Urolithin-A Promotes CD8+ T Cell-mediated Cancer Immunosurveillance via FOXO1 Activation.

Naïve T cells are key players in cancer immunosurveillance, even though their function declines during tumor progression. Thus, interventions capable of sustaining the quality and function of naïve T cells are needed to improve cancer immunoprevention.In this context, we studied the capacity of Urolithin-A (UroA), a potent mitophagy inducer, to enhance T cell-mediated cancer immunosurveillance.We discovered that UroA improved the cancer immune response by activating the transcription factor FOXO1 in CD8+ T cell. Sustained FOXO1 activation promoted the expression of the adhesion molecule L-selectin (CD62L) resulting in the expansion of the naïve T cells population. We found that UroA reduces FOXO1 phosphorylation favoring its nuclear localization and transcriptional activity. Overall, our findings determine FOXO1 as a novel molecular target of UroA in CD8+ T cells and indicate UroA as promising immunomodulator to improve cancer immunosurveillance. SIGNIFICANCE: Urolithin-A, a potent mitophagy inducer, emerges as a promising tool to enhance cancer immunosurveillance by activating the FOXO1 transcription factor in CD8+ T cells. This activation promotes the expansion of naïve T cells, offering a novel avenue for improving cancer immune response and highlighting UroA as a potential immunomodulator for bolstering our body's defenses against cancer.

Time-resolved quantitative proteome analysis of in vivo intestinal development.

Postnatal intestinal development is a very dynamic process characterized by substantial morphological changes that coincide with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. Time-resolved studies of intestinal development have so far been limited to investigation at the transcription level or to single or few proteins at a time. In the present study, we elucidate proteomic changes of primary intestinal epithelial cells from jejunum during early suckling (1-7 days of age), middle suckling (7-14 days), and weaning period (14-35 days) in mice, using a label-free proteomics approach. We show differential expression of 520 proteins during intestinal development and a pronounced change of the proteome during the middle suckling period and weaning. Proteins involved in several metabolic processes were found differentially expressed along the development. The temporal expression profiles of enzymes of the glycolysis were found to correlate with the increase in carbohydrate uptake at weaning, whereas the abundance changes of proteins involved in fatty acid metabolism as well as lactose metabolism indicated a nondiet driven preparation for the nutritional change at weaning. Further, we report the developmental abundance changes of proteins playing a vital role in the neonatal acquisition of passive immunity. In addition, different isoforms of several proteins were quantified, which may contribute to a better understanding of the roles of the specific isoforms in the small intestine. In summary, we provide a first, time-resolved proteome profile of intestinal epithelial cells along postnatal intestinal development.

Toward protein biomarkers for allergy: CD4+ T cell proteomics in allergic and nonallergic subjects sampled in and out of pollen season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Presence of age-associated low-grade inflammation does not worsen the body response to bacterial infection in old male rats.

In the field of frailty, there is an underlying hypothesis that chronic low-grade inflammation contributes to bad outcomes in response to a stressor. The host response to an Escherichia coli infection was assessed in 24 month old male rats exhibiting a chronic low-grade inflammation and in non-inflamed control rats. Mortality, weight loss and sarcopenia were the main outcomes measured. The presence of chronic low-grade inflammation did not affect post-infection mortality, body weight loss and tissue mass decreases. Infection-induced modifications of plasma acute phase proteins concentrations were not higher in low-grade inflamed than non-inflamed rats. Absolute synthesis rates of tissue proteins were independent of the initial inflammatory status, except for liver 10 days after infection. Altogether, age-associated chronic low-grade inflammation in male rats did not worsen the body response to bacterial infection. These results suggest that chronic low-grade inflammation is not an aggravating factor of the spiraling process leading to frailty.

Lactobacillus paracasei reduces intestinal inflammation in adoptive transfer mouse model of experimental colitis.

Studies showed that specific probiotics provide therapeutic benefits in inflammatory bowel disease. In vitro evidence suggested that Lactobacillus paracasei also called ST11 (CNCM I-2116) is a potent strain with immune modulation properties. However, little is known about its capacity to alleviate inflammatory symptoms in vivo In this context, the main objective of this study was to investigate the role of ST11 on intestinal inflammation using the adoptive transfer mouse model of experimental colitis. Rag2(-/-) recipient mice were fed with ST11 (10(9) CFU/day)a month prior toinduce colitis by adoptive transfer of naive T cells. One month later, in clear contrast to nonfed mice, weight loss was significantly reduced by 50% in ST11-fed mice. Further analysis of colon specimens revealed a significant reduction neutrophil infiltration and mucosal expression of IL1ß, IL-6, and IL12 proinflammatory cytokines, whereas no consistent differences in expression of antibacterial peptides or tight junction proteins were observed between PBS and ST11-fed mice. All together, our results demonstrate that oral administration of ST11 was safe and had a significant preventive effect on colitis. We conclude that probiotics such as Lactobacillus paracasei harbor worthwhile in vivo immunomodulatory properties to prevent intestinal inflammation by nutritional approaches.

Tolerance, safety, and effect on the faecal microbiota of an enteral formula supplemented with pre- and probiotics in critically ill children.

OBJECTIVES: The aim of this study was to demonstrate the tolerance and safety of an enteral formula containing prebiotics/probiotics, and its effect on the faecal microbiota in critically ill children. SUBJECTS AND METHODS: Ninety-four patients between 1 and 3 years old under mechanical ventilation requiring enteral feeding were randomised to receive either a test formula containing a synbiotic blend (composed of 2 probiotic strains [Lactobacillus paracasei NCC 2461 and Bifidobacterium longum NCC 3001], fructooligosaccharides [FOS], inulin, and Acacia gum), or a control formula. Patients remained in the intensive care unit for 7 days and were examined at day 14. Tolerance was assessed by overall caloric intake and time to reach caloric goal. Safety was assessed by abdominal distention, vomiting, and stool frequency. Microbiota was analysed by culture- and molecular-based methods. RESULTS: Overall caloric intake and time to reach caloric goal were similar between groups (noninferiority was shown). Abdominal distention, vomiting, and stool frequency were not affected by the supplementation with pre- and probiotics. Faecal bifidobacteria were higher in the test group at the end of the study. A similar trend was observed for total lactobacilli. L paracasei NCC 2461 and B longum NCC 3001 were detected in 80.4% and 17% of the test group patients, respectively. Enterobacteria levels remained unchanged during hospitalisation in the control group but diminished in the test group. CONCLUSIONS: The enteral formula supplemented with synbiotics was well tolerated by children in intensive care units; it was safe and produced an increase in faecal bacterial groups of previously reported beneficial effects.

Selection of bifidobacteria based on adhesion and anti-inflammatory capacity in vitro for amelioration of murine colitis.

Adhesion and anti-inflammatory properties of eight strains of bifidobacteria were tested using the intestinal epithelial cell lines Caco-2, T84, and HT29. Two strains were selected for further assessment of their anti-inflammatory capacity in two murine models of colitis. In vivo results confirmed the high anti-inflammatory capacity of a Bifidobacterium bifidum strain.

A diet containing whey protein, free glutamine, and transforming growth factor-beta ameliorates nutritional outcome and intestinal mucositis during repeated chemotherapeutic challenges in rats.

Anticancer chemotherapy often induces side effects such as mucositis. Recent data suggest that a diet, Clinutren Protect (CP), containing whey proteins, glutamine, and transforming growth factor-beta (TGFbeta)-rich casein limits intestinal mucositis and improves recovery after a single methotrexate (MTX) challenge in rats. Chemotherapy consists of alternating periods of treatment and rest. Thus, our study evaluated the effects of CP on nutritional outcome and intestinal mucositis in rats receiving repeated chemotherapeutic challenges. Thirty-six Sprague-Dawley rats received 3 cycles of MTX at 8-d intervals. Rats had free access to CP or control diet (Co) from 7 d before the first MTX injection until the end of the experiment at d 27. In Co, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein. L-Glutamine was replaced by L-alanine. Body composition was assessed by dual energy X-ray absorptiometry. Before MTX challenges, food intake and body weight were similar in both groups but became higher during MTX challenges in CP (P < 0.05). Fat mass decreased similarly in both groups. In contrast, the decrease of fat free mass between d -1 and d 27 was less pronounced in the CP group (-9.5 g) than in the Co group (-57.2 g) (P < 0.05). The intestinal damage score was lower in the CP group (0.6 +/- 0.3 vs. 2.1 +/- 0.6; P < 0.05). Fecal IgA increased over time in the CP group (P < 0.05) but not in the Co group. A diet containing whey proteins, glutamine, and TGFbeta improves nutritional outcome by limiting the reduction of fat free mass and reduces intestinal mucositis during repeated chemotherapeutic challenges in rats.

Lactobacillus paracasei CNCM I-2116 (ST11) inhibits substance P-induced skin inflammation and accelerates skin barrier function recovery in vitro.

Over the past few decades the number of people presenting reactive skin has increased in industrial countries. Skin inflammation mediated by neuropeptides and impaired skin barrier function are both underlying features of reactive skin conditions. Live microorganisms defined as probiotics have been successfully used to improve health status in humans. Beyond the effects on intestinal microbiota, some probiotic strains display potent immune-modulatory properties at the skin level. The aim of this study was to evaluate whether Lactobacillus paracasei CNCM-I 2116 (ST11) could modulate reactive skin-associated inflammatory mechanisms. The Caco-2/PBMC co-culture cell system was stimulated on the apical side with probiotics. The resulting medium collected from the basolateral compartment of the cell culture system, so called conditioned medium, was tested in ex vivo human abdominal plastic skin explant models of substance P-induced skin inflammation and skin barrier reconstruction. We show that ST11 was able to abrogate vasodilation, edema, mast cell degranulation and TNF-alpha release induced by substance P, compared to control. Moreover, using ex vivo skin organ culture, we show that ST11-conditioned medium induced a significantly faster barrier function recovery after SLS disruption, compared to control. These results support a beneficial role of ST11 on key biological processes associated with barrier function and skin reactivity.

Oral supplementation with Lactobacillus rhamnosus CGMCC 1.3724 prevents development of atopic dermatitis in NC/NgaTnd mice possibly by modulating local production of IFN-gamma.

Prevalence of allergies has increased during the last two decades. Alteration of the gut microbiota composition is thought to play a crucial role in development of atopic diseases. Oral administration of probiotics has been reported to treat and/or prevent symptoms of atopic diseases in infants, but the results are still controversial. We investigated the potential efficacy of dietary interventions by a probiotic strain on prevention and treatment of atopic dermatitis (AD) in a human-like AD model, NC/NgaTnd mice by perinatal administration. Pregnant NC/NgaTnd mice were orally treated with the probiotic strain Lactobacillus rhamnosus CGMCC 1.3724 (LPR), which was followed by treatment of pups until 12 weeks of age. LPR-treated mice exhibited significant lower clinical symptoms of dermatitis, reduced scratching frequency, lower levels of plasma total Immunoglobulin E and higher levels of interferon-gamma in skin biopsies, compared with untreated mice. The protective effect was also observed when mice started to be treated at weaning time (5 weeks of age) even with limited supplementation period of 2 weeks. However, treatment of mice with the probiotic starting 1 week after the onset of the disease (8 weeks of age) had limited effects. The usefulness of LPR for primary prevention of AD was supported.

Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice.

Fructooligosaccharides (FOS) are considered prebiotics because of their ability to promote growth of specific beneficial gut bacteria, such as bifidobacteria. Some studies reported potential immune-modulating properties. The aim of this study was to investigate the effect of FOS:inulin mix on murine response to Salmonella vaccine and evaluate the relevance toward protection against Salmonella infection. Balb/c mice were fed a diet containing 5% FOS:inulin mix or a control diet 1 wk before oral immunization with a suboptimal dose of live attenuated Salmonella typhimurium vaccine. Four weeks after vaccination, mice were infected with LD100 of virulent S. typhimurium. Specific blood Salmonella immunoglobulin G and fecal immunoglobulin A significantly increased in mice fed the diet containing prebiotics compared with control mice 4 wk postimmunization. Peritoneal macrophage phagocytic activity also significantly increased in FOS:inulin-fed mice at 1 wk postimmunization compared with control mice. No detectable effects were observed on the percentage of lymphoid cell subsets in the spleen. However, production of cytokines, interferon-gamma, interleukin-12, and tumor necrosis factor alpha, was numerically increased in spleen cell cultures stimulated with mitogens from FOS:inulin-fed mice 1 and 4 wk postimmunization. Salmonella translocation to lymphoid organs was not affected by feeding FOS:inulin. However, the improved response to Salmonella vaccine was concomitant with an increase in the survival rate of FOS:inulin-fed mice upon challenge with virulent Salmonella. No detectable effects were observed on the composition or the metabolic activity of the microbiota. Overall, the data suggest that a diet supplemented with FOS:inulin mix stimulates mucosal immunity and seems to improve efficacy of an oral vaccine.

Effect of Lactobacillus paracasei NCC2461 on antigen-specific T-cell mediated immune responses in aged mice.

Aging is associated with a reduced capacity to mount proper immune responses, in particular to vaccines. Probiotic lactic acid bacteria may improve the immune status of the elderly; however, there is little evidence showing an effect of these bacteria on humoral and cellular immune responses. In the present study, the immunomodulatory capacity of the probiotic Lactobacillus paracasei NCC2461 combined or not with a prebiotic composition, FOS/inulin, was examined in aged mice. Male C57BL/6J mice (21-months-old) were allocated to one of three groups fed ad libitum for 44 days with different diets: a normal diet (control), a normal diet plus NCC2461 given in the drinking water, or a diet containing FOS/inulin plus NCC2461 in the drinking water. All mice were immunized on day 15 and challenged on day 22 with keyhole limpet hemocyanin (KLH). T helper (Th)1 cell-dependent immune responses (anti-KLH immunoglobulin G(2a) [IgG(2a)] levels and delayed type hypersensitivity response) were increased significantly in NCC2461-supplemented mice when compared to controls. Supplementation with FOS/inulin did not further improve the immune-enhancing effect mediated by the probiotic. Splenocyte proliferation, T cell subsets, systemic total IgG levels, and mucosal total IgA responses were not affected. Interestingly, supplementation with NCC2461 modulated the intestinal microbiota composition by increasing the numbers of bifidobacteria and lactobacilli. In conclusion, oral intake of L. paracasei NCC2461 by aged mice enhanced the specific adaptive immune response to in vivo antigenic challenge without altering other cellular and humoral immune responses. The poor responsiveness to antigenic challenge, frequently observed in elderly people, may be improved by supplementation with L. paracasei NCC2461.

Prophylactic effect of oral administration of Lactobacillus johnsonii NCC533 (La1) during the weaning period on atopic dermatitis in NC/NgaTnd mice.

Bacterial exposure in infancy may be one of the determinants of atopic dermatitis (AD) morbidity in later life. Some clinical studies have shown that an intake of probiotics reduced the risks of AD in children; however, the timing and duration of administration for the prevention of AD still remain unclear. The aim of this study was to evaluate the effects on AD development of the administration of Lactobacillus johnsonii NC553 (La1) during the weaning period, using an animal model of human AD, NC/NgaTnd mice. La1 suspended in drinking water was administered to 4-week-old NC/NgaTnd mice for 4 weeks. Mice were kept up to 16 weeks of age in an air uncontrolled conventional condition. Clinical skin severity, scratching behaviour, histological features, and production of regulatory or inflammatory cytokines in spleens were analyzed. The results indicated that oral administration of La1 suppressed exacerbation of the clinical severity of dermatitis when compared to the controls. Scratching duration, which is the most important cause of skin damage, was also suppressed in mice fed with La1. La1 supplementation also suppressed epidermal hyperplasia and infiltration of inflammatory cells in skin. This study showed that exposure to La1 from the early stages might be beneficial to reduce the exacerbation of AD in children at high-risk of allergy.

Supplementation with oral probiotic bacteria protects human cutaneous immune homeostasis after UV exposure-double blind, randomized, placebo controlled clinical trial.

There is now strong evidence that probiotic bacteria can regulate inflammatory immune responses. Here, we analyzed whether oral supplementation with the probiotic bacterial strain Lactobacillus johnsonii (La1) could interfere with skin immune status following UV exposure. A randomized, double-blind, placebo controlled clinical trial was conducted with 54 healthy volunteers receiving either La1 or placebo, during six weeks prior to solar-simulated UV irradiation. Blister roofs and skin biopsies were recovered 1, 4 and 10 days after UV exposure from un-irradiated and irradiated skin and used for immunohistochemical analysis and mixed epidermal cell lymphocyte reaction (MECLR), respectively. La1 supplementation did not prevent the UV-induced phenotypic maturation of Langerhans cells (LCs) or the decrease in MECLR in irradiated skin samples, one day post-irradiation. On day 4, MECLR was still decreased in the placebo group, with a parallel reduction in the CD1a LC marker in irradiated epidermis. In contrast, the allostimulatory capacity of epidermal cells was totally recovered in the La1 group correlating with the normalization of CD1a expression within the epidermis. For the first time, the results provide evidence that ingested probiotic bacteria accelerate the recovery of skin immune homeostasis after UV-induced immunosuppression.

Patchy distribution of mucosal lesions in ileal Crohn's disease is not linked to differences in the dominant mucosa-associated bacteria: a study using fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis.

BACKGROUND: The mucosa-associated bacteria (MAB) are suspected of being involved in the pathogenesis of Crohn's disease. We analyzed and compared the MAB in noninflamed and inflamed ileal mucosa of Crohn's disease patients (n = 22). METHODS: Tissue samples from the inflamed ileal mucosa and from the adjacent noninflamed ileal mucosa were taken from surgical resection specimens. The MAB were investigated using fluorescence in situ hybridization with 7 group-specific probes and temporal temperature gradient gel electrophoresis (TTGE). RESULTS: Samples from both noninflamed and inflamed mucosa were obtained from 15 patients. The distribution of the bacterial populations was not different between noninflamed and inflamed mucosa. The Bacteroidetes phylum was dominant and accounted for 29% of MAB (0%-74%) in noninflamed tissues and 32% (0%-70%) in inflamed areas. The gamma Proteobacteria represented 12% (0%-70%) of MAB both in noninflamed and inflamed areas. The Clostridium coccoides group (Firmicutes phylum) represented 15% of MAB in noninflamed tissues versus 7% in inflamed areas. For most of the patients the similarity index between TTGE paired profiles was very high. CONCLUSION: The dominant MAB do not differ between noninflamed and inflamed ileal mucosa in Crohn's disease. This argues against a localized dysbiosis to explain the patchy distribution of mucosal lesions.

Prognostic values of alpha2-macroglobulin, fibrinogen and albumin in regards to mortality and frailty in old rats.

The study aimed to determine if acute phase proteins (APP) are markers of frailty in old rats. We evaluated in male Wistar rats at 96 weeks of age (n=72) whether single measurements of alpha(2)-macroglobulin, fibrinogen and albumin are predictive of mortality, body weight loss and inflammatory status during a 10-week follow-up period. Rats were clustered depending on levels of these APP at baseline. Rats with extremely high levels of alpha(2)-macroglobulin or fibrinogen (upper quartiles), or extremely low level of albumin (lower quartile), had an 11.6, 8.1 and 5.3-fold higher risk of mortality, respectively, than other rats. Body weight loss was negatively correlated with alpha(2)-macroglobulin, a trend was observed with fibrinogen (P=0.08) but not with albumin. Rats with fibrinogen levels >4.0 g/L or alpha(2)-macroglobulin levels >91 mg/L (respective top halves) at 96 weeks of age had higher levels of alpha(2)-macroglobulin and fibrinogen and lower levels of albumin throughout the follow-up period and higher levels of sTNFR-1 and lipopolysaccharide-binding protein at 106 weeks of age. Highest levels of alpha(2)-macroglobulin, fibrinogen and lowest albumin were predictive of mortality, whereas moderate levels of alpha(2)-macroglobulin and fibrinogen were, according to body weight loss and inflammatory status, markers of frailty in old rats.

Presence of low-grade inflammation in old rats does not worsen skeletal muscle loss under an endotoxemic and dietary stress.

The study aimed to determine if age-associated low-grade inflammation aggravates the response to a stress, especially regarding to sarcopenia. Initial inflammatory status in 22-month-old rats was based on plasma alpha(2)-macroglobulin and fibrinogen concentrations. The stress applied was a single intra-peritoneal injection of lipopolysaccharide followed by a 23-day period of malnutrition, i.e. a 4% casein diet distributed in quantity limited to 50% of spontaneous food intake. The response to the stress was analyzed in non-inflamed and low-grade inflamed rats and compared to non-inflamed and low-grade inflamed rats, which received the control treatment (i.e. no lipopolysaccharide injection and an 18% casein diet). The stress-induced body weight loss was higher in inflamed than non-inflamed rats, but the decrease in muscle weight was not worsened. Muscle protein turnover was not affected by the stress. Plasma alpha(2)-macroglobulin levels increased after the stress, whatever the initial inflammatory status. However, fibrinogen levels decreased more in inflamed than non-inflamed rats and albumin levels were not affected by the stress. Independently of the initial inflammatory status, the liver glutathione content was strongly depleted by the stress. These results extend and support our previous findings by demonstrating that age-associated low-grade inflammation does not aggravate sarcopenia in old rats.

Interaction of bifidobacteria with Caco-2 cells-adhesion and impact on expression profiles.

The aim of the present study was to study different strains of bifidobacteria for adhesion to Caco-2 intestinal epithelial cells (IECs) and to test for the mRNA response of these cells following interaction with bifidobacteria. Adhesion was tested at different pH conditions using model epithelia consisting of transwell cultures of fully differentiated Caco-2 cells. Microarrays were used to characterize changes in global expression profiles of Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) and challenged with non-pathogenic Escherichia coli D2241 or four different strains of bifidobacteria. Furthermore, cytokine mRNA of IECs in responses to challenge with Bifidobacterium bifidum S17 or E. coli D2241 was tested in PBMC-sensitised Caco-2 cells using RT-PCR. Bifidobacteria showed strain-specific adhesion to Caco-2. Shift of apical pH from 7 to 4.5 resulted in strain-specific changes of adhesion. Global expression profiles of PBMC-sensitised Caco-2 cells revealed differential expression of a significant number of genes only after challenge with E. coli D2241 while cells were essentially unresponsive to challenge with four strains of bifidobacteria showing different adhesion properties. Using a RT-PCR approach, in the same system a similar differential expression after challenge with E. coli D2241 or B. bifidum S17 was observed for various immune markers. The presented results suggest that Caco-2 cells might be specifically unresponsive to challenge with bifidobacteria irrespective of the level of adhesion.

Anti-inflammatory effects of bifidobacteria by inhibition of LPS-induced NF-kappaB activation.

AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs) in in vitro models both of the non-inflamed and inflamed intestinal epithelium. METHODS: A reporter gene system in HT-29 cells was used to measure levels of NF-kappaB activation after challenge with bifidobacteria or after bacterial pre-treatment following LPS challenge. IL-8 protein and pro-inflammatory gene expression was investigated using normal HT-29 cells. RESULTS: None of the bifidobacteria tested induced activation of nuclear factor kappaB (NF-kappaB) indicating that bifidobacteria themselves do not induce inflammatory events in IECs. However, six out of eight bifidobacteria tested inhibited lipopolysaccharide- (LPS-) induced NF-kappaB activation in a dose- and strain-dependent manner. In contrast, NF-kappaB activation in response to challenge with tumor necrosis factor-alpha (TNF-alpha) was affected by none of the tested bifidobacteria, indicating that the inhibitory effect of bifidobacteria is specific for LPS-induced inflammation in IECs. As shown with two of the six inhibition-positive bifidobacteria, LPS-induced inhibition of NF-kappaB activation was accompanied by a dose-dependent decrease of interleukin 8 (IL-8) secretion and by lower mRNA levels for IL-8, TNF-alpha, cyclooxygenase 2 (Cox-2), and intercellular adhesion molecule 1 (ICAM-1). CONCLUSION: Some strains of bifidobacteria are effective in inhibiting LPS-induced inflammation and thus might be appropriate candidates for probiotic intervention in chronic intestinal inflammation.