Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-kB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-kB activation and IL-8 production in IECs. PAF treatment (5 μM624 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P,0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs

Prolongation of carrageenan-induced inflammation in human colonic epithelial cells by activation of an NFκB-BCL10 loop

Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve tolllike receptor (TLR)4- and B-cell leukemia/lymphoma (BCL)10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, nonradioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48 h and increased RelB for 24 h following withdrawal of carrageenan after 12 h. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB‐BCL10 loop

Endowment Advisory Panels (1976): Memorandum 01

TGF-β1 is an important multifunctional cytokine with numerous protective effects on intestinal mucosa. The influence of TGF-β1 on serotonin transporter (SERT) activity, the critical mechanism regulating the extracellular availability of serotonin (5-HT), is not known. Current studies were designed to examine acute effects of TGF-β1 on SERT. Model human intestinal Caco-2 cells grown as monolayer's or as cysts in 3D culture and ex vivo mouse model were utilized. Treatment of Caco-2 cells with TGF-β1 (10 ng/ml, 60 min) stimulated SERT activity (~2 fold, P<0.005). This stimulation of SERT function was dependent upon activation of TGF-β1 receptor (TGFRI) as SB-431542, a specific TGF-βRI inhibitor blocked the SERT stimulation. SERT activation in response to TGF-β1 was attenuated by inhibition of PI3K and occurred via enhanced recruitment of SERT-GFP to apical surface in a PI3K dependent manner. The exocytosis inhibitor brefeldin A (2.5 μM) attenuated the TGF-β1-mediated increase in SERT function. TGF-β1 increased the association of SERT with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3) and promoted exocytosis of SERT. Caco-2 cells grown as cysts in 3D culture recapitulated the effects of TGF-β1 showing increased luminal staining of SERT. Ussing chamber studies revealed increase in 3H-5-HT uptake in mouse ileum treated ex vivo with TGF-β1 (10 ng/ml, 1h). These data demonstrate a novel mechanism rapidly regulating intestinal SERT via PI3K and STX3. Since decreased SERT is implicated in various gastro-intestinal disorders e.g IBD, IBS and diarrhea, understanding mechanisms stimulating SERT function by TGF-β1 offers a novel therapeutic strategy to treat GI disorders

Lipopolysaccharide-induced activation of NF-κB non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations

Background and Aims: B-cell lymphoma / leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods: Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results: By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 Serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either Serine 138 or Serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions: LPS-induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (Serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions

The Carrageenan Diet: Not Recommended

We read with great interest and concern recent reports of the spread of carrageenan-producing seaweeds in coral reefs in the Bay of Bengal, as well as in Butaritari, Kiribati, and other Pacific atolls [“Seaweed invader elicits angst in India,” P. Bagla, News of the Week, 6 June, p. 1271, and (1)]. The bio-invasion of carrageenan-producing, coral-destroying algae in remote Pacific atolls and marine reserves in the Gulf of Mannar Marine National Park is unfortunately a metaphor for the invasiveness of carrageenan in the Western diet and in other commonly used products. Although carrageenan exposure has been associated with development of inflammation in experimental models for decades (2), its use in processed foods (including infant formula and nutritional supplements) and other manufactured products (including pharmaceuticals, cosmetics, and toothpaste) continues to increase. In view of uncertainty about the safety of consumption of carrageenan-containing products, the efforts to cultivate carrageenan-producing seaweeds seem ill-advised

Carrageenan Reduces Bone Morphogenetic Protein-4 (BMP4) and Activates the Wnt/β-Catenin Pathway in Normal Human Colonocytes

Carrageenans are highly sulfated polysaccharides that are widely used as food additives in the Western diet, in order to improve the texture of processed foods. Although native and degraded carrageenans induce colonic ulcerations, polyps, and colorectal tumors in animal models, very little is known about the effects of carrageenan on human colonocytes. We evaluated effects of λ-carrageenan (λCGN) on the normal human colonocyte cell line NCM460, using a concentration of 1 μg/ml, about less than one tenth the average daily exposure to carrageenan in the Western diet. We measured secreted bone morphogenetic protein-4 (BMP4) in spent media and quantified its expression by quantitative RT-PCR. Wnt-related genes were measured by an oligonucleotide array. Cellular β-catenin was quantified by ELISA. We found a marked decline in secreted BMP4 (P \u3c 0.001) following exposure of NCM460 cells to λCGN for 24 hr. Quantitative RT-PCR for BMP4 transcripts revealed 24% and 45% inhibition of expression on days 2 and 4. cDNA gene expression array of Wnt signaling pathway target genes demonstrated significant changes, including 4.5-fold induction of Wnt 9A and suppression of Dickkopf 3 and RHOU genes. Measurement of β-catenin by ELISA revealed concomitant accumulation with increases of 67.8%, 61.6%, and 73.9% on days 1, 2, and 4, compared to untreated controls. We conclude that treatment of normal human colonocytes with λCGN activated the Wnt/β-Catenin cascade and suppressed the expression and secretion of BMP4, inducing significant changes in cellular pathways that are associated with both sporadic and juvenile polyps. CGN may influence development of intestinal polyps in vivo by these mechanisms