It was demonstrated that Wistar rats receiving a 10% cocoa diet for 3 weeks accumulate cocoa polyphenol metabolites in immune tissues, such as the thymus, lymph nodes, and spleen (28). In particular, the highest accumulation was in the thymus, where phenotypic changes were found due to the diet. In particular, cocoa intake resulted in an enhancement of the progression of immature thymocytes (those with low expression of the αβ T-cell receptor—TCR αβ⁻, and expressing or non-expressing the clusters of differentiation CD4 and CD8, i.e., TCRαβ^lowCD4⁻CD8⁻ or TCRαβ^lowCD4⁺CD8⁺) toward more mature stages (TCRαβ^highCD4⁺CD8⁻) (29) (Table 2). In spite of this increase in CD4⁺ (Th) cells in the thymus, the analysis of a secondary lymphoid tissue, such as the spleen, revealed that a 10% cocoa diet in young rats for 3 weeks increased the proportion of spleen B cells and decreased that of Th lymphocytes (18) (Table 2).

Lymph nodes were also affected by a cocoa diet. In particular, in mesenteric lymph nodes, a cocoa-enriched diet for 3 or 4 weeks in rats increased the proportion of innate cytotoxic lymphocytes, such as cells expressing γδ T-cell receptor (TCRγδ⁺) and NK cells, and also that of the Tc lymphocytes and B cells, whereas the proportion of Th cells decreased (30, 31) (Table 2). These effects were only produced by a 10% cocoa diet whereas a 4% cocoa dose was insufficient to influence the phenotype of mesenteric lymph nodes (30). Similarly, the intake of a 10% cocoa-enriched diet given to rats for 6 weeks decreased the proportion of TCRαβ⁺ cells but did not modify that of regulatory T cells (Treg) in inguinal lymph nodes in rats (32) (Table 2).

A more in-depth analysis of lymphocytes in mesenteric lymph nodes revealed that the increase of TCRγδ⁺ cells was attributed to the presence of a higher amount of CD8αα⁺ cells, a typical intestinal phenotype, which could be due to the migration of this cellular type from the intestine (34). The increase of Tc cells in mesenteric lymph nodes was accompanied by a higher proportion of activated cells (CD25⁺CD8⁺ cells) and cells expressing the αE-integrin (CD103⁺CD8⁺ cells) and a lower proportion of cells bearing L-selectin (CD62L⁺CD8⁺ cells) (31) (Table 2). CD103 is a subunit of the αE-integrin that can mediate cell adhesion and migration to the gut (35), whereas L-selectin is involved in lymphocyte rolling on the endothelium and the homing to secondary lymphoid tissues (36). These results could mean that the cocoa diet decreased the arrival of blood lymphocytes to mesenteric lymph nodes whereas it may favor intestinal cells entering. As cocoa compounds can reach the small intestine and even the colon (37, 38), they can affect the intestinal lymphocytes and promote their migration to mesenteric lymph nodes.

Overall, the increased proportion of CD8αα⁺TCRγδ⁺ cells, NK cells, and CD103⁺ Tc cells in mesenteric lymph nodes could be involved in cocoa’s influence on antibody immune response. TCRγδ⁺ cells have been associated with an attenuating effect on the synthesis of antibodies (39), and NK cells could also contribute to the regulation of antibody synthesis (40). Moreover, CD103⁺ cells have been associated with a regulatory function given that their proportion increased after treatment with immunosuppressive agents (41).

After feeding a cocoa-enriched diet, cocoa flavonoid metabolites are stored in the lymphoid tissues (thymus, lymph nodes, and spleen) as well as in the liver. In fact, epicatechin metabolites have been reported to be accumulated in concentrations twofold higher in the thymus, testes, and liver than in lymph nodes and spleen (28). With regard to the liver, the 10% cocoa intake in rats enhanced hepatic antioxidant capacity, without modifying hepatic superoxide dismutase and catalase activities (29).

The development of the acquired immune response implies the involvement of complex interactions between immune cells by means of particular surface molecules and the secretion of cytokines. The gene or protein expression of those molecules involved in the immune synapses, as well as cytokines and other molecules secreted by immune cells, can be evaluated.

In vitro studies carried out in lymphoid cell lines showed the ability of cocoa to reduce the synthesis of interleukin 2 (IL-2) involved in early T lymphocyte proliferation (42, 43). This cytokine is mainly produced by Th cells after antigen activation (44) and plays a crucial role in immune response, enhancing Tc cell, NK cell cytotoxic activities, T cell differentiation, and stimulating the proliferation and the antibody synthesis (45). These effects could be responsible for the cocoa downregulation of the antibody synthesis. However, the results obtained in vivo on IL-2 secretion or lymphocyte proliferation could not confirm such a mechanism (18, 21, 30) (Figure 1A). In particular, IL-2 secretion was not modified in spleen cells from rats fed 10% cocoa for 3 weeks, even though lymphocyte proliferation increased (18). On the other hand, higher or unmodified amounts of IL-2 secretion were detected after the stimulation of lymph node cells of rats fed a 10% cocoa diet for 3 or 9 weeks (21, 30). Therefore, the interaction of a cocoa diet in the initial phases of immune activation seems not to explain the attenuating effect on antibody synthesis. However, a recent study on the gene expression of mesenteric lymph node cells shows that certain molecules present on antigen-presenting cells (dendritic cells) were modified by this diet. In particular, a cocoa diet increased the gene expression of CD11c and OX40L (31) (Figure 1A). It has been suggested that, in a model of oral sensitization, a subset of dendritic cells (CD11c⁺, CD103⁺, and CD8⁺) that migrates and activates in the mesenteric lymph nodes seems responsible for the Th2 polarization in this model (46). OX40L–OX40 interaction has been related to follicular Th cells and promotes the generation of Th2 response during antigen presentation (47, 48), and it was increased in an oral sensitization process (31). Despite these results, the cocoa diet attenuated the antibody synthesis and, therefore, this diet must interact with downstream pathways of the Th2-immune responses that would eventually inhibit antibody synthesis.

In general, the cytokine pattern secreted by activated lymphocytes reveals the stimulation of Th1, Th2, Th17, or Treg cells (49). Interferon γ (IFN-γ) is the most representative cytokine in Th1 activation (50). No changes were detected either in serum levels of IFN-γ in rats fed cocoa for 4 weeks (32) or in the secretion of IFN-γ by activated splenocytes or lymph node cells from rats fed a cocoa diet for 3 weeks (18) or 4 weeks (31). Nevertheless, an increase of IFN-γ was observed in lymph node lymphocytes from rats fed a cocoa diet for 8 weeks (21) (Figure 1A). Therefore, it seems that a cocoa intake over longer periods promotes Th1 immune response.

More interesting results were found in IL-4, the most representative Th2 cytokine (51). A reduction of IL-4 was found in activated lymph node cells from rats fed cocoa for 3 weeks (30) and in splenocytes from rats fed cocoa for 3 or 8 weeks (18, 21). However, no changes in IL-4 were found after 4 weeks of nutritional intervention (31). On the other hand, this downregulation on IL-4 secretion did not match with previous data in vitro (42, 43, 52) but it partially explains the down-modulatory role of the cocoa diet on antibody synthesis (Figure 1A). IL-4 promotes IgE upregulation and increases intestinal permeability (53, 54); therefore, the decrease in IL-4, along with the TCRγδ⁺ cell increase induced by the cocoa diet, may be beneficial in reducing certain stages of hypersensitivity, such as food allergy. However, some reports focused on IL-10, a regulatory cytokine (55), did not detect any modification by the 10% cocoa diet (30, 31).

The effects of cocoa lowering IL-4 secretion in some lymphocytes populations agree with those found when the specific antibody-secreting cells after an immunization were enumerated. A significant decrease in the specific IgG-secreting cell numbers was reported by 5 and 10% cocoa diets, either in spleen or lymph node tissues, although no changes were observed in specific IgM-secreting cells (21) (Figure 1B). In summary, a cocoa-enriched diet plays an immune-regulatory role in the antibody immune response to an antigen that involves a lower number of specific antibody-secreting cells and, therefore, a decrease in antibody synthesis.

Several years ago, Ramiro-Puig et al. first demonstrated that a cocoa-enriched diet influences the GALT by means of the modulation of the intestinal secretory IgA (S-IgA) (30). Feeding just a 4% cocoa-enriched diet caused a decrease in the fecal S-IgA levels in the second week of the diet, but they were restored at the end of the third one. The 10% cocoa intervention caused lower fecal S-IgA secretion throughout the study (30), and this effect remained when the diet was maintained for 7 weeks (19). However, when a dose–effect study was performed with diets containing 2, 5, or 10% cocoa, the 2% diet was not enough to modify intestinal immunoglobulins (20). With regards to the gut wash—a typical sample used to evaluate intestinal immunoglobulins that consists of incubating the intestine with saline buffer at 37°C in a shaker for a few minutes to allow the release of the mucosa-linked antibodies—a lower secretion of S-IgA and S-IgM was detected (19, 20, 30, 56). These results evidence a lack of the S-IgM compensatory mechanism in certain states of S-IgA deficiency (57), probably because cocoa is also acting on S-IgM. Other studies have confirmed the previous attenuating effect of a 10% cocoa diet on S-IgA levels both in fecal samples and in gut washes (31, 58). Moreover, the immunoglobulin content has also been determined in intestinal tissues, such as Peyer’s patches (PP) and mesenteric lymph nodes; and, in both tissues, the 10% cocoa diet for 3 weeks was able to decrease the levels of IgA and IgM (56).

The downregulation of intestinal immunoglobulins produced by a cocoa diet may be due to the influence of some cocoa compounds on the complex immune response developed in the GALT. This immune compartment includes inductive sites (PP and mesenteric lymph nodes) and effector sites [lamina propria (LPL) and intraepithelial lymphocytes (IEL)] (59). As explained, cocoa intake induced some changes in mesenteric lymph nodes, but the cocoa effect is not only restricted to that particular compartment. Therefore, further studies were then focused on looking in more depth the effect of a cocoa diet on PP as well as LPL and IEL.

The attenuation of serum or intestinal immunoglobulin synthesis may be the result of multitude pathways, but the reduction of mucosal IgA observed after cocoa dietary intervention may possibly involve specific mechanisms located at the intestinal site.

The rat intake of 10% cocoa for 4 weeks modified the composition of lymphocytes in the PP and in the intraepithelial compartment whereas no modifications were seen in LPL (33) (Table 2). With regard to PP, cocoa-enriched diets were able to reduce the proportion of TCRαβ⁺ T cells and to increase the proportion of B lymphocytes and TCRγδ⁺ cells (30, 33), results that agree with changes detected in the mesenteric lymph nodes (30, 31). Analyzing in depth TCRαβ⁺ cells in the intestine, the cocoa diet decreased the proportion of Th cells and increased that of natural killer T cells (NKT). In addition, after cocoa intake, PP also had higher proportions of CD4⁺CD25⁺ cells, CD4⁺CD103⁺ cells, CD8⁺CD103⁺ cells, and CD4⁺CD62L⁺ cells. Apart from the influence of cocoa intake on PP composition, the intraepithelial compartment was also affected by this diet. In IEL from the small intestine of rats fed cocoa, there was a higher percentage of TCRγδ⁺ cells (both CD8αα⁺ and CD8αβ⁺) and NK cells (33).

In summary, in the GALT, the lower production of intestinal antibodies was accompanied by a relative increase in B cell numbers and a relative decrease in TCRαβ⁺ or Th cell numbers in the inductive sites (mesenteric lymph nodes and PP). These results suggest that the antibody synthesis in B cells might be depleted by a lower stimulation from Th cells and/or a higher regulatory effect induced by cells, such as TCRγδ⁺, NK, NKT, CD4⁺CD25⁺, CD4⁺CD103⁺, CD8⁺CD103⁺, and CD4⁺CD62L⁺, which is in agreement with the role of some of these cells in the regulation of the antibody synthesis (25, 40, 60). In whatever way the activation and differentiation of intestinal B cells was attenuated, a depletion of the high-capacity IgA-secretory cells was produced as reported when they were counted by Enzyme-Linked ImmunoSpot in PP (30) or by an immunofluorescence analysis in the small intestine lamina propria (33). These results agree with a lower IgA gene expression in PP and small intestine seen after 4 and 7 weeks of cocoa intake (19, 33).

The gene expression of molecules involved in the intestinal immune response can shed some light on the mechanisms induced by cocoa on the regulation of the intestinal immune system. In this context, the mRNA levels of IgA, transforming growth factor (TGF) β1, IL-6, CD40, C–C chemokine receptor (CCR) 9, retinoic acid receptor (RAR) α, and RARβ have been reported in GALT tissues, such as mesenteric lymph nodes, PP, and small intestine after 3 or 7 weeks of a cocoa diet (19, 20).

CD40 is involved in the interaction between B and Th cells to begin the antibody immune response (61), and cocoa intake did not modify the expression of this molecule in any of the tissues considered (19, 20) suggesting that cocoa had no influence in this phase of the antibody synthesis. The main pathway that brings differentiation of B cells into IgA-secreting cells takes place in PP or mesenteric lymph nodes (62) and depends on cytokines, such as TGF-β1 and IL-6, among others (63). The 10% cocoa diet significantly decreased the TGF-β1 expression in the small intestine after 3 and 4 weeks (20, 33), although no changes were found after 7 weeks of nutritional intervention (19) (Figure 2). On the contrary, the longest nutritional intervention, but not the shortest one, was able to downregulate the IL-6 synthesis in mesenteric lymph nodes (19, 20). Therefore, the effect on these two cytokines, TGF-β1 and IL-6, involved in the S-IgA secretion at different periods (64), might be partly responsible for the downregulatory effect of cocoa. Neither TGF-β1 nor IgA gene expressions were downregulated by the 5% cocoa diet (20), which also caused a reduction in intestinal S-IgA, indicating that additional mechanisms may be interfering in the intestinal S-IgA content.

The next stage occurs when the activated B cells leave the inductor sites (PP and mesenteric lymph nodes) and home to the effector sites (i.e., lamina propria), where the differentiation into IgA plasma cells takes place (65). Intestinal homing is regulated, among others, by chemokine-mediated interactions including the chemokine receptor CCR9, which binds to CCL25 and the CCL28 chemokines in the intestine (66). While 3 weeks of diet did not modify the expression of the CCR9 receptor nor the CCL25 but did increase the expression of CCL28 in the small intestine (20), 7 weeks of cocoa diet resulted in a downregulation of CCR9 and CCL28 gene expression in the same compartment (19) (Figure 2). In addition, retinoic acid produced by intestinal dendritic cells also plays a key role in gut homing (66) through the interaction with nuclear RAR (67). The gene expression of both RARα and RARβ was not modified in the intestinal tissue of rats fed cocoa for 3 weeks (20) but both decreased after 7 weeks (19) (Figure 2). Overall, these results could indicate that after being fed a cocoa diet over a long time there was an impairment of the arrival of IgA-secreting cells to the intestine because of the lack of gut-homing receptors observed. However, they do not explain the early decrease in S-IgA that was observed.

Finally, delivering IgA into the intestinal lumen depends on the transmembrane epithelial protein polymeric immunoglobulin receptor (pIgR) (68). This receptor was not modified by the cocoa intake (20), thus indicating that cocoa-induced S-IgA reduction did not occur as a consequence of a decreased transport across the epithelium.

Apart from these IgA-secreting mechanisms that depend on T-cell activation, IgA⁺ B cells can be alternatively generated in a T-cell-independent manner involving toll-like receptor (TLR) signaling.

The gene and protein expression of other TLR has been modified both in the inductive sites of GALT (PP and mesenteric lymph nodes) and the effector sites (intestinal wall) after cocoa intake (19, 20, 33). In this context, among other components present in the cocoa, flavonoids have been suggested as dietary factors able to modulate TLR-mediated signaling pathways (69). TLR pathways can be modulated by flavonoids at different levels, and there are evidences of several flavonoids interfering at gene/protein expression level, in subsequent activation pathways such as the myeloid differentiation primary response 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon beta (TRIF), and even downstream-associated signal transduction cascades (i.e., MAPK) (69). In this sense, alternative mechanisms in TLR regulation by cocoa flavonoids have been also suggested such as the direct modulation of their intracellular negative regulators such as the interleukin-1 receptor associated kinase (IRAK), toll interacting protein (TOLLIP), etc. To date, in vitro studies demonstrate the upregulation of IRAK-M by procyanidin dimer B2 (70), similarly to the effect described by other flavonoids like epigallocatechin-galate in TOLLIP expression (70). Anyway, the synergistic action of cocoa on all these TLR-activating signaling could also contribute to the attenuation of S-IgA synthesis.

Cocoa intake for 4 weeks reduced the proportion of TLR4⁺ cells in the IEL compartment (33) which agrees with a decrease of TLR4 mRNA in small intestine observed in previous studies (Figure 2). Nonetheless, higher TLR4 gene expression was found in PP (19). The TLR4 is the receptor of bacterial endotoxin lipopolysaccharide, and its signaling has implications for IgA production (71), becoming another pathway to attenuate intestinal S-IgA synthesis.

Toll-like receptors are expressed preferentially in tissues that are in constant contact with microorganisms (72, 73), and changes in the TLR expression induced by flavonoids could reflect changes in the intestinal microbiota and/or in its relation with intestinal immune cells (69, 74). Accordingly, several studies have shown that cocoa (58, 75, 76), cocoa flavonoids (58, 77), or cocoa fiber (78) induce changes in gut microbiota composition. Moreover, a lower proportion of IgA-coated bacteria have been observed after cocoa intake (79).

In summary, it could not be discarded that the influence of cocoa on GALT was partially mediated by its effect on the intestinal microbiota, which can lead to differential TLR activation and, therefore, may also influence the lowering IgA effect of cocoa.

Recently, an analysis of an untargeted ¹H NMR spectroscopy-based metabolomic approach in 24-h urine samples have been carried out in order to correlate urine cocoa metabolites with cocoa effects on immunity and the gut microbiota (80). The results of this analysis demonstrate that cocoa intake, besides affecting microbiota composition, also alters the host and bacterial metabolism concerning energy and amino acid pathways leading to a particular metabolic signature that correlates with the S-IgA lowering effect of cocoa. Accordingly, a different pattern of intestinal and serum short-chain fatty acids, with increasing amounts of butyric acid, has been reported (78).

Finally, and in order to have a broader view of the molecules involved in the intestinal immune response modulated by cocoa, the changes in colonic gene expression by a microarray analysis after a cocoa nutritional intervention has been carried out (81). This study shows that a cocoa diet downregulated an extensive number of genes, many of them involved in the biological processes related to the immune system and inflammation. Specifically, the most downregulated gene after cocoa intake was tachykinin 4 (81), described as the promoter of B lineage cells (82), which could explain the attenuating effect of cocoa on antibody synthesis, despite the fact that the proportion of B cells did not decrease but, on the contrary, increased in some lymphoid tissues. Moreover, other genes involved in pathways related to the mast cell-mediated immunity, its activation, and its degranulation were downregulated (81), pointing out the possible role of cocoa in inducing tolerance in allergic processes as observed in some studies next reported.

The mucosal immune system is interconnected (59). Due to cocoa’s influence on the intestinal immune system, it became of interest to know whether this effect was also extended to other mucosal compartments, such as the salivary glands. The IgA and IgM content in the salivary glands (submaxillary and parotid salivary glands) was quantified after a 10% cocoa intake in rats for 3 weeks. The cocoa diet induced a decrease in the IgA and IgM content in both glands (56). This attenuating effect was associated with a drastic reduction in the IgA gene expression together with a lower expression of some molecules involved in the maturation and differentiation of B cells, such as IL-6 and TGF-β1 (56), as previously observed in the small intestinal samples (19, 20). However, in agreement with what was detected at intestinal level, no changes were detected in pIgR gene expression in the salivary glands. Therefore, in conclusion, this study shows that cocoa intake not only has an influence on the gut intestinal compartment and the systemic immunity but also on other mucosal sites in rats.

Rheumatoid arthritis is a symmetric, polyarticular, systemic, and autoimmune inflammatory disease in which multiple factors, including genetic, immune, and environmental ones are involved (83). Diet components such as n-3 fatty acids, vitamins D and K, and antioxidants are protective compounds against rheumatoid arthritis (84). In this context, diets containing 5 or 10% cocoa were tested on adjuvant arthritis, a model of rheumatoid arthritis widely used for the screening of anti-inflammatory drugs (85). In this animal model, cocoa diet decreased the synthesis of antibodies against the pathology inducer (Table 3) and was also able to decrease the proportion of Th cells in both blood and regional lymphoid tissues (86). This latter effect is important because, as anti-CD4 therapy has been shown to prevent or ameliorate adjuvant arthritis (87, 88), the cocoa-induced decrease in Th cells could be beneficial to the arthritic process. Moreover, a 10% cocoa diet avoided the Th/Tc imbalance and the reduction of the proportion of NKT cells produced by the disease (86). However, the effect of cocoa on hind-paw inflammation was very poor (86), which did not agree with the protective effect of other flavonoids in a similar inflammatory model when given by oral (quercetin) or by intraperitoneal routes (quercetin, rutin, hesperidin, and morin) (89–91). Nevertheless, a cocoa extract inhibited mice ear edema (92) and acute paw edema in rat (93, 94). Moreover, cocoa flavonoids such as epicatechin, catechin, and procyanidin B2, among others, are able to attenuate the synthesis of inflammatory mediators, such as tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1, IL-6, and IL-8 (95–100).

The influence of a 10% cocoa diet was also analyzed in collagen-induced arthritis, another model of arthritis. This inflammatory model requires T- and B-cell responses to autologous collagen (102). B cells from animals with collagen-induced arthritis produce a strong specific immune response against triple helical epitopes of collagen type II (103). Anti-collagen autoantibodies bind to the joint cartilage, activate the complement cascade, and mediate the inflammatory attack on the joints, thus contributing to the disease development (104). Susceptible Louvain rats were fed with a 10% cocoa diet for 2 weeks before arthritis induction and during the latency period (2 weeks after induction), and thereafter with a 5% cocoa diet until the end of the study (an additional 2 weeks). In this case, the cocoa-enriched diet was able to reduce the synthesis of specific antibodies against type II collagen, differentially according to their isotype (Table 3), decrease the Th lymphocyte proportion in regional lymph nodes, and reduce the release of inflammatory mediators from peritoneal macrophages. However, these immunomodulatory effects were not enough to reduce the hind-paw swelling in arthritic animals (32). It must be taken into account that the decrease in anti-collagen antibody concentration in that rat strain was only observed at the end of the study, and it was in a lesser extent and more slowly than that expected and observed in healthy rats as shown before. In a similar context, other authors reported the beneficial effect of isolated flavonoids in improving the paw swelling in animals in long-term studies (105–107). Otherwise a nutritional intervention with the flavonoid genistein had no success (108).