Chantal Matar

Role of Intestinal Epithelial Cells in Immune Effects Mediated by Gram-Positive Probiotic Bacteria: Involvement of Toll-Like Receptors

ABSTRACT The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4+ T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens.

Immunomodulating capacity of kefir

Kefir is a fermented milk produced by the action of lactic acid bacteria, yeasts and acetic acid bacteria, trapped in a complex matrix of polysaccharides and proteins. Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet. However, published human or animal feeding trials to substantiate this view are not numerous. The aim of this work was to determine the immunomodulating capacity of kefir on the intestinal mucosal immune response in mice and to demonstrate the importance of dose and cell viability on this response. BALB/c mice were fed with commercial kefir ad libitum (diluted 1/10, 1/50, 1/100 or 1/200) or pasteurized kefir (diluted 1/6, 1/10, 1/50, 1/100) for 2, 5 or 7 consecutive days. At the end of each feeding period, the bacterial translocation assay was performed in the liver. Small intestine structure was studied by haematoxilin-eosin staining and light microscopy. The number of IgA+ and IgG+ cells was also determined. For the functional doses chosen, cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma) were determined. Kefir and pasteurized kefir were able to modulate the mucosal immune system in a dose-dependent manner. Kefir was administred 10-times more diluted than pasteurized kefir, but it induced an immunomodulation of similar magnitude, indicating the importance of cell viabilty. The results suggest that a Th1 response was controlled by Th2 cytokines induced by kefir feeding. Pasteurized kefir would induce both Th2 and Th1 responses. This is the first study in vivo regarding the mechanisms involved in the immunomodulating capacity of the oral administration of kefir containing viable or heat-inactivated bacteria at different doses.

Immunomodulating effects of milks fermented by Lactobacillus helveticus and its non-proteolytic variant

The effect of milks fermented by Lactobacillus helveticus and its non-proteolytic variant on mucosal and tumoral immunity was studied. Milks fermented by Lb. helveticus wild type or its non-proteolytic variant were administered orally to mice for different periods (3, 5 and 7 d). The immune response was assessed by analysing the activity of the peritoneal macrophages, the number of cells secreting IgA associated with the gut-associated lymphoid tissue and with the bronchial-associated lymphoid tissue. The number of cells was determined by direct immunofluorescence. The antitumour activity was monitored by studying the regression of the subcutaneously implanted fibrosarcomas. After 3 d feeding of milk fermented by Lb. helveticus wild type, the number of sIgA increased significantly at both the intestinal and bronchial levels, indicating that a cellular migration had occurred. This effect was not noticeable when milk fermented by Lb. helveticus Protease (-) was orally administered. Both fermented milks (wild type or its variant) exhibited an effect on the activity of the peritoneal macrophages, which might be indirectly correlated to the regression of the fibrosarcoma. Although the mechanism by which the lactic acid bacteria enhance the immune system is not clear, this study clearly suggests that the bioactive compounds released during milk fermentation might contribute to the immunoenhancing properties of these products. By releasing biopeptide, lactic acid bacteria have important implications in modulation of the hosts immune response, more specifically its cellular immune response.

Induction of a humoral immune response following an Escherichia coli O157:H7 infection with an immunomodulatory peptidic fraction derived from Lactobacillus helveticus-fermented milk.

ABSTRACT Numerous beneficial effects have been attributed to probiotic lactic acid bacteria (LAB), such as the stimulation of the immune system, the prevention of enteric infections by enteropathogens, and the regression of immunodependent tumors. It has been shown that biologically active metabolites released during fermentation, in particular biopeptides, could act as immunomodulatory agents. However, no studies have been conducted to evaluate the implication of these bioactive peptides in the induction of a protective immune response against enteric infections. The present study aimed to evaluate the possible immunomodulatory and anti-infectious effects of a peptidic fraction released in milk fermented by Lactobacillus helveticus. The immune response in the mucosa-associated lymphoid tissue was monitored following an administration of the potentially bioactive peptidic fraction. The total immunoglobulin A (IgA) immune response was evaluated after an Escherichia coli O157:H7 infection in a BALB/c murine model. Immunohistochemical and enzyme-linked immunosorbent assays revealed an increase in the number of IgA-secreting B lymphocytes in the intestinal lamina propria and an enhanced total secretory and systemic IgA response. Cytokine profiling also revealed stimulation of a Th2 response in mice fed the peptidic fraction, whereas infected controls demonstrated a proinflammatory Th1 response. These results indicate that bioactive peptides released during fermentation by LAB could contribute to the known immunomodulatory effects of probiotic bacteria.

Effects of milk fermented by Lactobacillus helveticus R389 on a murine breast cancer model

IntroductionAntitumour activity is one of the health-promoting effects attributed to the lactic acid bacteria and their products of fermentation. Previous studies in mice demonstrated that bioactive compounds released in milk fermented by Lactobacillus helveticus R389 contribute to its immunoenhancing and antitumour properties. The aim of the present work was to study the effects of the consumption of milk fermented by L. helveticus R389 or its proteolytic-deficient variant, L. helveticus L89, on a murine hormone-dependent breast cancer model.MethodsMice were fed with milk fermented by L. helveticus R389 or L. helveticus L89, during 2 or 7 days. The tumour control group received no special feeding. At the end of the feeding period, the mice were challenged by a subcutaneous injection of tumour cells in the mammary gland. Four days post-injection, the mice received fermented milk on a cyclical basis. The rate of tumour development and the cytokines in serum, mammary gland tissue and tumour-isolated cells were monitored. Bcl-2-positive cells in mammary glands and cellular apoptosis in tumour tissue were also studied.ResultsSeven days of cyclical administration of milk fermented by either bacterial strain delayed or stopped the tumour development. Cytokines demonstrated that L. helveticus R389 modulated the immune response challenged by the tumour. IL-10 and IL-4 were increased in all the samples from this group. In comparison with the tumour control, all test groups showed a decrease of IL-6, a cytokine involved in oestrogen synthesis. Seven days of cyclical feeding with milk fermented by L. helveticus R389 produced an increase in the number of apoptotic cells, compared with all other groups.ConclusionThis study demonstrated that 7 days of cyclical administration of milk fermented by both strains of L. helveticus diminishes tumour growth, stimulating an antitumour immune response. Compounds released during milk fermentation with L. helveticus R389 would be implicated in its immunoregulatory capacity on the immune response in mammary glands and tumour, which were correlated with the cytokines found at the systemic level. The milk fermented by L. helveticus R389 was able to modulate the relationship between immune and endocrine systems (by IL-6 diminution), which is very important in oestrogen-dependent tumour and induced cellular apoptosis.

MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells

BackgroundA subset of breast cancer cells displays increased ability to self-renew and reproduce breast cancer heterogeneity. The characterization of these so-called putative breast tumor-initiating cells (BT-ICs) may open the road for novel therapeutic strategies. As microRNAs (miRNAs) control developmental programs in stem cells, BT-ICs may also rely on specific miRNA profiles for their sustained activity. To explore the notion that miRNAs may have a role in sustaining BT-ICs, we performed a comprehensive profiling of miRNA expression in a model of putative BT-ICs enriched by non-attachment growth conditions.ResultsWe found breast cancer cells grown under non-attachment conditions display a unique pattern of miRNA expression, highlighted by a marked low expression of miR-30 family members relative to parental cells. We further show that miR-30a regulates non-attachment growth. A target screening revealed that miR-30 family redundantly modulates the expression of apoptosis and proliferation-related genes. At least one of these targets, the anti-apoptotic protein AVEN, was able to partially revert the effect of miR-30a overexpression. Finally, overexpression of miR-30a in vivo was associated with reduced breast tumor progression.ConclusionsmiR30-family regulates the growth of breast cancer cells in non-attachment conditions. This is the first analysis of target prediction in a whole family of microRNAs potentially involved in survival of putative BT-ICs.

The application of probiotics in cancer

Lactic acid bacteria (LAB) are present in many foods such as yoghurt and are frequently used as probiotics to favour some biological functions in the host. Many investigators have evaluated the therapeutic effects of yoghurt and LAB commonly used in yoghurt production against diseases such as cancer, infection, and gastrointestinal disorders. The increase of immune cell activity in the prevention of cancer by LAB consumption has also been described. Another possible explanation for the preventive effect of probiotics on carcinogenesis is their effect on other bacteria in the intestine. Probiotics may suppress the growth of bacteria that convert procarcinogens into carcinogens, thereby reducing the amount of carcinogens in the intestine. The present review is focused on two types of cancer in which milk fermented by LAB may show a beneficial effect: colon cancer and breast cancer.

Effects of the oral administration of the products derived from milk fermentation by kefir microflora on immune stimulation

Nutritional status has a major impact on the immune system. Probiotic effects ascribed to fermented dairy products arise not only from whole microorganisms but also from metabolites (peptides, exopolysaccharides) produced during the fermentation. We recently demonstrated the immunomodulating capacity of kefir in a murine model. We now aimed at studying the immunomodulating capacity in vivo of the products derived from milk fermentation by kefir microflora (PMFKM) on the gut. BALB/c mice received the PMFKM for 2, 5 or 7 consecutive days. IgA+ and IgG+ cells were determined on histological slices of the small and large intestine. IL-4, IL-6, IL-10, IL-12, IFNgamma and TNFalpha were determined in the gut, intestinal fluid and blood serum. IL-6 was also determined in the supernatant of a primary culture of small intestine epithelial cells challenged with PMFKM. PMFKM up-regulated IL-6 secretion, necessary for B-cell terminal differentiation to IgA secreting cells in the gut lamina propria. There was an increase in the number of IgA+ cells in the small and large intestine. The increase in the number of IgA+ cells was accompanied by an increase in the number of IL-4+, IL-10+ and IL-6+ cells in the small intestine. Effects of PMFKM in the large intestine were less widely apparent than the ones observed at the small intestine lamina propria. All cytokines that increased in the small intestine lamina propria, also did so in blood serum, reflecting here the immunostimulation achieved in the gut mucosa. We observed that the PMFKM induced a mucosal response and it was able to up and down regulate it for protective immunity, maintaining the intestinal homeostasis, enhancing the IgA production at both the small and large intestine level. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health status of the host.

Fermented Canadian lowbush blueberry juice stimulates glucose uptake and AMP-activated protein kinase in insulin-sensitive cultured muscle cells and adipocytes

Extracts of the Canadian lowbush blueberry (Vaccinium angustifolium Ait.) have recently been demonstrated to possess significant antidiabetic potential, in accordance with the traditional use of this plant as an antidiabetic natural health product. Fermentation of blueberry juice with the Serratia vaccinii bacterium is known to modify the phenolic content and increase antioxidant activity. The present study evaluated the effects of fermented blueberry juice on glucose uptake, adipogenesis, and the signaling pathways that regulate glucose transport in muscle cells and adipocytes. A 6-hour treatment with fermented juice potentiated glucose uptake by 48% in C2C12 myotubes and by 142% in 3T3-L1 adipocytes, in the presence or absence of insulin, whereas nonfermented juice had no effect on transport. Fermented juice dramatically inhibited triglyceride content during adipogenesis of 3T3-L1 cells. Chlorogenic acid and gallic acid, both major phenolic components of fermented juice, had no effect on glucose uptake. Western blot analysis of the insulin-independent AMP-activated protein kinase revealed increased phosphorylation resulting from a 6-hour treatment. This activation or the increase in glucose uptake could not be explained by increased cytosolic calcium. Fermentation with S. vaccinii is concluded to confer antidiabetic activities to blueberry juice. Although the active principles and their mechanisms of action remain to be identified, transformed blueberry juice may nevertheless represent a novel complementary therapy and a source of novel therapeutic agents against diabetes mellitus.

Milk fermentation products of L. helveticus R389 activate calcineurin as a signal to promote gut mucosal immunity

BackgroundFermented milks containing probiotic bacteria are a way of delivering bioactive constituents to targets in the gastrointestinal tract. We reported previously that the fermentation of milk at constant pH 6 by L. helveticus R389 increased its content of peptide fractions, and the oral administration of the non-bacterial fraction (FMSpH6) to mice increased total secretory IgA in the intestinal lumen and enhanced the number of IgA and various cytokines producing cells as well as the secretion of IL-6 by small intestine epithelial cells. We also demonstrated that this FMSpH6 was effective for the prevention of Salmonella typhimurium infection in mice. In this work, we studied in mice the impact of the oral administration of the supernatant of milk fermented by L. helveticus R389 on the gut physiology by measuring parameters such as calcium channels and E-cadherin expression, the activation of the biological signal calcineurin and mast and goblet cells, as a way to determine some mechanisms involved in the immunomodulating effects of the milk fermentation products, observed in previous studies. We analyzed the impact of the supernatant of milk fermented by L. helveticus R389 at pH6-controlled on the expression of calcineurin and on the reinforcement of the ephitelial barrier, measuring parameters such as calcium channels and E-cadherin expression and in the reinforcement of the non-specific immunity determining mast cells and goblet cells associated to the gut.ResultsWe observed an enhanced expression of TRPV6 channels in the duodenum, indicating an improved capacity for dietary Ca2+ uptake. We demonstrated an enhanced expression of calcineurin in the small intestine, able to upregulate immune parameters such as IL-2 and TNF production, with an increase in the number of these cytokines secreting cells. We determined an increase in the number of mucosal mast cells and goblet cells, which would mean an improved state of mucosal surveillance at sites of infection.ConclusionThe oral administration of the supernatant of milk fermented by L. helveticus R389 enhanced the gut mucosal immunity by improving the mechanisms that reinforce the epithelial and non-specific barriers and the gut functioning at sites of infection, with an improvement in the expression of the enzyme calcineurin, an important signal in the network that activates the gut immune system. The results of this work contribute to revealing the mechanisms underlying the immunomodulation of the gut immune function by fermented milks with probiotic bacteria.