Interleukin 15 and CD4⁺ T cells cooperate to promote small intestinal enteropathy in response to dietary antigen.

BACKGROUND & AIMS: CD4(+) T cells specific for dietary gluten and interleukin 15 (IL15) contribute to the pathogenesis of celiac disease. We investigated whether and how they interact to damage the intestine using mice that overexpress human IL15 in the intestinal epithelium and have CD4(+) T cells specific for ovalbumin, a dietary antigen. METHODS: We crossed mice with CD4(+) T cells specific for ovalbumin (OTII) with mice that overexpress human IL15 under an intestine-specific promoter (B6 × IL15Tge). The offspring (OTII × IL15Tge mice) received control or ovalbumin-containing diets until 3 months of age. Enteropathy was monitored by weight, ratio of villous:crypt length, and the number of intestinal lymphocytes. Phenotype, cytokine production, and degranulation of mucosal and spleen lymphocytes were analyzed by multicolor flow cytometry or enzyme-linked immunosorbent assay. Regulatory T-cell function and CD8(+) T-cell activation were analyzed in co-culture assays. RESULTS: Exposure to ovalbumin reduced growth and led to enteropathy in OTII × IL15Tge mice but not in control OTII × B6 littermates. Enteropathy was associated with expansion of mucosal granzyme B(+) CD8(+) T cells, and developed despite increased frequency of functional ovalbumin-specific regulatory T cells. Ovalbumin-activated CD4(+) T cells secreted IL2, which along with IL15 stimulated expansion of noncognate intestinal cytotoxic CD8(+) T cells, which did not respond to regulatory T cells and induced epithelial damage. CONCLUSIONS: We observed that in mice given food antigen, cooperation between IL15 and CD4(+) T cells is necessary and sufficient to activate CD8(+) T cells and damage the small intestine. We propose that this process is involved in the development of celiac disease.

Interleukin-15-dependent NKp46+ innate lymphoid cells control intestinal inflammation by recruiting inflammatory monocytes.

With the goal in mind to define how interleukin-15 (IL-15) contributes to acute intestinal inflammation, we have used a mouse model of ileitis induced by oral infection with Toxoplasma gondii. We observed that a crosstalk between IL-15 and interleukin-18 (IL-18) promoted intestinal recruitment of inflammatory monocytes, where these cells participated in parasite control but also in tissue damage. A stromal source of IL-15 controlled the development of lamina propria NKp46(+)NK1.1(+) cells, whereas IL-18 produced during T. gondii infection stimulated their production of the chemokine CCL3. In turn, CCL3 attracted inflammatory monocytes via their chemokine receptor CCR1, which was indispensable for their recruitment into the inflamed gut. Collectively, these results identify the IL-15-dependent subset of intestinal NKp46(+) cells as an important source of CCL3, which can amplify intestinal inflammation via the recruitment of CCR1(+) inflammatory monocytes. Preliminary evidence suggests that this pathway might operate in Crohn's disease.

Distribution of epicatechin metabolites in lymphoid tissues and testes of young rats with a cocoa-enriched diet.

An increasing number of scientific studies support that flavanol-rich foods and beverages such as cocoa can promote human health, and are beneficial agents for the prevention of some diseases. Our previous studies showed that long-term cocoa intake enhances the antioxidant status in lymphoid organs and also modulates lymphocyte functionality in healthy young rats. Cocoa polyphenolic antioxidants seem to be the best candidates for those effects. However, data regarding polyphenol metabolites in tissues after a long-term cocoa intake are scarce. In the present study we mainly focus on the uptake and accumulation of epicatechin metabolites in lymphoid organs, including the thymus, spleen and mesenteric lymphoid nodes, as well as in the liver and testes after a diet rich in cocoa. Ten young weaned Wistar rats were fed randomly with a 10 % (w/w) cocoa diet or a control diet for 3 weeks, corresponding to their infancy and youth. Tissues were treated with a solid-phase extraction and analysed by liquid chromatography-tandem MS. The major compounds recovered in these tissues were glucuronide derivatives of epicatechin and methylepicatechin. The highest concentration of these metabolites was found in the thymus, testicles and liver, followed by lymphatic nodes and spleen. The high amount of epicatechin metabolites found in the thymus supports our previous findings showing its high antioxidant capacity compared with other tissues such as the spleen. Moreover, this is the first time that epicatechin metabolites have been found in high concentrations in the testes, confirming other studies that have suggested the testes as an important site of oxidation.

Cocoa: antioxidant and immunomodulator.

Cocoa, a product consumed since 600 BC, is now a subject of increasing interest because of its antioxidant properties, which are mainly attributed to the content of flavonoids such as ( - )-epicatechin, catechin and procyanidins. Moreover, recent findings suggest a regulatory effect of cocoa on the immune cells implicated in innate and acquired immunity. Cocoa exerts regulatory activity on the secretion of inflammatory mediators from macrophages and other leucocytes in vitro. In addition, emerging data from in vivo studies support an immunomodulating effect. Long-term cocoa intake in rats affects both intestinal and systemic immune function. Studies in this line suggest that high-dose cocoa intake in young rats favours the T helper 1 (Th1) response and increases intestinal gammadelta T lymphocyte count, whereas the antibody-secreting response decreases. The mechanisms involved in this activity are uncertain; nonetheless, because redox-sensitive pathways control immune cell function, the action of cocoa flavonoids on modulating cell signalling and gene expression deserves investigation.

Influence of a cocoa-enriched diet on specific immune response in ovalbumin-sensitized rats.

Previous studies in young rats have reported the impact of 3 weeks of high cocoa intake on healthy immune status. The present article describes the effects of a longer-term cocoa-enriched diet (9 weeks) on the specific immune response to ovalbumin (OVA) in adult Wistar rats. At 4 weeks after immunization, control rats produced anti-OVA antibodies, which, according their amount and isotype, were arranged as follows: IgG1 > IgG2a > IgM > IgG2b > IgG2c. Both cocoa diets studied (4% and 10%) down-modulated OVA-specific antibody levels of IgG1 (main subclass associated with the Th2 immune response in rats), IgG2a, IgG2c and IgM isotypes. Conversely, cocoa-fed rats presented equal or higher levels of anti-OVA IgG2b antibodies (subclass linked to the Th1 response). Spleen and lymph node cells from OVA-immunized control and cocoa-fed animals proliferated similarly under OVA stimulation. However, spleen cells from cocoa-fed animals showed decreased interleukin-4 secretion (main Th2 cytokine), and lymph node cells from the same rats displayed higher interferon-gamma secretion (main Th1 cytokine). These changes were accompanied by a reduction in the number of anti-OVA IgG-secreting cells in spleen. In conclusion, cocoa diets induced attenuation of antibody synthesis that may be attributable to specific down-regulation of the Th2 immune response.

Neuroprotective effect of cocoa flavonoids on in vitro oxidative stress.

BACKGROUND: Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways. AIM OF THE STUDY: To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line. METHODS: We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis. RESULTS: Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%. CONCLUSIONS: Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation.

Clinical benefit and preservation of flavonols in dark chocolate manufacturing.

The consumption of high-cacao-content chocolate has been associated with positive health benefits ascribed to flavanol [corrected] antioxidants derived from the ground, fermented cocoa seeds of Theobroma cacao. However, flavanols [corrected] impart a bitter, astringent flavor to foodstuffs, frequently masked in chocolates and confections by aggressive processing and adulteration with other flavors. Recent reports have implied that not all varieties of dark chocolate are created equally, and significant caveats exist regarding its potential health benefits. It is perhaps not surprising that extensive processing, dilution, and the addition of flavor modifiers may improve the palatability of chocolate, but could have negative nutritional and clinical benefits. This article examines the chemical composition of chocolate and the clinical data associated with the consumption of flavonoid-rich cocoa. We review the steps in chocolate manufacturing that directly affect the antioxidant levels in chocolate products, and the caveats associated with claims of health benefits from the consumption of dark chocolate.

Intestinal immune system of young rats influenced by cocoa-enriched diet.

Gut-associated lymphoid tissue (GALT) maintains mucosal homeostasis by counteracting pathogens and inducing a state of nonresponsiveness when it receives signals from food antigens and commensal bacteria. We report for the first time the influence of continuous cocoa consumption on GALT function in rats postweaning. Weaned Wistar rats were fed cocoa-enriched diets (4% or 10% food intake) for 3 weeks. The function of the primary inductive sites of GALT, such as Peyer's patches (PP) and mesenteric lymph nodes (MLN), was evaluated through an analysis of IgA-secretory ability and lymphocyte composition (T, B and natural killer cells), activation (IL-2 secretion and IL-2 receptor alpha expression) and proliferation. T-helper effector cell balance was also established based on cytokine profile (interferon gamma, IL-4 and IL-10) after mitogen activation. A 10% cocoa intake induced significant changes in PP and MLN lymphocyte composition and function, whereas a 4% cocoa diet did not cause significant modifications in either tissues. Cocoa diet strongly reduced secretory IgA (S-IgA) in the intestinal lumen, although IgA's secretory ability was only slightly decreased in PP. In addition, the 10% cocoa diet increased T-cell-antigen receptor gammadelta cell proportion in both lymphoid tissues. Thus, cocoa intake modulates intestinal immune responses in young rats, influencing gammadelta T-cells and S-IgA production.

Cocoa-enriched diet enhances antioxidant enzyme activity and modulates lymphocyte composition in thymus from young rats.

Cocoa is a rich source of flavonoids, mainly (-)-epicatechin, (+)-catechin, and procyanidins. This article reports the effect of continuous cocoa intake on antioxidant capacity in plasma and tissues, including lymphoid organs and liver, from young rats. Weaned Wistar rats received natural cocoa (4% or 10% food intake) for three weeks, corresponding to their infancy. Flavonoid absorption was confirmed through the quantification of epicatechin metabolites in urine. Total antioxidant capacity (TAC) and the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase, were examined. Cocoa intake enhanced TAC in all tissues especially in thymus. Moreover, thymus SOD and catalase activities were also dose-dependently increased by cocoa. It was also analyzed whether the enhanced antioxidant system in thymus could influence its cellular composition. An increase in the percentage of thymocytes in advanced development stage was found. In summary, cocoa diet enhances thymus antioxidant defenses and influences thymocyte differentiation.