The anticancer mechanisms of exopolysaccharide from Weissella cibaria D-2 on colorectal cancer via apoptosis induction.

Exopolysaccharide (EPS) from Weissella cibaria has been devoted to the study of food industry. However, the anticancer activity of W. cibaria derived EPS has not yet been investigated. In this study, we obtained the EPS from W. cibaria D-2 isolated from the feces of healthy infants and found that D-2-EPS, a homopolysaccharide with porous web like structure, could effectively inhibit the proliferation, migration, invasion and induce cell cycle arrest in G0/G1 phase of colorectal cancer (CRC) cells. In HT-29 tumor xenografts, D-2-EPS significantly retarded tumor growth without obvious cytotoxicity to normal organs. Furthermore, we revealed that D-2-EPS promoted the apoptosis of CRC cells by increasing the levels of Fas, FasL and activating Caspase-8/Caspase-3, indicating that D-2-EPS might induce apoptosis through the extrinsic Fas/FasL pathway. Taken together, the D-2-EPS has the potential to be developed as a nutraceutical or drug to prevent and treat colorectal cancer.

Exopolysaccharide from Weissella confusa J4-1 inhibits colorectal cancer via induction of cell cycle arrest.

Exopolysaccharide (EPS), a bioproduct of lactic acid bacteria (LAB), has various health-promoting biological activities that may be beneficial for cancer therapy. This in vivo and in vitro study aimed to elucidate the anti-colorectal cancer (CRC) capacity of a homopolysaccharide EPS obtained from Weissella confusa J4-1 (EPSJ4-1) isolated from the faeces of healthy infants. We confirmed that EPSJ4-1 contained glucose and effectively suppressed the proliferation, migration, and invasion of CRC cells. EPSJ4-1 treatment significantly retarded the growth of HT-29 tumour xenografts without causing cytotoxicity to normal organs. EPSJ4-1 exerts an inhibitory effect on cell proliferation by inducing G0/G1 phase cell cycle arrest in CRC cells. Furthermore, EPSJ4-1 upregulated p21 levels and downregulated mutant p53 and cyclin kinase 2 levels. This is the first study to demonstrate the antitumour effects of EPS from W. confusa on CRC via cell cycle arrest and inhibition of cell migration and invasion, suggesting that EPSJ4-1 has the potential to be developed as a nutraceutical or pharmaceutical drug to prevent and treat CRC.

An Efficient Strategy Based on Liquid-Liquid Extraction with Three-Phase Solvent System and High Speed Counter-Current Chromatography for Rapid Enrichment and Separation of Epimers of Minor Bufadienolide from Toad Meat.

This study presents an efficient strategy based on liquid-liquid extraction with three-phase solvent system and high speed counter-current chromatography for rapid enrichment and separation of epimers of minor bufadienolide from toad meat. The reflux extraction conditions were optimized by response surface methodology first, and a novel three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water (3:6:5:5, v/v) was developed for liquid-liquid extraction of the crude extract. This integrative extraction process could enrich minor bufadienolide from complex matrix efficiently and minimize the loss of minor targets induced by repeated extraction with different kinds of organic solvents occurring in the classical liquid two-phase extraction. As a result, four epimers of minor bufadienolide were greatly enriched in the middle phase and total content of these epimers of minor bufadienolide was increased from 3.25% to 46.23%. Then, the enriched four epimers were separated by HSCCC with a two-phase solvent system composed of chloroform/methanol/water (4:2:2, v/v) successfully. Furthermore, we tested Na+,K+-ATPase (NKA) inhibitory effect of the four epimers. 3ß-Isomers of bufadienolide showed stronger (>8-fold) inhibitory activity than 3α-isomers. The characterization of minor bufadienolide in toad meat and their significant difference of inhibitory effect on NKA would promote the further quantitative analysis and safety evaluation of toad meat as a food source.

Resistin-like molecule α promotes pathogenic Th17 cell responses and bacterial-induced intestinal inflammation.

Resistin-like molecule (RELM)α belongs to a family of secreted mammalian proteins that have putative immunomodulatory functions. Recent studies have identified a pathogenic role for RELMα in chemically induced colitis through effects on innate cell populations. However, whether RELMα regulates intestinal adaptive immunity to enteric pathogens is unknown. In this study, we employed Citrobacter rodentium as a physiologic model of pathogenic Escherichia coli-induced diarrheal disease, colitis, and Th17 cell responses. In response to Citrobacter, RELMα expression was induced in intestinal epithelial cells, infiltrating macrophages, and eosinophils of the infected colons. Citrobacter-infected RELMα(-/-) mice exhibited reduced infection-induced intestinal inflammation, characterized by decreased leukocyte recruitment to the colons and reduced immune cell activation compared with wild-type (WT) mice. Interestingly, Citrobacter colonization and clearance were unaffected in RELMα(-/-) mice, suggesting that the immune stimulatory effects of RELMα following Citrobacter infection were pathologic rather than host-protective. Furthermore, infected RELMα(-/-) mice exhibited decreased CD4(+) T cell expression of the proinflammatory cytokine IL-17A. To directly test whether RELMα promoted Citrobacter-induced intestinal inflammation via IL-17A, infected WT and IL-17A(-/-) mice were treated with rRELMα. RELMα treatment of Citrobacter-infected WT mice exacerbated intestinal inflammation and IL-17A expression whereas IL-17A(-/-) mice were protected from RELMα-induced intestinal inflammation. Finally, infected RELMα(-/-) mice exhibited reduced levels of serum IL-23p19 compared with WT mice, and RELMα(-/-) peritoneal macrophages showed deficient IL-23p19 induction. Taken together, these data identify a proinflammatory role for RELMα in bacterial-induced colitis and suggest that the IL-23/Th17 axis is a critical mediator of RELMα-induced inflammation.

Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.

Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) alpha/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-alpha-deficient (Retnla(-/-)) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-alpha. After challenge with Schistosoma mansoni (Sm) eggs, Retnla(-/-) mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg-challenged Retnla(-/-) mice exhibited elevated expression of pathogen-specific CD4(+) T cell-derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM-alpha could bind to macrophages and effector CD4(+) Th2 cells and inhibited Th2 cytokine production in a Bruton's tyrosine kinase-dependent manner. Additionally, Retnla(-/-) AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM-alpha in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4(+) T cell responses.

TSLP regulates intestinal immunity and inflammation in mouse models of helminth infection and colitis.

Intestinal epithelial cells (IECs) produce thymic stromal lymphopoietin (TSLP); however, the in vivo influence of TSLP-TSLP receptor (TSLPR) interactions on immunity and inflammation in the intestine remains unclear. We show that TSLP-TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. Monoclonal antibody-mediated neutralization of TSLP or deletion of the TSLPR in normally resistant mice resulted in defective expression of Th2 cytokines and persistent infection. Susceptibility was accompanied by elevated expression of interleukin (IL) 12/23p40, interferon (IFN) gamma, and IL-17A, and development of severe intestinal inflammation. Critically, neutralization of IFN-gamma in Trichuris-infected TSLPR(-/-) mice restored Th2 cytokine responses and resulted in worm expulsion, providing the first demonstration of TSLPR-independent pathways for Th2 cytokine production. Additionally, TSLPR(-/-) mice displayed elevated production of IL-12/23p40 and IFN-gamma, and developed heightened intestinal inflammation upon exposure to dextran sodium sulfate, demonstrating a previously unrecognized immunoregulatory role for TSLP in a mouse model of inflammatory bowel disease.

Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine.

Alterations in the composition of intestinal commensal bacteria are associated with enhanced susceptibility to multiple inflammatory diseases, including those conditions associated with interleukin (IL)-17-producing CD4(+) T helper (Th17) cells. However, the relationship between commensal bacteria and the expression of proinflammatory cytokines remains unclear. Using germ-free mice, we show that the frequency of Th17 cells in the large intestine is significantly elevated in the absence of commensal bacteria. Commensal-dependent expression of the IL-17 family member IL-25 (IL-17E) by intestinal epithelial cells limits the expansion of Th17 cells in the intestine by inhibiting expression of macrophage-derived IL-23. We propose that acquisition of, or alterations in, commensal bacteria influences intestinal immune homeostasis via direct regulation of the IL-25-IL-23-IL-17 axis.

Goblet cell-derived resistin-like molecule beta augments CD4+ T cell production of IFN-gamma and infection-induced intestinal inflammation.

The secreted goblet cell-derived protein resistin-like molecule beta (RELMbeta) has been implicated in divergent functions, including a direct effector function against parasitic helminths and a pathogenic function in promoting inflammation in models of colitis and ileitis. However, whether RELMbeta influences CD4(+) T cell responses in the intestine is unknown. Using a natural model of intestinal inflammation induced by chronic infection with gastrointestinal helminth Trichuris muris, we identify dual functions for RELMbeta in augmenting CD4(+) Th1 cell responses and promoting infection-induced intestinal inflammation. Following exposure to low-dose Trichuris, wild-type C57BL/6 mice exhibit persistent infection associated with robust IFN-gamma production and intestinal inflammation. In contrast, infected RELMbeta(-/-) mice exhibited a significantly reduced expression of parasite-specific CD4(+) T cell-derived IFN-gamma and TNF-alpha and failed to develop Trichuris-induced intestinal inflammation. In in vitro T cell differentiation assays, recombinant RELMbeta activated macrophages to express MHC class II and secrete IL-12/23p40 and enhanced their ability to mediate Ag-specific IFN-gamma expression in CD4(+) T cells. Taken together, these data suggest that goblet cell-macrophage cross-talk, mediated in part by RELMbeta, can promote adaptive CD4(+) T cell responses and chronic inflammation following intestinal helminth infection.

Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis.

Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic microorganisms in the gastrointestinal (GI) tract, but the influence of IECs on the development and regulation of immunity to infection is unknown. Here we show that IEC-intrinsic IkappaB kinase (IKK)-beta-dependent gene expression is a critical regulator of responses of dendritic cells and CD4+ T cells in the GI tract. Mice with an IEC-specific deletion of IKK-beta show a reduced expression of the epithelial-cell-restricted cytokine thymic stromal lymphopoietin in the intestine and, after infection with the gut-dwelling parasite Trichuris, fail to develop a pathogen-specific CD4+ T helper type 2 (T(H)2) response and are unable to eradicate infection. Further, these animals show exacerbated production of dendritic-cell-derived interleukin-12/23p40 and tumour necrosis factor-alpha, increased levels of CD4+ T-cell-derived interferon-gamma and interleukin-17, and develop severe intestinal inflammation. Blockade of proinflammatory cytokines during Trichuris infection ablates the requirement for IKK-beta in IECs to promote CD4+ T(H)2 cell-dependent immunity, identifying an essential function for IECs in tissue-specific conditioning of dendritic cells and limiting type 1 cytokine production in the GI tract. These results indicate that the balance of IKK-beta-dependent gene expression in the intestinal epithelium is crucial in intestinal immune homeostasis by promoting mucosal immunity and limiting chronic inflammation.