Immunoregulatory Mechanisms Underlying Prevention of Colitis-Associated Colorectal Cancer by Probiotic Bacteria

Background: Inflammatory bowel disease (IBD) increases the risk of colorectal cancer. Probiotic bacteria produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anticarcinogenic effects. This study aimed to investigate the cellular and molecular mechanisms underlying the efficacy of probiotic bacteria in mouse models of cancer. Methodology/Principal Findings: The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in mouse models of inflammation-driven colorectal cancer. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen and colonic lamina propria lymphocytes (LPL) were phenotypically and functionally characterized. Mice treated with CLA or VSL#3 recovered faster from the acute inflammatory phase of disease and had lower disease severity in the chronic, tumor-bearing phase of disease. Adenoma and adenocarcinoma formation was also diminished by both treatments. VSL#3 increased the mRNA expression of TNF-a, angiostatin and PPAR c whereas CLA decreased COX-2 levels. Moreover, VSL#3-treated mice had increased IL-17 expression in MLN CD4+ T cells and accumulation of Treg LPL and memory CD4+ T cells. Conclusions/Significance: Both CLA and VSL#3 suppressed colon carcinogenesis, although VSL#3 showed greater anticarcinogeni

IL-3 and oncogenic Abl regulate the myeloblast transcriptome by altering mRNA stability

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. The degree to which IL-3 acts at the posttranscriptional level is largely unknown. We have conducted global mRNA decay profiling and bioinformatic analyses in 32Dcl3 myeloblasts indicating that IL-3 caused immediate early stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Stabilized transcripts were enriched for AU-Response elements (AREs), and an ARE-containing domain from the interleukin-6 (IL-6) 3′-UTR rendered a heterologous gene responsive to IL-3-mediated transcript stabilization. Many IL-3-stabilized transcripts had been associated with leukemic transformation. Deregulated Abl kinase shared with IL-3 the ability to delay turnover of transcripts involved in proliferation or differentiation blockade, relying, in part, on signaling through the Mek/ Erk pathway. These findings support a model of IL-3 action through mRNA stability control and suggest that aberrant stabilization of an mRNA network linked to IL-3 contributes to leukemic cell growth. © 2009 Ernst et al

Probiotic Bacteria Produce Conjugated Linoleic Acid Locally in the Gut That Targets Macrophage PPAR γ to Suppress Colitis

Inflammatory bowel disease (IBD) therapies are modestly successful and associated with significant side effects. Thus, the investigation of novel approaches to prevent colitis is important. Probiotic bacteria can produce immunoregulatory metabolites in vitro such as conjugated linoleic acid (CLA), a polyunsaturated fatty acid with potent anti-inflammatory effects. This study aimed to investigate the cellular and molecular mechanisms underlying the anti-inflammatory efficacy of probiotic bacteria using a mouse model of colitis. The immune modulatory mechanisms of VSL#3 probiotic bacteria and CLA were investigated in a mouse model of DSS colitis. Colonic specimens were collected for histopathology, gene expression and flow cytometry analyses. Immune cell subsets in the mesenteric lymph nodes (MLN), spleen, blood and colonic lamina propria cells were phenotypically and functionally characterized. Fecal samples and colonic contents were collected to determine the effect of VSL#3 and CLA on gut microbial diversity and CLA production. CLA and VSL#3 treatment ameliorated colitis and decreased colonic bacterial diversity, a finding that correlated with decreased gut pathology. Colonic CLA concentrations were increased in response to probiotic bacterial treatment, but without systemic distribution in blood. VSL#3 and CLA decreased macrophage accumulation in the MLN of mice with DSS colitis. The loss of PPAR γ in myeloid cells abrogated the protective effect of probiotic bacteria and CLA in mice with DSS colitis. Probiotic bacteria modulate gut microbial diversity and favor local production of CLA in the colon that targets myeloid cell PPAR γ to suppress colitis

Intestinal Epithelial Serum Amyloid A Modulates Bacterial Growth In Vitro and Pro-Inflammatory Responses in Mouse Experimental Colitis

<p>Abstract</p> <p>Background</p> <p>Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis.</p> <p>Methods</p> <p>Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live <it>Escherichia coli</it>.</p> <p>Results</p> <p>Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured <it>E. coli</it>. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls.</p> <p>Conclusions</p> <p>Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..</p

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation

Activator protein-1 signalling pathway and apoptosis are modulated by poly(ADP-ribose) polymerase-1 in experimental colitis

Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA injury in the nucleus of eukaryotic cells and has been implicated in intestinal barrier dysfunction during inflammatory bowel diseases. In this study we investigated whether PARP-1 may regulate the inflammatory response of experimental colitis at the level of signal transduction mechanisms. Mice genetically deficient of PARP-1 (PARP-1(−/−)) and wild-type littermates were subjected to rectal instillation of trinitrobenzene sulphonic acid (TNBS). Signs of inflammation were monitored for 14 days. In wild-type mice, TNBS treatment resulted in colonic ulceration and marked apoptosis, which was associated with decreased colon content of the antiapoptotic protein Bcl-2, whereas the proapoptotic Bax was unchanged. Elevated levels of plasma nitrate/nitrite, metabolites of nitric oxide (NO), were also found. These inflammatory events were associated with activation of c-Jun-NH(2) terminal kinase (JNK), phosphorylation of c-Jun and activation of the nuclear transcription factor activator protein-1 (AP-1) in the colon. In contrast, PARP-1(−/−) mice exhibited a significant reduction of colon damage and apoptosis, which was associated with increased colonic expression of Bcl-2 and lower levels of plasma nitrate/nitrite when compared to wild-type mice. Amelioration of colon damage was associated with a significant reduction of the activation of JNK and reduction of the DNA binding of AP-1. The data indicate that PARP-1 exerts a pathological role in colitis possibly by regulating the early stress-related transcriptional response through a positive modulation of the AP-1 and JNK pathways

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation