Cocoa Butter Intake Regulates Gut Immunity through the Release and Transport of IL-1β, IL-6 and IL-10: Activation of Negative Feedback Control System with Inflammatory and Anti-Inflammatory Cytokines

Proper food intake is important for maintaining good health in humans. Chocolate exerts anti-inflammatory and cardioprotective effects; however, the mechanisms underlying these effects remain unclear. Therefore, in this study, we aimed to investigate the effects of cocoa butter intake on gut and mucosal immunity in rats. The effects of cocoa butter intake on jejunal-originated mesenteric lymph flow, the concentration of cells, and interleukin (IL)-1β, IL-6 and IL-10 levels in the lymph were investigated via in vivo rat experiments. To evaluate the role of macrophages, the effects of clodronate, a macrophage depletion compound, on the cocoa butter-induced responses were examined. To clarify the cellular mechanisms of the responses, the effects of linoleic acids, the main essential long-chains fatty acids absorbed with cocoa butter, on the immunological responses of cells isolated from jejunal villi, in the presence or absence of toll-like receptors-2 and -4 inhibitor and myeloid differentiation response (MyD) 88 inhibitor. Cocoa butter intake increased the lymph flow, cell density, and IL-1β, IL-6 and IL-10 levels in the mesenteric lymph. The concentration of released IL-6 was the highest. The release of IL-10 showed a marked difference between the preparations. Clodronate significantly enhanced the release of all cytokines into the lymph. Toll-like receptor-2 and -4 inhibitor significantly suppressed the linoleic acid-mediated releases of IL-1β, IL-6, and IL-10 from the isolated cells. In addition, the Toll-like receptor 4 agonist released the IL-1β, IL-6, and IL-10 from the cells. In contrast, the MyD 88 inhibitor significantly decreased the release of IL-1β and IL-6, while only slightly the release of IL-10 from the cells. These findings suggest that cocoa butter intake regulates the gut and mucosal immunity via the release and transport of IL-1β, IL-6, and IL-10 in jejunal villi and into mesenteric lymph in a negative feedback system with inflammatory and anti-inflammatory cytokines.

Finally, we will be taken care to apply the findings obtained with rat experiments for human beings because the rat jejunum exists specialized lymphoid tissues in the walls like payer patch in the human ileum (2, 3, 4).

Keywords: Cocoa Butter, Absorption, Jejunum, IL-1β, IL-6, IL-10

Abbreviations: Interleukin, IL; Toll-Like Receptors, TLR; Enzyme-Linked Immunosorbent Assay, ELISA; Regulatory T Cells, Treg; Physiological Saline Solution, PSS; Antibody, Ab; Phosphate- Buffered Saline, PBS; Standard Errors Of The Mean, SEM; Not Significant, NS

Food intake is commonly known to be very important for keeping health care in human subjects. Thus, the traditional Japanese health care system recommends that a suitable volume of water be consumed every day, e.g., by drinking green tea or eating misosoup [1]. We previously demonstrated that water intake increased rat mesenteric lymph flow and the flux of long-chain fatty acid and innate lymphoid cell 3 (ILC-3)-secreted interleukin-22 (IL-22) through the lymph vessels [2, 3]. In addition, the water intake-mediated upregulation of podoplanin in the jejunal villi contributes to maintain higher tissue colloid osmotic pressure in the jejunal villi, resulting in the production of a large amount of jejunal-originated mesenteric lymph flow [4].

In contrast, there are marked differences of food intake custom in the world between Asia, and American and Europe. Especially, American and European peoples eat commonly bread, potato, and meat with oil. In addition, the 18th to 20th Century in the America and northern Europe, the early chocolate drink called as “Drink of the Gods” for keeping health care. It has been evolved its current pleasurable white chocolate. Currently, chocolate is noted to have anti-inflammatory and cardioprotective effects for maintaining good health [5,6]. In addition, Mediterranean has usually food intake with olive oil for keeping health care [7]. It is known that the eating the food with olive oil may prevent coronary heart disease, obesity, and allergies [7]. However, there is no or little scientific evidence to explain the effectiveness of cocoa butter or olive oil intake for keeping health care. Therefore, to clarify the roles of cocoa butter and olive oil in gut mucosal immunity, in this study we aimed to evaluate the effects of cocoa butter (the compositions of essential long-chain fatty acids: linoleic acid 3.3 %, arachidonic acid 0.3%; the compositions of main long-chain fatty acids: stearic acid 33.3 %, oleic acid 31.8 %, palmitic acid 27.9 %) or olive oil (the compositions of essential long-chain fatty acids: linoleic acid 13.6 %, arachidonic acid 1.4%; the compositions of main long-chain fatty acids: stearic acid 3.0 %, oleic acid 58.0 %, palmitic acid 14.4 %) intake on rat jejunal-originated lymph flow and the concentrations of cells, and cytokines IL-1β, IL-6 and IL-10 levels in the lymph. Because the IL-1β and IL-6 are well known to be inflammatory cytokines, whereas IL-10 plays an anti-inflammatory role in gut mucosal immunity [8-11]. The cytokines also play key roles of innate immunity in the body [8-11]. The effects of pretreatment with clodronate, which produces selective macrophage depletion in vivo [12], on the cocoa butter-mediated changes in the mesenteric lymph flow and the concentrations of cells, IL-1β, IL-6 and IL-10 levels in the lymph were also investigated. In addition, immunohistochemical studies for the distribution of macrophage using markers of macrophage CD 68 and F4/80 [13, 14] in rat jejunum were conducted following the pretreatment with clodronate. Finally, to clarify the cellular mechanisms underlying the effects of cocoa butter intake on changes in the IL-1β, IL-6 and IL-10 levels in the lymph, the in vitro experiments were conducted. Thus, the effects of the linoleic acid, which is the main essential fatty acid contained with cocoa butter, olive oil, and the stored long-chain fatty acids in the jejunal villi absorbed with lipid foods [2], on the concentrations of IL-1β, IL-6 and IL-10 in the cell medium were investigated. The cells isolated from the lamina propria of jejunal villi, which included T and B lymphocytes, macrophages, and dendritic cells. Next, the effects of the pretreatment with a TLR 2 and 4 inhibitor or a MyD 88 inhibitor on the linoleic acid-induced changes in the levels of IL-1β, IL-6 and IL-10 in the medium of isolated cells were investigated, because the TLR 4 is located on the surface of macrophages and the MyD88 is a key substance for the TLR 4-mediated cell signaling in the macrophage [8-11].

All experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of Shinshu University (1st April, 2019).

Forty Male Sprague–Dawley rats (10–12-weeks-old; Japan SLC, Tokyo, Japan) were fed a standard pellet diet containing linoleic acid (MF, Oriental Yeast, Tokyo, Japan) and provided water ad libitum. The animals were fasted overnight to reduce the effect of feeding on the mesenteric lymph flow. The rats were anesthetized with isoflurane and then placed on an operating table in a supine position. A catheter was inserted into the femoral vein to inject the physiological saline solution (PSS; Otsuka Pharma, Tokyo, Japan) used in the experiments. PSS was intravenously administered to maintain the normal physiological conditions in the rats before starting the experiments. To minimize the hemodynamic changes in the jejunal microcirculation, intravenous infusion of PSS was stopped during the experiments [2]. To collect the lymph from the jejunal-originated mesenteric lymph vessels, the abdomen was opened by cutting along the midline, and mesenteric adipose and connective tissues were removed to expose the mesenteric lymph node located outside the jejunum and the efferent lymph vessel. A heparinized small polyethylene catheter (0.5–0.6 mm) was inserted centrifugally into an efferent lymph vessel.

The compositions of lipids in the transported lymph through the mesenteric lymph vessels in the control that was fasted overnight with water provided ad libitum and after cocoa butter intake were analyzed using gas chromatography mass spectrometry (GC-MS, QP2010SE; Shimazu Co., Kyoto, Japan) and a GC column for analyzing the composition of fatty acids (Agilent J&W GC Column DB-23, Agilent Tech Co., California, USA).

To evaluate the effect of cocoa butter intake (the compositions of essential long-chain fatty acids: linoleic acid 3.3 %, arachidonic acid 0.3%; the compositions of main long-chain fatty acids: stearic acid 33.3 %, oleic acid 31.8 %, palmitic acid 27.9 %; Cocoa Co. Ltd., Tokyo, Japan) on the jejunal-originated mesenteric lymph flow and the concentrations of the cells, cytokines, IL-1β, IL-6, and IL-10, in the lymph, the cocoa butter (0.3 mL) with a small amount of emulsifier was administered through a needle catheter inserted into the stomach through the mouth. The lymph was collected over a set period (60 min) and the lymph volume was measured. In addition, as control experiment of intake food which makes the whole digestive system work, we investigated the effects of olive oil intake (0.3 mL) (the compositions of essential long-chain fatty acids: linoleic acid 13.6 %, arachidonic acid 1.4%; the compositions of main long-chain fatty acids: stearic acid 3.0 %, oleic acid 58.0 %, palmitic acid 14.4 %; FUJIFILM Co. Ltd., Tokyo) on the concentrations of IL-1β, IL-6, and IL-10, in the lymph.

The reason to use 0.3 mL cocoa butter in the experiments is not cytotoxic and safety, because the used dose is equivalent to around 139 g commercial chocolate intake in human subject with 60 Kg in body weight [7].

To investigate the effects of macrophages in the jejunal villi on the cocoa butter intake-mediated changes in the levels of IL-1β, IL-6, and IL-10 in the lymph, we investigated the effects of 24 h intraperitoneal administration of the macrophage depletion substance, clodronate-containing liposomes (catalog no,7767; Funakoshi, Tokyo, Japan) on the cocoa butter-mediated changes in the concentrations of the cytokines transported into the mesenteric lymph.

To clarify the cellular mechanisms of cocoa butter intake-mediated responses of cytokines, the effects of 2 min treatment with linoleic acid (catalog no. 125-05821, Fujifilm, Osaka, Japan) which is the main essential long-chain fatty acids digested and absorbed with lipid foods including cocoa butter in the jejunum, on the changes in the levels of IL-1β, IL-6, and IL-10 were investigated in the medium of isolated cells. In vitro experiments were conducted using cells isolated from the jejunal villi excluding the epithelial cell layers, which included with lymphocytes, macrophages, and dendritic cells. We also evaluated the effects of a TLR-2 and -4 inhibitor (5 x 10-6 M, pretreatment with 5min; catalog no. BP2-26245; Novusbio, Centennial, USA) or a MyD 88 inhibitor (5 x 10-6 M, pretreatment with 5min, catalog no. BP2-29328, Novusbio, Centennial, USA) on the linoleic acid-mediated changes in the levels of IL-1β, IL-6, and IL-10 in the isolated cells medium. Because the TLR 4 is located on the surface of macrophages and the MyD88 is a key substance for the TLR 4-mediated cell signaling in the macrophage [7-10].

The solvents of the TLR-2 and -4 inhibitor and MyD 88 were sterilized with distilled water and phosphate buffer solution (PBS), respectively.

The concentration of cells was measured with Türk reagent using diluted mesenteric lymph. The diluted lymph was dropped onto a Burker-Türk counter, and the number of cells on the plate was counted under a photomicroscope [2].

First, the obtained mesenteric lymph was centrifuged and the supernatants were used to measure the concentrations of IL-1β, IL-6, and IL-10. The concentrations of cytokines IL-1β, IL-6, and IL-10 in the lymph and isolated cell medium were measured using enzyme-linked immunosorbent assay (ELISA) kits: a rat IL-1β ELISA quantitative kit (catalog no. RLB00; R&D Systems, Minneapolis, MN, USA), IL-6 ELISA kit (catalog no. R6000B; R&D Systems, Minneapolis, MN, USA) and a mouse/rat IL-10 ELISA kit (catalog no. KE20003; Rosemont, IL, USA), respectively.

According to the method used previously (2, 3, 4), twenty Male Sprague-Dawley rats (10- to 12-week-old; Japan SLC, Tokyo) were fasted overnight and provided water ad libitum. The rats were anesthetized with isoflurane and then their small intestines were isolated. The jejuna were isolated from the upper one-third length of the small intestine. Each preparation was washed with excess PSS to remove the stool and mucus, followed by incubation for 30 min at 37 °C with vigorous shaking in a Ca2+- and Mg2+-free Hank’s balanced salt solution supplemented with 5 mM ethylenediaminetetraacetic acid and 1 mM dithiothreitol to remove the epithelial layer. To isolate cells from the lamina propria, the remaining jejuna were chopped into small pieces with a scalpel and digested for 30 min at 37 °C with gentle shaking in the Roswell Park Memorial Institute1640 medium supplemented with 5 % fetal bovine serum, 0.5 mg/mL collagenase IV (Sigma, St. Louis, USA), and 50 U/mL DNase (Wako, Osaka, Japan). Cells were subjected to 40–70% Percoll gradient centrifugation, and the cells from the middle layer were harvested and used for experiments with linoleic acid and/or the TLR-2 and -4 inhibitor or MyD 88 inhibitor.

To evaluate the effects of the intraperitoneal administration of clodronate-containing liposomes (20 mg) on the distribution of macrophages in the jejunal villi, the immunoreactivities of selective markers of the macrophages in the gut, CD 68 (catalog no. bs-0649R; Bioss, Boston, USA) or F4/80 (catalog no. 28463-1-AP; Proteintech, Rosemont, USA), in the lamina propria of jejunal villi were investigated [13]. The jejunum, loaded with or without clodronate for 24 h, was rapidly isolated and fixed with 4% paraformaldehyde in PBS overnight. The fixed samples were sectioned, and the slices were washed three times with PBS and incubated for 2 h at room temperature with primary polyclonal antibodies to 1:5000 diluted FITC-labelled CD68 or F4/80. After washing three times in PBS, the tissue slices were mounted with Pro-Long Gold antifade reagent and 4′-6-diamidino-2–2-phenylindole (DAPI, catalog no P36935; Invitrogen, Waltham, USA) to counterstain the cell nuclei. The slices were examined under a fluorescence microscope (KEYENSE, BZ9000, Osaka, Japan) and photographed.

To quantify the immunoreactivity data taken with the same brightness, high-resolution digital photomicrographs were processed using the Scion Image analysis program [15]. The constant area of each photomicrograph was outlined on a gray-scale image at the same density and processed for density measurements. The results were expressed in arbitrary units (mean density per pixel).

All chemicals were obtained from Wako (Tokyo, Japan). Heparin sulfate was purchased from Mochida Pharmaceutical Co. (Tokyo, Japan). Drug concentrations were described as the final concentrations in PBS.

All results are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using the Student’s t-test for paired or unpaired results or one-way analysis of variance, followed by Duncan’s post hoc test, as appropriate. Differences were considered statistically significant at p < 0.05.

A representative photomicrograph for the effects of intragastric administration of cocoa butter (0.3 mL), on the lymph volume collected over set periods of 60 mins from rat jejunum-originated lymph vessel is shown in Figure 1a. The control is shown before the administration of cocoa butter. The lymph volume collected over 120 - 240 min periods after the administration of cocoa butter was markedly increased compared to that collected at earlier time points. The color of lymph collected over 120 – 240 min periods clearly changed to be white. The summarized data are shown in Figure 1b.

In addition, the concentrations of cells through the lymph vessels tentatively decreased around 0~60 min after cocoa butter administration. On the other hand, in the lymph collected over 120 - 240 min after the administration, the concentrations of the cells in the lymph significantly increased (Figure 1C).

We analyzed the composition of lipids in the jejunal-originated lymph. In the control condition before the fasted overnight, the rats were fed with the standard pellet diet included with linoleic acid. The main lipids in the lymph were confirmed to be three long-chain fatty acids; linoleic acid 23.9 + 0.9 %, palmitic acid 23.0 + 0.4 %, and arachidonic acid 16.4 + 0.8 %. The linoleic acid is the highest concentration of essential fatty acids in the lymph.

Approximately 0 - 120 min after the administration, the administration of cocoa butter produced no or little change in the composition of lipids in the lymph (linoleic acid 22.6 + 1.0 %, palmitic acid 21.8 + 0.8 %, and arachidonic acid 14.9 + 1.2 %). In contrast, over 120 - 240 min periods after the administration, the total flux of lipids in the lymph increased significantly (the control 2.5 + 0.9 μg/hr; n=4, 120 - 180 min 13.8 + 1.9 μg/hr; n=4, 180 – 240 min 9.8 + 1.1 μg/hr; n=4, each value vs the control, p < 0.01). Especially, oleic acid and stearic acid in the lymph increased rapidly (in the control, 7.3 + 0.9 and 11.2 + 1.0 %, n=4; 180 - 240 min, 23.1 + 2.1 and 18.2 + 2.5 %; n=4, each value p < 0.01 vs the control). The linoleic acid was 14.8 + 1.9 % (n=4), but the total flux of linoleic acid through the lymph increased significantly over 120 - 240 min periods of the administration (the control, 0.7 + 0.1 μg/hr; n=4, 120 - 180 min 2.1 + 0.5 μg/hr; n=4, 180 - 240 min 1.5 + 0.5 μg/hr; n=4, each value p < 0.01 vs the control).

The cocoa butter intake (0.3 mL) increased the concentrations of inflammatory cytokine IL-1β in the lymph collected over 60 min periods are shown in Figure 2a-1. The control is shown before the administration of cocoa butter. Low concentrations of IL-1β increased time-dependently following cocoa butter intake.

In contrast, high concentration levels of the inflammatory cytokine IL-6 were released time-dependently into the mesenteric lymph following cocoa butter intake. The summarized data are presented in Figure 2b-1.

Furthermore, the anti-inflammatory cytokine IL-10 was also released time-dependently into the lymph following the cocoa butter intake. The summarized data are shown in Figure 2c-1.

In some preparations (n=4), the cocoa butter intake produced no release of IL-10 until 240 min after the administration. There is marked heterogeneity along the rats used in the cocoa butter intake-mediated release of IL-10 in the lymph.

The olive oil-mediated increase of IL-1β level in the lymph was not detected until 240 min after the administration (Figure 2a-2).

The control is shown before the administration of olive oil.

In contrast, the olive oil intake caused time-dependently the increase of IL-6 level in the mesenteric lymph, being lower concentration compared with cocoa butter intake (Figure 2b-2). The slight concentration of IL-10 in the lymph increased with the olive oil intake, being not significant compared with the control (Figure 2c-2).

A representative photomicrograph of the effects of cocoa butter intake in a rat pretreated with clodronate (20 mg, 24 hr) on the jejunal-originated lymph volume collected over 60 min periods is presented in Figure 3a-1. The control is shown before the administration of cocoa butter. Compared with that presented in Figure 1a-1, the lymph volume collected after the intragastric administration of cocoa butter significantly decreased. The summarized data are shown in Figure 3a-2.

The pretreatment with clodronate also significantly decreased the concentrations of cells in the lymph collected over 60 min periods. The summarized data are presented in Figure 3a-3.

Compared with the findings presented in Figure 2a-2, the pretreatment with clodronate (20 mg, 24 hr) reduced the cocoa butter intake-mediated increases in the concentration of IL-1β in the mesenteric lymph collected over 60 min periods (Figure 4a). The control is shown before the administration of cocoa butter.

Surprisingly, the pretreatment with clodronate (20 mg, 24 hr) released IL-6 in the lymph in the control condition before the administration of cocoa butter (Figure 4b). The cocoa butter intake significantly accelerated time-dependently the increases in the concentration of IL-6 in the lymph collected over approximately 120-240 min (Figure 4b).

Similar to effect of clodronate on IL-6 release, the pretreatment with clodronate produced a release of IL-10 into the lymph in the control condition (Figure 4c). Following the cocoa butter intake, the IL-10 concentration tended to increase slightly, but not significantly, in the lymph collected over approximately120-240 min in all preparations (Figure 4c).

Representative photomicrographs of CD 68-positive cells in the control tissues and the tissues pretreated with clodronate (20 mg, 24 hr) are shown in Figure 5a-1. In the control without clodronate, marked CD 68 immunoreactivity was observed in the lamina propria of jejunal villi. Compared with the control, the lower CD 68 immunoreactivity was observed in the preparation pretreated with clodronate. Consistent with these findings, the F4/80 immunoreactivity in the lamina propria of jejunal villi was similarly reduced by the pretreatment with clodronate (Fig. 5b-1). The CD 68 and F4/80 immunoreactivities in the lamina propria of jejunal villi were quantitatively analyzed using density measurement. The data were summarized in Figure 5a, b-2. The expression level of CD 68 and F4/80 in immunohistochemistry images were significantly reduced by the pretreatment with clodronate.

The control is shown before the administration of linoleic acid and TLR 2&4 inhibitor. Approximately 2 min of incubation of linoleic acid with the isolated cells significantly released IL-1β.

The effect of pretreatment with the TLR 2 and 4 inhibitor on the linoleic acid (20 μL)-mediated increase of IL-1β level are demonstrated in Figure 6Aa. The pretreatment with the TLR 2 and 4 inhibitor significantly decreased the linoleic acid-mediated release of IL-1β. Similar to the response of IL-1β, the 2 min treatment with linoleic acid significantly released IL-6 from the isolated cells, respectively (Figure 6Ab). The pretreatment with the TLR 2 and 4 inhibitor also significantly reduced the linoleic acid-mediated releases in IL-6 and IL-10, respectively.

In all control experiments without linoleic acid and the inhibitors, no significant changes in the cytokine levels in the cell medium were observed.

The effects of MyD 88, a key substance of cell signaling in macrophage, on the linoleic acid-mediated releases of IL-1β, IL-6 and IL-10 from the isolated cells. The control is shown before the administration of linoleic acid and the MyD 88 inhibitor. The pretreatment with MyD 88 significantly decreased the releases of IL-1β (Figure 6Ba) and IL-6 (Figure 6Bb). In contrast, the linoleic acid-mediated release of IL-10 was reduced slightly, but not significant (Figure 6Bc). In addition, in the control experiment a small amount of IL-10 release was observed (17.2 + 4.5 %, n=4). The solvent of MyD 88 was used PBS.

The findings obtained in the experiments were summarized as follows; (1) The intragastric administration of cocoa butter in rats increased the jejunal-originated mesenteric lymph flow and the concentration of cells in the lymph approximately 120~240 min after the administration. (2) Cocoa butter intake led to significant release and transport of IL-1β, IL-6 and IL-10 into the mesenteric lymph. However, there was a marked variation in the IL-10 response in the rats. (3) The clodronate-induced macrophage depletion in the jejunal villi significantly reduced the cocoa butter-mediated increases of the mesenteric lymph flow and the concentration of cells in the lymph. (4) Pretreatment with clodronate itself accelerated to release and transport IL-6 and IL-10 into the lymph. (5) Pretreatment with clodronate also increased the cocoa butter intake-mediated release and transport of IL-6 in the lymph approximately 120 - 240 min after the intake. (6) In contrast, in the presence of clodronate the concentrations of IL-1β and IL-10 in the lymph tended to increase slightly, but not significantly, with the cocoa butter intake. (7) The olive oil intake as the control of food intake increased time-dependently the concentration of IL-6 in the lymph, being similar to the response with cocoa butter intake. However, the olive oil intake caused a slight increase of IL-10 and no change in IL-1β in the lymph. (8) The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased by the pretreatment with clodronate. (9) Linoleic acid was physiologically stored in the jejunal villi with digestion and absorption of lipid foods included cocoa butter. (10) Linoleic acid significantly released approximately 2 min IL-1β, IL-6 and IL-10 in the medium of cells isolated from the lamina propria of rat jejunal villi. However, there was a large variation in the IL-10 response. (11) The TLR 2 and 4 inhibitor significantly reduced the linoleic acid-mediated releases of IL-1β, IL-6 and IL-10. However, there also was heterogeneity of the IL-10 response between the cell preparations. (12) MyD 88 significantly decreased the linoleic acid-mediated releases of IL-1β and IL-6, and slightly but not significant IL-10.

In the present study, the effects of olive oil intake on the responses of cytokine levels in the mesenteric lymph were adopted as the control food intake and then the effects of composition long-chain fatty acids on the cytokine levels were not evaluated. Therefore, we have not yet investigated that how the differences of between cocoa butter- and olive oil-mediated responses of IL-1β, IL-6 and IL-10 dependent with each composition of saturated and unsaturated long-chain fatty acids are existed. In addition, the effects of linolenic acid on the responses of IL-1β, IL-6 and IL-10 level in the isolated cells are now evaluated comparing with the linoleic acid-mediated responses. In preliminary study, linolenic-mediated releases of IL-1β, IL-6 and IL-10 have tended to be smaller than those produced with linoleic acid. However, we will be in the future, needed to conduct rapidly such kinds of studies.

The cocoa butter-mediated time-dependent increases of the mesenteric lymph flow and cells in the lymph may be related to the digestion and absorption time of lipids, being longer than those of water. The findings that the total flux of lipids through the lymph and the white color change of the lymph increased significantly over 120 – 240 periods of the administration of cocoa butter support strongly the physiological properties in the mesenteric lymph. Miura et, al. [16] also demonstrated that olive oil absorption accelerated the transport of lymphocytes through intestinal collecting lymphatics in 120 min.

The treatment with clodronate significantly decreased the mesenteric lymph flow and cells in the lymph. The findings may be related to the hypothesis that the clodronate-induced macrophage depletion (Figure 5). The depletion may release many cellular substances of macrophage included with cytokines and then interact with tissue components in the jejunal villi. The interaction may obstruct the movement of soluble substances with tissue fluid and lipoprotein ligated with albumin through the lamina propria, resulting in the clodronate-mediated decreases of the lymph flow and cells in the lymph (Figure 3).

Another important finding of the study is that the clodronate-induced macrophage depletion in jejunal villi led to the release of IL-6 and IL-10 into the lymph in the control condition without cocoa butter intake. The treatment with clodronate significantly accelerated the cocoa butter intake-mediated release and transport of IL-6 into the lymph. However, the treatment with clodronate also tended, but not significant, to increase the cocoa butter intake-mediated releases of IL-1β and IL-10. In addition, 2 min treatment with linoleic acid released IL-1β, IL-6, and IL-10 in the cells isolated from jejunal villi.

Based on these findings, the linoleic acid stored and/or absorbed with cocoa butter intake mainly stimulates the macrophages, resulting the production and release of inflammatory cytokines IL-1β and IL-6. In fact, the cocoa butter intake stimulated to transport linoleic acid into the mesenteric lymph. The released cytokines of IL-1β and IL-6 and linoleic acid itself may accelerate the release of anti-inflammatory cytokine IL-10 in the macrophages and lymphocytes with negative feedback control system in the gut mucosal immunity, resulting in maintaining health care in human subjects [7]. The hypothesis may be agreed with the present finding that there were marked heterogeneity responses of the linoleic acid-mediated release of IL-10 and MyD 88 inhibitor-mediated reduction of the IL-10 release, which may be related to production and release of IL-10 in many kinds of cells in the jejunal villi.

Consistent with our findings, oral administration of linoleic acids for 10 days was found to modulate the production of inflammatory mediators by rat macrophages [17]. Furthermore, dietary cocoa butter resulted in an increase in the levels of IL-6 at 6 hr after the absorption in healthy woman [8].

It is noteworthy that our study is the first to evaluate acute effects of cocoa butter intake on the release and transport of IL-1β, IL-6 and IL-10 in rat jejunal-originated lymph in vivo, as well as the acute (around ~ 2 min) effect of linoleic acids on the releases of IL-1β, IL-6 and IL-10 into the medium of cells isolated from the lamina propria of jejunal villi. In addition, we clearly demonstrated that the macrophages located in the jejunal villi may contribute mainly to the cocoa butter intake-mediated releases of IL-1β, IL-6 and IL-10. Consistent with these findings, the macrophages in the jejunal villi are known to produce the IL-1β, IL-6 and IL-10 (18-20). On the other hand, IL-10 is also produced and released from regulatory T cells (Treg) and helper T2 cells (Th2) distributed in the lamina propria of jejunal villi [21, 22]. Agreement with the evidence, the finding that the pretreatment with clodronate itself released the IL-6 and IL-10 in the control condition may be related to the evidence that the depleted macrophage led to leak the cytokines in the jejunal villi. Similarly, a very slight release of IL-1β (~ 0.3 pg/ml) was observed following the pretreatment with clodronate. In addition, the pretreatment with clodronate accelerated the cocoa butter intake -mediated release of IL-6 in the jejunal preparations (~15,000 pg/ml vs ~8,000 pg/ml without clodronate). The large number off macrophages containing large amounts of IL-6 may contribute to the observed cocoa butter intake-mediated higher IL-6 release.

On the other hand, the evidence that the macrophages, Treg and Th2 in the jejunal villi contain IL-10 may be, in part related to the marked heterogeneity of the cocoa butter intake-mediated IL-10 release between the preparations. However, the larger concentration of IL-10 contained mainly into the macrophages may be related to the observed cocoa butter intake-mediated IL-10 release in all preparations pretreated with clodronate.

However, concerning the regulation for releases of inflammatory versus anti-inflammatory cytokines, what important parameters of the ration omega 6/omega 3 and the presence / absence of the omega 9 oleic acid have decided the cytokine releases. Such kinds of studies will be also in the future needed.

Surprisingly, the pretreatment with TLR 2 and 4 inhibitor caused a significant reduction of the 2 min treated linoleic acid-mediated releases of IL-1β, IL-6 and IL-10 in the isolated cells. These findings may suggest that the activation of TLR 2 and 4 on the macrophages and lymphocytes in interaction with the fatty acid, contributes, in part to the release of IL-1β, IL-6 and IL-10 from the cells. In agreement with, the pretreatment with MyD 88 inhibitor significantly inhibited the linoleic acid-mediated releases of IL-1β and IL-6. In fact, the TLR 4 is found in the macrophages [23, 24]. However, the detail mechanisms underlying the linoleic acid-mediated release of cytokines from isolated cells remain unclear. This point may be a major limitation of our manuscript. However, in preliminary experiments, we confirmed a TLR 4 agonist induced the releases of IL-1β, IL-6 and IL-10 in the cells isolated from jejunal villi. These findings may suggest that cocoa butter intake stimulates the TLR 4 on the macrophages in the lamina propria, resulting in facilitation of the MyD 88-mediated exocytosis of the cytokines in the macrophage. However, the detailed mechanisms of the MyD 88-mediated exocytosis of the cytokines will be in the future clarified using specific agonists and antagonists of Ca2+ and phosphorylation.

Cocoa butter intake releases and transports inflammatory cytokines IL-1β and IL-6, and anti-inflammatory cytokine IL-10 into rat jejunal-originated mesenteric lymph vessels. Linoleic acid, absorbed with cocoa butter and stored in the jejunal villi, stimulates to release IL-1β, IL-6 and IL-10 from macrophages and lymphocytes from lamina propria of jejunal villi through the activation of TLR 2&4 and MyD 88 in the cells. Thus, cocoa butter intake regulates gut mucosal immunity through the activation of the release and transport for inflammatory and anti-inflammatory cytokines with negative feedback system, resulting in keeping for health care in human subjects.

This study and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Shinshu University (1st April, 2019).

All authors approved the final version of the manuscript and the publication of this manuscript.

All relevant data are available from the corresponding author on request.

The authors declare that the research was conducted in the absence of the commercial or financial relationships that could be constructed as a potential conflict of interest.

The Department of Innovation of Medical and Health Sciences Research at Shinshu University School of Medicine was established and supported financially by the donation of BOURBON, Co., Ltd, Kashiwazaki, Niigata, Japan and Aizawa Hospital, Matsumoto, Nagano, Japan. The authors declare that this study received funding from BOURBON Co. Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

T.O. designed the experiments, analyzed the data, constructed all figures, and wrote the manuscript. Y.K., M.H., T-W. A., and N.A. designed the experiments, analyzed the data, and revised the manuscript. K.A., R.K., M.T., N.K., D.M., Y.Y., and M.K., performed the experiments, and analyzed data.

We thank Editage for English language editing and their support to non-native English speakers.