Immunomodulation properties of multi-species fermented milks.

Dairy propionibacteria (PAB) are used as a ripening starter in combination with Lactic acid bacteria (LAB) for dairy products such as Swiss-type cheese. LAB and PAB have also been studied for their probiotic properties but little is still known about their individual and/or synergistic beneficial effects within dairy matrices. In the context of a rising incidence of Inflammatory Bowel Diseases, it has become crucial to evaluate the immunomodulatory potential of bacteria ingested in large numbers via dairy products. We therefore selected different strains and combinations of technological LAB and PAB. We determined their immunomodulatory potential by IL-10 and IL-12 induction, in human peripheral blood mononuclear cells, on either single or mixed cultures, grown on laboratory medium or directly in milk. Milk was fermented with selected anti-inflammatory strains of LAB or PAB/LAB mixed cultures and the resulting bacterial fractions were also evaluated for these properties, together with starter viability and optimum technological aspects. The most promising fermented milks were evaluated in the context of TNBS- or DSS-induced colitis in mice. The improvement in inflammatory parameters evidenced an alleviation of colitis symptoms as a result of fermented milk consumption. This effect was clearly strain-dependent and modulated by growth within a fermented dairy product. These findings offer new tools and perspectives for the development of immunomodulatory fermented dairy products for targeted populations.

In vitro characterization of the digestive stress response and immunomodulatory properties of microorganisms isolated from smear-ripened cheese.

Thirty-six microorganisms (twenty-one bacteria, twelve yeasts and three fungi) were isolated from surface-ripened cheeses and subjected to in vitro digestive stress. The approach mimicked gastric and/or duodenal digestion. Lactobacillus rhamnosus GG, Escherichia coli Nissle 1917 and Saccharomyces boulardii were used as reference strains. We studied the microorganisms grown separately in culture medium and then included (or not) in a rennet gel. The microorganisms' immunomodulatory abilities were also assessed by profiling cytokine induction in human peripheral blood mononuclear cells (PBMCs). The loss of viability was less than 1 log CFU/mL for yeasts under all conditions. In contrast, Gram-negative bacteria survived gastric and/or duodenal stress well but most of the Gram-positive bacteria were more sensitive (especially to gastric stress). Inclusion of sensitive Gram-positive bacteria in rennet gel dramatically improved gastric survival, when compared with a non-included cultured (with a 4 log CFU/mL change in survival). However, the rennet gel did not protect the bacteria against duodenal stress. The PBMC cytokine assay tests showed that the response to yeasts was usually anti-inflammatory, whereas the response to bacteria varied from one strain to another.

Gut microbiota limits heavy metals burden caused by chronic oral exposure.

Environmental exposure to pollutants such as heavy metal(s) is responsible for various altered physiological functions which are detrimental for health. The gut microbiota is critical for intestinal homeostasis but its role on xenobiotic handling is not fully understood, especially when continuous sub-chronic exposure is addressed. We first confirmed the essential role of the intestinal microbiome to limit heavy metal body burden by using germ-free mice following 6-weeks oral exposure. Significant increases of cadmium and lead absorption and dissemination in blood and target organs were measured in germ-free mice when compared with conventional specific pathogen free (SPF) mice. Besides the "barrier" function of the luminal microbiota, this may involve specific host-genes such as metallothioneins, which are differentially expressed in the gastrointestinal tract of each group of mice. Considering genes relevant for divalent metal transporters and oxidative pathways, significant differences in basal gene expression were measured between control and germ-free mice. Moreover, the magnitude of induction of these genes upon stimulation by heavy metals varied greatly depending on the dose and type of metal as well as the microbial status of the animal. Collectively, these data illustrate the complex host-microbes interplay occurring with environmental pollutants inside the gut.

Chronic ingestion of cadmium and lead alters the bioavailability of essential and heavy metals, gene expression pathways and genotoxicity in mouse intestine.

Chronic ingestion of environmental heavy metals such as lead (Pb) and cadmium (Cd) causes various well-documented pathologies in specific target organs following their intestinal absorption and subsequent accumulation. However, little is known about the direct impact of the non-absorbed heavy metals on the small intestine and the colon homeostasis. The aim of our study was to compare the specific bioaccumulation and retention of Cd and Pb and their effect on the essential metal balance in primary organs, with those occurring specifically in the gastrointestinal tract of mice. Various doses of Cd (5, 20 and 100 mg l(-1)) and Pb (100 and 500 mg l(-1)) chloride salts were provided in drinking water for subchronic to chronic exposures (4, 8 and 12 weeks). In contrast to a clear dose- and time-dependent accumulation in target organs, results showed that intestines are poor accumulators for Cd and Pb. Notwithstanding, changes in gene expression of representative intestinal markers revealed that the transport-, oxidative- and inflammatory status of the gut epithelium of the duodenum, ileum and colon were specifically affected by both heavy metal species. Additionally, in vivo comet assay used to evaluate the impact of heavy metals on DNA damage showed clear genotoxic activities of Cd, on both the upper and distal parts of the gastrointestinal tract. Altogether, these results outline the resilience of the gut which balances the various effects of chronic Cd and Pb in the intestinal mucosa. Collectively, it provides useful information for the risk assessment of heavy metals in gut homeostasis and further disease's susceptibility.

Spores from two distinct colony types of the strain Bacillus subtilis PB6 substantiate anti-inflammatory probiotic effects in mice.

BACKGROUND & AIMS: Reducing symptoms of inflammatory bowel diseases (IBD) by dietary supplements represents more than ever an attractive clinical approach. Since the use of spore forming bacteria may offer interesting advantages, the aim of the study was to address the anti-inflammatory potential of Bacillus >subtilis strain PB6 (ATCC - PTA 6737) spores, provided as a powder preparation. METHODS: The immunomodulatory potential of strain PB6 was first characterized in vitro on human immunocompetent cells for both the commercial spore powder (Anaban™) and two phenotypic variants of the vegetative form. Assessment of the in vivo anti-inflammatory capacity of the standard spore powder and a variant spore powder preparation was performed using a mouse model of acute, TNBS-induced colitis. Performance was compared with the drug prednisolone, and was based on blinded macroscopic and histological scores, blood inflammatory markers and measurements of infiltration of mucosal neutrophils. RESULTS: Strain PB6 induced substantial levels of IL-10 but very low levels of IL-12, TNFα and IFNγ on human PBMC. Both spore powders prevented colitis as shown by significant reductions of near all inflammatory read-outs. CONCLUSION: B. subtilis strain PB6, provided as a preparation of spores, shows pre-clinical anti-inflammatory effects, suggesting further evaluation in a clinical intervention trial, e.g. with IBD type patients.

Contribution of surface ß-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii.

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-ß-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of ß-glucan. To evaluate this hypothesis, gtfF genes of three ß-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of ß-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of ß-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of ß-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, ß-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.

Promising immunomodulatory effects of selected strains of dairy propionibacteria as evidenced in vitro and in vivo.

Immunomodulatory properties of 10 dairy propionibacteria, analyzed on human peripheral blood mononuclear cells (PBMCs), revealed a highly strain-dependent induction of anti-inflammatory cytokine interleukin 10 (IL-10). Two selected strains of Propionibacterium freudenreichii showed a protective effect against two models of colitis in mice, suggesting a probiotic potential predicted by immune-based selection criteria for these cheese starter bacteria.

Probiotic yeasts: anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice.

AIM: To evaluate the in vitro immunomodulation capacity of various non-pathogenic yeast strains and to investigate the ability of some of these food grade yeasts to prevent experimental colitis in mice. METHODS: In vitro immunomodulation was assessed by measuring cytokines [interleukin (IL)-12p70, IL-10, tumor necrosis factor and interferon gamma] released by human peripheral blood mononuclear cells after 24 h stimulation with 6 live yeast strains (Saccharomyces ssp.) and with bacterial reference strains. A murine model of acute 2-4-6-trinitrobenzene sulfonic acid (TNBS)-colitis was next used to evaluate the distinct prophylactic protective capacities of three yeast strains compared with the performance of prednisolone treatment. RESULTS: The six yeast strains all showed similar non-discriminating anti-inflammatory potential when tested on immunocompetent cells in vitro. However, although they exhibited similar colonization patterns in vivo, some yeast strains showed significant anti-inflammatory activities in the TNBS-induced colitis model, whereas others had weaker or no preventive effect at all, as evidenced by colitis markers (body-weight loss, macroscopic and histological scores, myeloperoxidase activities and blood inflammatory markers). CONCLUSION: A careful selection of strains is required among the biodiversity of yeasts for specific clinical studies, including applications in inflammatory bowel disease and other therapeutic uses.

Probiotic properties of non-conventional lactic acid bacteria: immunomodulation by Oenococcus oeni.

The widely used probiotic bacteria belong to the genera Lactobacillus and Bifidobacterium and have in most cases been isolated from the human gastrointestinal tract. However, other "less conventional" bacteria, from allochthonous or extremophilic origin, sharing similar structural or functional features, may also confer specific health benefits to a host. Firstly, we explored the in vitro immuno-modulatory or immune-stimulatory activities of 25 wine lactic acid bacteria belonging to Oenococcus oeni and Pediococcus parvulus. While cytokines released by peripheral blood mononuclear cells (PBMCs) stimulated by P. parvulus strains, showed little variation, O. oeni strains induced strain-specific cytokine patterns. Some O. oeni strains were then further analyzed under various conditions for growth, dose and culture medium. In a second phase, we evaluated the oral tolerance and safety of two strains of O. oeni in mice fed a high dose of bacteria for a week. Finally, evidence was gathered on the in vivo anti-inflammatory potential of a selected O. oeni strain using an experimental 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model. Although results did not match the anti-inflammatory levels obtained with certain conventional probiotics, strain IOEB 9115 significantly lowered colonic injury and alleviated colitis symptoms. The 'natural' tolerance towards acid, ethanol, and phenolic compounds of O. oeni strains combined with a measureable immunomodulatory potential, suggest a possible use of selected strains isolated from wine as live probiotics.

Differential crosstalk between epithelial cells, dendritic cells and bacteria in a co-culture model.

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic bacteria, but the influence of IECs in the regulation of the associated mucosal immune system remains largely unknown. The network of dendritic cells (DCs) in the vicinity of IECs is known to play a crucial role in the regulation of gut homeostasis. We investigated the cross-talk between murine IECs (m-IC(cl2) cell line), bone marrow derived DCs and different bacteria using an in vitro Transwell co-culture model. IECs responded poorly to different gram-positive lactic acid bacteria (LAB) and to a Staphylococcus aureus strain. In contrast two Escherichia coli strains, including the probiotic strain Nissle 1917, strongly activated IECs, as evidenced by the induction of different chemokines. While a differential maturation of DCs is observed upon direct stimulation with the various bacteria, DC maturation across the epithelial barrier was only observed upon challenge of the apical surface of the IECs with both E. coli strains and LPS. These results suggested that the capacity of bacteria to induce pro-inflammatory signals through the epithelial barrier is not linked to their pathogenic or commensal status, but seem to be dependent on the presence of specific surface factors. As already reported, we confirmed that m-IC(cl2) cells are highly susceptible to LPS. It is highly possible, at least in this model, that free LPS is the "specific factor" key to activate IEC or BMDC. Moreover, IECs are broadly unresponsive to gram-positive bacterial components, notably TLR-2 ligands, in contrast to gram-negative bacterial components. These results suggest that the gut epithelium will sense the commensal bacteria in a different way, and seems to be unresponsive to gram positive bacteria in particular to LAB.

Protection against Yersinia pseudotuberculosis infection conferred by a Lactococcus lactis mucosal delivery vector secreting LcrV.

Herein, we sought to evaluate the potential of a recombinant Lactococcus lactis strain secreting the Yersinia pseudotuberculosis low-calcium response V (LcrV) antigen for mucosal vaccination against Yersinia infections. We showed that the recombinant strain induced specific systemic and mucosal antibody and cellular immune responses after intranasal immunization and protected mice against both oral and systemic Y. pseudotuberculosis infections. This constitutes the first proof of principle for a novel anti-Yersinia mucosal vaccination strategy using recombinant lactic acid bacteria.

Therapeutic potential of Yersinia anti-inflammatory components.

Microbial pathogens have developed various stratagems for modulating and/or circumventing the host's innate and adaptive immunity. Hence, certain virulence factors can be viewed as potential therapeutic agents for human immunopathological diseases. This is the case for virulence plasmid-encoded proteins from pathogenic Yersiniae that inhibit the host's inflammatory response by interfering with various cellular signaling pathways.

A key role of dendritic cells in probiotic functionality.

BACKGROUND: Disruption of the intestinal homeostasis and tolerance towards the resident microbiota is a major mechanism involved in the development of inflammatory bowel disease. While some bacteria are inducers of disease, others, known as probiotics, are able to reduce inflammation. Because dendritic cells (DCs) play a central role in regulating immune responses and in inducing tolerance, we investigated their role in the anti-inflammatory potential of probiotic lactic acid bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Selected LAB strains, while efficiently taken up by DCs in vitro, induced a partial maturation of the cells. Transfer of probiotic-treated DCs conferred protection against 2, 4, 6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Protection was associated with a reduction of inflammatory scores and colonic expression of pro-inflammatory genes, while a high local expression of the immunoregulatory enzyme indolamine 2, 3 dioxgenase (IDO) was observed. The preventive effect of probiotic-pulsed DCs required not only MyD88-, TLR2- and NOD2-dependent signaling but also the induction of CD4+ CD25+ regulatory cells in an IL-10-independent pathway. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that selected probiotics can stimulate DC regulatory functions by targeting specific pattern-recognition receptors and pathways. The results not only emphasize the role of DCs in probiotic immune interactions, but indicate a possible role in immune-intervention therapy for IBD.

Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria.

AIM: To investigate the correlation between in vitro and in vivo immunomodulation potential of the probiotic strain and its ability to prevent experimental colitis in mice. METHODS: In vitro immunomodulation was assessed by measuring interleukin (IL)-12p70, IL-10, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) release by human peripheral blood mononuclear cells (PBMCs) after 24 h stimulation with 13 live bacterial strains. A murine model of acute TNBS-colitis was next used to evaluate the prophylactic protective capacity of the same set of strains. RESULTS: A strain-specific in vivo protection was observed. The strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offered the best protection in the in vivo colitis model. In contrast, strains leading to a low IL-10/IL-12 cytokine ratio could not significantly attenuate colitis symptoms. CONCLUSION: These results show that we could predict the in vivo protective capacity of the studied lactic acid bacteria (LAB) based on the cytokine profile we established in vitro. The PBMC-based assay we used may thus serve as a useful primary indicator to narrow down the number of candidate strains to be tested in murine models for their anti-inflammatory potential.

Enhanced antiinflammatory capacity of a Lactobacillus plantarum mutant synthesizing modified teichoic acids.

Teichoic acids (TAs), and especially lipoteichoic acids (LTAs), are one of the main immunostimulatory components of pathogenic Gram-positive bacteria. Their contribution to the immunomodulatory properties of commensal bacteria and especially of lactic acid bacteria has not yet been investigated in detail. To evaluate the role of TAs in the interaction between lactic acid bacteria and the immune system, we analyzed the antiinflammatory properties of a mutant of Lactobacillus plantarum NCIMB8826 affected in the TA biosynthesis pathway both in vitro (mononuclear cells stimulation) and in vivo (murine model of colitis). This Dlt- mutant was found to incorporate much less D-Ala in its TAs than the WT strain. This defect significantly impacted the immunomodulation reactions induced by the bacterium, as shown by a dramatically reduced secretion of proinflammatory cytokines by peripheral blood mononuclear cells and monocytes stimulated by the Dlt- mutant as compared with the parental strain. Concomitantly, a significant increase in IL-10 production was stimulated by the Dlt- mutant in comparison with the WT strain. Moreover, the proinflammatory capacity of L. plantarum-purified LTA was found to be Toll-like receptor 2-dependent. Consistent with the in vitro results, the Dlt- mutant was significantly more protective in a murine colitis model than its WT counterpart. The results indicated that composition of LTA within the whole-cell context of L. plantarum can modulate proinflammatory or antiinflammatory immune responses.

Clinical and immunological effects of a 6 week immunotherapy cycle with murabutide in HIV-1 patients with unsuccessful long-term antiretroviral treatment.

In an effort to evaluate the potential of non-specific immunotherapy in restoring global immunity, we have examined the clinical tolerance and biological effects of a 6 week administration of the immunomodulator, murabutide, in chronically infected HIV-1 patients. Forty-two subjects, presenting weak immune reconstitution and ineffective virus suppression following long-term highly active antiretroviral therapy (HAART), were randomized to receive, or not, murabutide 7 mg/day on five consecutive days/week. Clinical and immunological parameters were monitored before and after the immunotherapy period. Administration of murabutide was generally well tolerated, although some grade III adverse events, reversible on treatment cessation, were observed. Interestingly, in comparison with pre-inclusion levels, at 1 week after the immunotherapy cycle, only murabutide recipients presented a significant increase in CD4 cells, platelet counts, and in the percentage of patients with undetectable viral loads (<50 copies/mL). Statistical significance between the two groups was only evident with the latter parameter. Some of these clinical changes were maintained even up to 12 weeks after murabutide administration, and were accompanied by an increased ability to mount cellular responses to active immunization with a recall antigen, and by a significant increase in the percentage of patients presenting positive lymphoproliferative responses to the viral antigen gp160. These results warrant further evaluation of extended periods or cycles of murabutide immunotherapy as adjunct to HAART.