Polyphenol-rich pomegranate fruit extract (POMx) suppresses PMACI-induced expression of pro-inflammatory cytokines by inhibiting the activation of MAP Kinases and NF-κB in human KU812 cells

<p>Abstract</p> <p>Background</p> <p>Mast cells and basophils are multifunctional effector cells and contain plentiful secretary granules in their cytoplasm. These cell types are involved in several inflammatory and immune events and are known to produce an array of mediators including a broad spectrum of cytokines. Pomegranate fruit is rich in anthocyanins and hydrolysable tannins; a group of polyphenolic compounds shown to be potent antioxidant with anti-inflammatory activity. However, no studies have been undertaken to investigate whether a polyphenol-rich pomegranate fruit extract (POMx) inhibits the inflammatory activity of activated human mast cells and basophils. The aim of this study was to examine whether POMx modulates inflammatory reactions using human basophilic cell line KU812.</p> <p>Methods</p> <p>KU812 cells were stimulated with phorbol-12-myristate 13-acetate plus calcium inophore A23187 (PMACI). The inhibitory effect of POMx on pro-inflammatory cytokine gene expression and production by stimulated KU812 cells was measured by quantitative RT-PCR, and cytokine-specific ELISA assays, respectively. Western blotting was used to analyze the effect of POMx on the activation of mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)-κB in PMACI stimulated KU812 cells. Effect on the activity of NF-κB was determined using Luciferase reporter assay. Significance of differences from control values were analyzed by means of standard statistical methods.</p> <p>Results</p> <p>POMx significantly decreased PMACI stimulated inflammatory gene expression and production of interleukin (IL)-6 and IL-8 in KU812 cells. The inhibitory effect of POMx on the pro-inflammatory cytokines was MAPK subgroups c-jun N-terminal kinase (JNK)- and extracellular-regulated kinase (ERK) dependent. In addition, POMx suppressed the NF-κB activation induced by PMACI by inhibiting IκB-degradation in human basophil cells. POMx also suppressed the powerful induction of NF-κB promoter-mediated luciferase activity in transiently transfected KU812 cells.</p> <p>Conclusion</p> <p>These novel pharmacological actions of POMx provide new suggestion that POMx or POMx-derived compounds may be of therapeutic use for the treatment of inflammatory diseases by suppressing mast cells/basophils activation.</p

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

Specific effects of Bcl10 serine mutations on phosphorylations in canonical and non-canonical pathways of NF-κB activation following carrageenan

To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation

The probiotic Lactobacillus plantarum counteracts TNF-α-induced downregulation of SMCT1 expression and function

The major short-chain fatty acid (SCFA) butyrate is produced in the colonic lumen by bacterial fermentation of dietary fiber. Butyrate serves as primary fuel for the colonocytes and also ameliorates mucosal inflammation. Disturbed energy homeostasis seen in inflamed mucosa of inflammatory bowel disease patients has been attributed to impaired absorption of butyrate. Since sodium-coupled monocarboxylate transporter 1 (SMCT1, SLC5A8) has recently been shown to play a role in Na+-coupled transport of monocarboxylates, including SCFA, such as luminal butyrate, we examined the effects of proinflammatory TNF-α on SMCT1 expression and function and potential anti-inflammatory role of probiotic Lactobacillus species in counteracting the TNF-α effects. Rat intestinal epithelial cell (IEC)-6 or human intestinal Caco-2 cells were treated with TNF-α in the presence or absence of Lactobacilli culture supernatants (CS). TNF-α treatments for 24 h dose-dependently inhibited SMCT1-mediated, Na+-dependent butyrate uptake and SMCT1 mRNA expression in IEC-6 cells and SMCT1 promoter activity in Caco-2 cells. CS of L. plantarum (LP) stimulated Na+-dependent butyrate uptake (2.5-fold, P < 0.05), SMCT1 mRNA expression, and promoter activity. Furthermore, preincubating the cells with LP-CS followed by coincubation with TNF-α significantly attenuated the inhibitory effects of TNF-α on SMCT1 function, expression, and promoter activity. In vivo, oral administration of live LP enhanced SMCT1 mRNA expression in the colonic and ileal tissues of C57BL/6 mice after 24 h. Efficacy of LP or their secreted soluble factors to stimulate SMCT1 expression and function and to counteract the inhibitory effects of TNF-α on butyrate absorption could have potential therapeutic value