by strong IL-12-inducing strains also produced high levels of interferon (IFN)-β. When combining two strains, low IL-12 inducers inhibited this IFN-β production as well as IL-12 and Th1-skewing chemokines. The IFN-β induction was mediated through c-Jun N-terminal kinase (JNK) irrespective of the stimulating strain. The inhibitory bacteria induced higher levels of the transcription factor c-Jun dimerization protein (JDP)-2, thereby counteracting the effect of JNK. Our data demonstrate that lactobacilli can be divided into two groups of bacteria featuring contrasting effects, while all bifidobacteria exhibit uniform effects. This underlines the importance of selecting the proper strain(s) for probiotic purposes.