Jody Backer

Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins and to assess whether these factors retain their bioactivity when administered to IL-10-deficient mice. B. infantis conditioned medium (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF-alpha and IFN-gamma. Transepithelial resistance (TER), tight junction proteins [claudins 1, 2, 3, and 4, zonula occludens (ZO)-1, and occludin] and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10-deficient mice in Ussing chambers. A separate group of IL-1-deficient mice was treated with BiCM for 4 wk and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF-alpha- and IFN-gamma-induced drops in TER and rearrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF-alpha and IFN-gamma. Oral BiCM administration acutely reduced colonic permeability in mice whereas long-term BiCM treatment in IL-10-deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN-gamma secretion. In conclusion, peptide bioactive factors from B. infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.

UNLABELLED: A breakdown in intestinal barrier function and increased bacterial translocation are key events in the pathogenesis of sepsis and liver disease. Altering gut microflora with noninvasive and immunomodulatory probiotic organisms has been proposed as an adjunctive therapy to reduce the level of bacterial translocation and prevent the onset of sepsis. The purpose of this study was to determine the efficacy of a probiotic compound in attenuating hepatic and intestinal injury in a mouse model of sepsis. Wild-type and interleukin-10 (IL-10) gene-deficient 129 Sv/Ev mice were fed the probiotic compound VSL#3 for 7 days. To induce sepsis, the mice were injected with lipopolysaccharide (LPS) and D-galactosamine (GalN) in the presence and absence of the peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662. The mice were killed after 6 hours, and their colons were removed for the measurement of the cytokine production and epithelial function. The functional permeability was assessed by the mannitol movement and cyclic adenosine monophosphate-dependent chloride secretion in tissue mounted in Ussing chambers. The livers were analyzed for bacterial translocation, cytokine production, histological injury, and PPARgamma levels. The tissue levels of tumor necrosis factor alpha, interferon gamma, IL-6, and IL-12p35 ribonucleic acid were measured by semiquantitative reverse transcription polymerase chain reaction. Mice injected with LPS/GalN demonstrated a breakdown in colonic barrier function, which correlated with enhanced proinflammatory cytokine secretion, bacterial translocation, and significant hepatic injury. A pretreatment with oral probiotics prevented the breakdown in intestinal barrier function, reduced bacterial translocation, and significantly attenuated liver injury. The inhibition of PPARgamma with GW9662 abrogated the protection induced by probiotics. CONCLUSION: Orally administered probiotics prevented liver and intestinal damage in a mouse model of sepsis through a PPARgamma-dependent mechanism.

Colonic epithelial cells are constantly exposed to high levels of bacterial DNA in the intestinal lumen and must recognize and respond appropriately to pathogens, while they maintain a tolerance to nonpathogenic commensal bacterial strains. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9). The aim of this study was to investigate TLR9 expression and localization in colonic epithelial cells under basal conditions and in response to bacterial DNA. HT-29 cells were exposed to DNA from various strains of commensal and pathogenic microbes. TLR9 mRNA expression was determined by real-time reverse transcription-PCR, and interleukin-8 (IL-8) secretion was measured by an enzyme-linked immunosorbent assay. Localization of TLR9 was determined by flow cytometry in HT-29 cells and by immunofluorescence in HT-29 cells and mouse colonic tissue. Immunofluorescence and flow cytometric analyses demonstrated that there was intracellular and surface expression of TLR9 in HT-29 cells under basal conditions. Exposure of cells to DNA from pathogenic strains of Salmonella and Escherichia coli resulted in a significant increase in TLR9 mRNA expression. Salmonella enterica serovar Dublin DNA increased surface TLR9 protein and IL-8 secretion. There was no change in mRNA levels or localization of TLR9 in response to Bifidobacterium breve. Chloroquine did not block IL-8 secretion in response to S. enterica serovar Dublin DNA. TLR9 was expressed on the colonic apical surface in wild-type mice but not in germfree mice. These results demonstrate that intestinal epithelial cells recognize pathogenic bacterial DNA and respond by increasing surface localization and expression of TLR9, suggesting that the epithelial inflammatory response to pathogenic DNA is mediated at least in part by increased TLR9 expression.

Bcl-2, a member of the apoptosis-regulating family of proteins confers a survival advantage on cells by inhibiting apoptosis. Bcl-2 expression is estrogen-responsive and high in various tumors. Overexpression of Bcl-2 has been associated with the loss of contact inhibition, unregulated growth and foci formation in culture. In this study, we have examined the effects of bcl-2 overexpression and expression on cell-cell adhesion in MCF-7 and MDCK epithelial cell lines respectively. Overexpression of Bcl-2 in estrogen receptor-positive MCF-7 mammary carcinoma cells led to decreased cell surface E-cadherin and the disruption of junctional complexes concurrent with intracellular redistribution of their components. Particularly noticeable, was the partial nuclear localization of the tight junction-associated protein ZO-1 which coincided with upregulation of ErbB2. The expression of this EGF co-receptor is regulated by the ZO-1-associated transcription factor ZONAB. Growth in estrogen-depleted media led to downregulation of Bcl-2 expression and upregulation and membrane localization of all junctional proteins. Similar disruption in junctions, accompanied by decreased transepithelial resistance, was observed when Bcl-2 was expressed in MDCK cells. These results strongly suggest that Bcl-2 expression decreases the level of functional E-cadherin thereby interfering with junction formation. The inhibition of junction formation decreases cell-cell adhesion leading to the loss of contact inhibition, which, in vivo, can lead to unregulated growth and tumorigenesis.