A diet enriched with cocoa prevents IgE synthesis in a rat allergy model.

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF)-α and the interleukin (IL)-10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.

Effect of a cocoa flavonoid-enriched diet on experimental autoimmune arthritis.

Previously we established that a cocoa-enriched diet in young rats reduces specific antibody production and the T helper (Th) lymphocyte proportion in lymphoid tissues. The aim of the present study was to ascertain the modulatory ability of a cocoa flavonoid-enriched diet on collagen-induced arthritis (CIA), which is mediated by anti-collagen autoantibody response and Th lymphocyte activation. Female Louvain (LOU) rats were fed with a cocoa-enriched diet, beginning 2 weeks before CIA induction. Hind-paw swelling and serum cytokine and anti-collagen antibody concentrations were determined. Anti-collagen antibody-secreting cell counts and lymphocyte subset proportions were established in inguinal lymph nodes (ILN). Reactive oxygen species (ROS), nitric oxide (NO) and TNFα produced by peritoneal macrophages were determined. Although arthritic cocoa-fed rats showed a similar hind-paw swelling time course as the arthritic animals fed a standard diet, the cocoa intake was able to decrease specific IgG2a, IgG2b and IgG2c titres. Moreover, cocoa intake in CIA rats reduced ROS production, TNFα and NO release from peritoneal macrophages, and decreased the Th:cytotoxic T cell ratio in ILN. In conclusion, a cocoa flavonoid-enriched diet in LOU rats with CIA produced no effect on hind-paw swelling but was able to modulate the specific antibody response and also the Th lymphocyte proportion, as well as the synthesis of pro-inflammatory mediators from peritoneal macrophages. Therefore, a cocoa-enriched diet could be a good adjuvant therapy in disorders with oxidative stress or autoimmune pathogenesis.

Effect of cocoa-enriched diets on lymphocytes involved in adjuvant arthritis in rats.

Cocoa and its flavonoids have potential anti-inflammatory properties in vitro and in acute inflammation models in vivo. The aim of the present study was to ascertain the effects of two cocoa-enriched diets on adjuvant arthritis (AA) in rats, considering not only clinical and biochemical inflammatory indices, but also antibody response and lymphocyte composition. Female Wistar rats were fed with a 5 or 10 % cocoa-enriched diet beginning 2 weeks before arthritis induction and until the end of the study. AA was induced by an intradermal injection of heat-killed Mycobacterium butyricum suspension. The hind-paw swelling (plethysmometry), serum anti-mycobacterial antibody concentration (ELISA), blood and inguinal lymph node lymphocyte subset percentage (flow cytometry), and IL-2, interferon γ and PGE2 released from splenocytes (ELISA) were assessed. Although the cocoa diets had no significant effect on hind-paw swelling, a tendency to reduce it was observed at the end of the study. Cocoa-enriched diets were able to decrease the serum anti-mycobacterial antibody concentration and the splenocyte PGE2 production, as well as the proportion of T-helper (Th) lymphocytes in blood and regional lymph nodes, which probably includes cells responsible for the arthritic process. The cocoa diets prevented a decrease in the proportion of regulatory T-cells in blood and a disequilibrium between inguinal lymph node natural killer (NK) CD8⁺ and NK CD8⁻ subsets. In conclusion, the cocoa-enriched diets during AA were not able to significantly decrease joint inflammation but modified Th-cell proportions and prevented specific antibody synthesis.

The suckling rat as a model for immunonutrition studies in early life.

Diet plays a crucial role in maintaining optimal immune function. Research demonstrates the immunomodulatory properties and mechanisms of particular nutrients; however, these aspects are studied less in early life, when diet may exert an important role in the immune development of the neonate. Besides the limited data from epidemiological and human interventional trials in early life, animal models hold the key to increase the current knowledge about this interaction in this particular period. This paper reports the potential of the suckling rat as a model for immunonutrition studies in early life. In particular, it describes the main changes in the systemic and mucosal immune system development during rat suckling and allows some of these elements to be established as target biomarkers for studying the influence of particular nutrients. Different approaches to evaluate these immune effects, including the manipulation of the maternal diet during gestation and/or lactation or feeding the nutrient directly to the pups, are also described in detail. In summary, this paper provides investigators with useful tools for better designing experimental approaches focused on nutrition in early life for programming and immune development by using the suckling rat as a model.

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

BACKGROUND: Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. RESULTS: The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. CONCLUSIONS: Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Premature delivery influences the immunological composition of colostrum and transitional and mature human milk.

Human breast milk is the ideal nutrition for the newborn, and in addition to its nutritional contribution, necessary for infant growth and development, it contains various immune bioactive factors that confer some of the numerous beneficial effects of breastfeeding. The current study analyzed the concentrations of IgA, growth factors such as epidermal growth factor (EGF), TGFß1, and TGFß2, cytokines IL-6, IL-8, IL-10, IL-13, and TNFα, and TNF-receptor I (TNF-RI) in colostrum and transitional and mature milk from mothers with mature, premature, and very premature infants. Human milk samples were collected from mothers delivering at term (T), preterm (PT), and very preterm (VPT). Milk from all the mothers was collected at 3 different time points after delivery corresponding to colostrum and transitional and mature milk. After obtaining milk whey, IgA, EGF, TGFß1, and TGFß2 were determined by ELISA and IL-6, IL-8, IL-10, IL-13, TNFα and TNF-RI by cytometric bead array immunoassay. The colostrum of the PT group was extremely rich in most of the factors studied, but higher concentrations than in the T group were only found for IL-6 (P = 0.051), TGFß1, and TGFß2 (P < 0.05). Conversely, the colostrum of the VPT group had lower concentrations of IgA, IL-8, IL-10, and TNFα than those in the T group (P < 0.05). Results suggest that maternal lactogenic compensatory mechanisms accelerating the development of immature breast-fed preterm infants may take effect only after wk 30 of gestation.

Long-term feeding of the cis-9,trans-11 isomer of conjugated linoleic acid reinforces the specific immune response in rats.

Several effects on the immune system have been ascribed to the cis9,trans11 conjugated linoleic acid (CLA) isomer. We studied whether feeding a diet enriched with an 80:20 CLA isomer mix of cis9,trans11 and trans10,cis12 CLA from gestation to adulthood affects the capacity of adult rats to achieve a specific immune response. Pregnant Wistar rats were fed a 1% CLA diet or a control diet beginning on d 7 of gestation. Weaned pups received the same diet as dams until they were 15 wk old. Rats from both groups were immunized with ovalbumin (OVA) when they were 9 wk old. Dietary CLA enhanced splenocyte OVA-specific proliferation by approximately 50% (P < 0.05) and decreased the mitogen-induced proliferative responses of these cells by approximately 10-20% (P < 0.05). The diminished splenocyte proliferative response was accompanied by a lower interleukin-2 secretion (P < 0.05). Long-term CLA supplementation did not increase serum, spleen, or mesenteric lymph node production of OVA-specific antibodies (Ab) or the number of spleen anti-OVA Ab-secreting cells. Interestingly, dietary CLA increased intestinal anti-OVA IgA production by approximately 75% (P < 0.05). In conclusion, a 1% CLA diet administered from gestation to adulthood enhanced specific systemic cell-mediated immunity as well as the mucosal IgA immune response, whereas it downregulated the polyclonal activation of the immune system. These data support the long-term effects of dietary cis9,trans11 CLA isomer on the immune system.

Higher immunoglobulin production in conjugated linoleic acid-supplemented rats during gestation and suckling.

Conjugated linoleic acid (CLA) has been reported to exert beneficial physiological effects on body composition and the immune system. However, little information is available on the influence of CLA on immune function during early life periods. The present study evaluates the effect of feeding an 80:20 mixture of cis-9, trans-11- and trans-10, cis-12-CLA isomers during gestation and suckling on the systemic immune response of weaned Wistar rats. Pups received dietary CLA from dams through the placental barrier and during suckling by breast milk (group A) or by oral administration (group B). Pups from group C only received CLA during suckling by oral administration. Group D constituted the reference group. Milk from dams fed the CLA diet had a high content of CLA and higher IgA and IgG concentrations than rats fed the standard diet. The plasma of pups from groups A, B and C showed six, twelve and nine times higher content of the cis-9, trans-11-CLA isomer than that of the group D pups. Rats from group A exhibited higher serum IgG concentrations than rats from the rest of the groups (22.14 (SEM 2.14) v. about 5 mg/ml; P < 0.05), whereas rats from groups A and B showed approximately 2-fold higher splenocyte IgM production than rats from groups C and D. However, CLA supplementation did not influence significantly the splenocyte proliferative response or cytokine secretion. Supplementation during gestation and suckling with an 80:20 cis-9, trans-11-trans-10, cis-12 CLA mix enhances the production of the main in vivo and in vitro Ig isotypes in Wistar rats.

Intestinal intraepithelial NK and NKT cell ontogeny in Lewis rats.

Natural killer T (NKT) cells have been described in the liver and spleen of adult rats, but their presence and function in other tissues and in early life remains uncertain. This study was designed to determine the proportion of NK cells and NKT cells among small intestine intraepithelial (IE) lymphocytes in suckling rats and adult animals by flow cytometry. Very few intestinal IE-NKT cells (NKR-P1A+ TCRalphabeta+) were present in adult rats ( approximately 1%), but a high proportion of this population was found during early life ( approximately 40% of IE lymphocytes in 9-day-old rats), with a marked age-decreasing pattern. Most of these cells presented the CD8alphabeta+ phenotype. Intestinal IE-NK cells (NKR-P1A+ TCRalphabeta-) were also present in a relatively high proportion during the suckling period ( approximately 30% of IE lymphocytes). Thus, a predominance of both NK and NKT cell subpopulations in small intestine epithelium is characteristic in the early life of rats and may have a protective role during the suckling period.

Supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea.

Group A rotaviruses (RV) are the most common causative agents of acute gastroenteritis in children <2 y. The present study was designed to establish the effect of a bovine whey protein concentrate (WPC) in a RV infection model in suckling rats. From d 3 of life, suckling Lewis rats received daily supplements of WPC, WPC plus lactoferrin (LF), standard infant formula (SIF), or water (RV-infected group and an untreated, uninfected reference group). On d 8 of life, heterologous simian RV SA-11 was inoculated orally in the WPC-RV, WPC+LF-RV, SIF-RV, and RV groups. WPC and WPC+LF reduced diarrhea incidence from approximately 90% in RV group to approximately 60% in WPC-RV and WPC+LF-RV groups (P < 0.05), whereas the area under the curve (AUC) of severity along time diminished from approximately 10 AUC in the RV group to approximately 6 AUC in both supplemented groups (P < 0.05). Serum levels of anti-RV antibodies, splenocyte proliferation, and interferon-gamma secretion after specific stimulation were significantly lower in the WPC-RV and WPC+LF-RV groups than in the SIF-RV and RV groups. In the intraepithelial intestinal compartment, RV infection increased the proportion of typical mucosal T cells (IE-T CD8alphaalpha+); however, this modification was controlled by WPC and WPC+LF supplementation. In general, for most of the parameters studied, the SIF-RV and RV groups did not differ. In summary, daily supplementation with WPC or WPC+LF in early life considerably reduces the severity of RV-induced acute gastroenteritis and modulates the immune response against the pathogen.

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.

Phenotypic and functional characteristics of rat spleen lymphocytes during suckling.

The aim of this study is to characterize the rat spleen lymphoid tissue during the suckling period by means of lymphocyte composition and their functionality. Lymphocyte phenotype was determined by immunofluorescence and flow cytometry. The proliferative ability and the antibody secretion activity were considered as functional markers. During the first 2 weeks of life, rat spleen mainly contained B cells (CD45RA+ or Igkappa+). In this period, T (TCRalphabeta+CD4+, TCRalphabeta+CD8+ and TCRgammadelta+CD8+) and NK/NKT (NKR-P1A+) cell proportions were far less than those of adult rats. Moreover, the spleen immune functionality proved to be very low. In the second half of the suckling period, CD4+ and CD8+ cells in the spleen increased in number and proportion, with immature cells progressively displaced by phenotypic mature lymphocytes containing CD3, TCRalphabeta, CD5 and CD2 molecules on their surface. Additionally, although B and T lymphocyte developed their proliferative ability during this period, it was not fully developed at weaning.

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.

Flavonoids from Theobroma cacao down-regulate inflammatory mediators.

In the present study, we report the effects of a cocoa extract on the secretion and RNA expression of various proinflammatory mediators by macrophages. Monocyte chemoattractant protein 1 and tumor necrosis factor alpha (TNFalpha) were significantly and dose-dependently diminished by cocoa extract, and this effect was higher than that produced by equivalent concentrations of epicatechin but was lower than that produced by isoquercitrin. Interestingly, cocoa extract added prior to cell activation resulted in a significantly greater inhibition of TNFalpha secretion. Both cocoa extract and epicatechin decreased TNFalpha, interleukin (IL) 1alpha, and IL-6 mRNA expression, suggesting that their inhibitory effect on cytokine secretion is produced, in part, at the transcriptional level. Cocoa extract also significantly decreased NO secretion in a dose-dependent manner and with a greater effect than that produced by epicatechin. In conclusion, our study shows that cocoa flavonoids not only inhibit NO release from macrophages but also down-regulate inflammatory cytokines and chemokines.

Motor activity as an unbiased variable to assess anaphylaxis in allergic rats.

The release of mediators by mast cells triggers allergic symptoms involving various physiological systems and, in the most severe cases, the development of anaphylactic shock compromising mainly the nervous and cardiovascular systems. We aimed to establish variables to objectively study the anaphylactic response (AR) after an oral challenge in an allergy model. Brown Norway rats were immunized by intraperitoneal injection of ovalbumin with alum and toxin from Bordetella pertussis. Specific immunoglobulin (Ig) E antibodies were developed in immunized animals. Forty days after immunization, the rats were orally challenged with the allergen, and motor activity, body temperature and serum mast cell protease concentration were determined. The anaphylaxis induced a reduction in body temperature and a decrease in the number of animal movements, which was inversely correlated with serum mast cell protease release. In summary, motor activity is a reliable tool for assessing AR and also an unbiased method for screening new anti-allergic drugs.

Alterations of motor activity circadian rhythm in rats with adjuvant arthritis.

Adjuvant arthritis in rats produces alterations in the motor activity circadian rhythm. Specifically arthritic animals show a decrease in the total daily motor activity and an advance in the acrophase of the rhythm. Slight changes are also observed in the power content of the circadian harmonic as well as in the amplitude.

CD4 expression decrease by antisense oligonucleotides: inhibition of rat T CD4+ cell reactivity.

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

Cocoa flavonoid-enriched diet modulates systemic and intestinal immunoglobulin synthesis in adult Lewis rats.

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

The effects of cocoa on the immune system.

Cocoa is a food relatively rich in polyphenols, which makes it a potent antioxidant. Due to its activity as an antioxidant, as well as through other mechanisms, cocoa consumption has been reported to be beneficial for cardiovascular health, brain functions, and cancer prevention. Furthermore, cocoa influences the immune system, in particular the inflammatory innate response and the systemic and intestinal adaptive immune response. Preclinical studies have demonstrated that a cocoa-enriched diet modifies T cell functions that conduce to a modulation of the synthesis of systemic and gut antibodies. In this regard, it seems that a cocoa diet in rats produces changes in the lymphocyte composition of secondary lymphoid tissues and the cytokines secreted by T cells. These results suggest that it is possible that cocoa could inhibit the function of T helper type 2 cells, and in line with this, the preventive effect of cocoa on IgE synthesis in a rat allergy model has been reported, which opens up new perspectives when considering the beneficial effects of cocoa compounds. On the other hand, cocoa intake modifies the functionality of gut-associated lymphoid tissue by means of modulating IgA secretion and intestinal microbiota. The mechanisms involved in these influences are discussed here. Further research may elucidate the cocoa compounds involved in such an effect and also the possible medical approaches to these repercussions.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake.

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RARα and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL­6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RARα and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RARα and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.