*3.2. Triglyceride Rich-Lipoproteins Modulate Maturation and Activation Markers in Human Monocyte-Derived Dendritic Cells*

In gaining deeper insight into the role of postprandial TRLs in DC maturation and activation, marker gene expressions during human monocyte differentiation into moDCs were studied. Postprandial TRLs at 100 μg TGs/mL added to the differentiation medium that contained GM-CSF and IL-4 for 6 days did not induce cytotoxicity (data not shown). In previous reports, postprandial TRLs were not cytotoxic to human monocyte-derived osteoclasts [9] and macrophages [15] at similar concentrations. As shown in Figure 2, both transcriptional activity of *CD123* (Figure 2a) and *CCR7* (Figure 2b), key regulators of DC maturation, were upregulated by TRL-MUFAs and TRL-PUFAs but more markedly by TRL-SFAs, suggesting that postprandial fatty acids present in TRLs, in a saturation degree-dependent manner, modulate the DC maturation process. To further explore the possible pro-inflammatory or tolerogenic effects of postprandial TRLs in human moDCs, gene expression of CD80 (Figure 2c) and CD86 (Figure 2d) pro-inflammatory activation markers and PD-L1 (Figure 2e) and PD-L2 (Figure 2f) tolerogenic activation markers were analyzed. Postprandial TRLs affected the activation status of moDCs. Interestingly, TRL-SFAs upregulated the postprandial-TRL-induced transcriptional activity of CD80 and CD86 pro-inflammatory gene markers in moDCs. In contrast, TRL-MUFAs and TRL-PUFAs induced an upregulation in PD-L1 and PD-L2 gene expression, whereas TRL-SFAs did not induce any changes compared to control gene expression.

**Figure 2.** In vitro expression of dendritic cell (DC) gene markers in monocyte-derived DCs (moDCs) after stimulation with TRL-SFAs, TRL-MUFAs and TRL-PUFAs (TRL, triglyceride-rich lipoprotein) at 100 μg of TGs/mL for 6 days and in presence of GM-CSF and interleukin-4 (IL-4). DC maturation markers: (**a**) CD123 and (**b**) CCR7. DC pro-inflammatory activation markers: (**c**) CD80 and (**d**) CD86. DC tolerogenic activation markers: (**e**) PD-L1 and (**f**) PD-L2. Control means non-treated cells in the presence of GM-CSF and IL-4. Values are presented as means ± SD (*n* = 6) and those marked with different letters are significantly different (*p* < 0.05).

## *3.3. Fatty Acid-Enriched Meals Modulate Serum IL-12p70 and IL-10 Postprandial Secretion and Triglyceride-Rich Lipoproteins Regulate Cytokine Levels and Gene Expression in Human Monocyte-Derived Dendritic Cells*

We also investigated the secretion levels of pro-inflammatory IL-12p70 and tolerogenic IL-10 in postprandial serum of healthy volunteers. After meal ingestion, postprandial serum levels of IL-12p70 (Figure 3a) were increased by the SFA meal but not by the MUFA or PUFA meal when compared to the control meal with no fat. Remarkably, IL-12p70 AUCTOTAL was particularly increased by the SFA meal (*p* = 0.0125, Figure 3b) over postprandial 6 h. In contrast, IL-10 levels were postprandially lower after the SFA meal (Figure 3c) and higher after the MUFA and PUFA meal ingestion. Thus, AUCTOTAL values for serum IL-10 were significantly lower (*p* = 0.0039) only after the ingestion of the high-fat meal enriched in SFAs and higher after the ingestion of the MUFA- (*p* = 0.0054) and PUFA- (*p* = 0.0170) enriched meals in healthy volunteers when compared to the control meal with no fat (Figure 3d).

**Figure 3.** (**a**) Serum and (**b**) area under the curve of pro-inflammatory IL12p70 levels and (**c**) serum and (**d**) area under the curve of tolerogenic IL10 levels at fasting and at postprandial period after the administration of a control meal (with no fat) or high-fat meals enriched in SFAs, MUFAs, or MUFAs + omega-3 LCPUFAs (PUFAs) in healthy subjects. Values are presented as means ± SD (*n* = 6) and those marked with different letters are significantly different (*p* < 0.05). AUC: area under the curve.

In line with these results, in vitro experiments showed that TRL-SFAs promoted the secretion and transcriptional activity of *IL12p70* (Figures 4a and 4b, respectively). On the other hand, no changes were observed on the IL-10 secretion in moDCs (Figure 4c); however, TRL-SFAs downregulated *IL10* transcriptional activity in moDCs (Figure 4d). Contrary to SFAs, TRL-MUFAs and TRL-PUFAs upregulated tolerogenic *IL10* transcriptional activity in moDCs.

**Figure 4.** In vitro expression and secretion of proinflammatory and tolerogenic cytokines in moDCs after stimulation with TRL-SFAs, TRL-MUFAs, and TRL-PUFAs at 100 μg of TGs/mL for 6 days and in presence of GM-CSF and IL-4. (**a**) IL-12p70 secretion and (**b**) mRNA expression. (**c**) IL-10 secretion and (**d**) mRNA expression. Values are presented as means ± SD (*n* = 6) and those marked with different letters are significantly different (*p* < 0.05).

## *3.4. Triglyceride-Rich Lipoproteins Induce Lipid Accumulation and ApoB48R Transcriptional Activity in Monocyte-Derived Dendritic Cells in a Fatty Acid-Dependent Manner*

Finally, we want to establish whether lipid accumulation would function to induce moDC activation. TRL-SFAs induced higher increase in TG accumulation (Figure 5a) and *ApoB48R* transcriptional activity (Figure 5b) compared those induced by TRL-MUFAs and TRL-PUFAs in moDCs. In addition, intracellular TG correlated with the expression of the ApoB48R (*R*<sup>2</sup> 0.9998, *p* = 0.0093, Figure 5c) and *CD80 (R*<sup>2</sup> 0.9991, *p* = 0.0192, Figure 5d) activation marker, suggesting that dietary FAs present in TRLS, in a saturation degree-dependent manner, intervene in the activation process of moDCs.

**Figure 5.** (**a**) Intracellular TGs accumulation and (**b**) In vitro *apoB48R* mRNA induced in moDCs after stimulation with TRL-SFAs, TRL-MUFAs and TRL-PUFAs at 100 μg of TGs/mL for 6 days and in presence of GM-CSF and IL-4. (**c**) Correlation between *apoB48R* mRNA and (**d**) *CD80* mRNA (DC activation marker) with intracellular TGs in moDCs. Control means non-treated cells in presence of GM-CSF and IL-4. Values are presented as means ± SD (*n* = 6) and those marked with different letters are significantly different (*p* < 0.05). TGs: triglycerides.

#### **4. Discussion**

The literature often suggests that lifestyle and traditional dietary habits unique to the Mediterranean region play a role in the prevention of oxidative- and inflammatory-related pathologies, such as cardiometabolic diseases and cancer [23]. Olive oil, the main dietary fat in the Mediterranean diet, due to its content of oleic acid (MUFA) and minor constituents, modulate different processes linked to chronic low-grade inflammation [24]. This view is in contrast to diets rich in SFAs, such as the "meat-based" or "Westernized" diets, which are inductive of inflammatory states [25]. One of the key processes of inflammation is the maturation and activation of circulating myeloid cells. These leukocytes are the first immune cells that respond quickly to injury and their activation, if permanent or chronic, may cause the increase of the inflammatory response, the perpetuation of the inflammatory state and the development of obesity or autoimmune disease [26,27]. DCs are professional antigen-presenting cells within the immune system, that are uniquely capable of priming naïve T cells, and once activated they have a pivotal ability to induce primary innate and adaptive immune response [28]. However, little is known about the effect of dietary fatty acids on human moDC [29]. The experimental use of primary human DCs is limited by their rarity in peripheral blood (less than 1% of MNCs), so to avoid this, in vitro moDCs are generally selected as a pragmatic model [30].

The postprandial period, the state that comprises and follows a meal, has an important, yet underrated, role in the onset of several pathologies. After fatty meal intake, dietary fatty acids are largely integrated into nascent TRLs, which are liberated from the small intestine into the bloodstream. It has been previously reported that dietary fatty acids have divergent postprandial effects on chronic disease-related events [31], suggesting that acute outcomes in response to dietary SFA-, MUFA- or PUFA-adjustment may be helpful to lightly attenuate, even for preventing, diet-related chronic diseases [10]. It is essential to mention that the postprandial period is defined by a large number of metabolic transformations that comprise the raise of circulating TRLs. In response to fatty meal intake, earlier human studies have demonstrated the association of activated myeloid cells with postprandial hyperlipidemia [32,33]. Notwithstanding the relevance, studies during the postprandial period on interaction between human postprandial TRLs and moDCs are still unknown. Our results show for the first time that, after SFA-enriched meal intake, postprandial hypertriglyceridemia is associated with an increase of serum GM-CSF in healthy subjects. This effect was regulated by the main fatty acids in dietary fats, being significantly raised after the ingestion of an SFA-enriched meal when compared to the ingestion of MUFA-enriched meals. In line with these results, McFarlin et al. demonstrated that high-calorie meals significant increased postprandial GM-CSF and G-CSF levels in humans [34].

In gaining deeper insight into the role of dietary fatty acids on differentiation of moDCs, we obtained fresh monocytes from the fasting blood samples of healthy volunteers. Then, cells were differentiated into moDCs (GM-CSF + IL4 treatment for 6 days) in the absence or the presence of TRL-SFAs, TRL-MUFAs, and TRL-PUFAs isolated from postprandial serum samples of the same volunteers. In these experimental setting of autologous interaction, we observed an upregulation of DC maturation (CD123 and CCR7) and pro-inflammatory activation (CD80 and CD86) markers, and a downregulation of tolerogenic activation (PD-L1 and PD-L2) markers in human moDCs in response to postprandial TRL-SFAs. These effects support the notion that dietary saturated fats promote pro-inflammatory functions in mature DCs through metabolic pathways involving lipoproteins. In line with these results, Nicholas et al. demonstrated that palmitic acid-stimulated moDCs upregulated the expression of CD83 and CD86 [35]. Additionally, palmitic acid induced TLR4-dependent secretion of IL-1β, generated reactive oxygen species, and activated the NFκB canonical pathway in moDCs [35]. Importantly, our study showed a significant attenuation of incremental DC maturation and activation following the treatment with TRL-MUFAs and TRL-PUFAs, suggesting that the replacement of dietary SFAs by MUFAs (in combination or not with omega-3 long-chain PUFAs) could be helpful to prevent excessive DC-associated with postprandial events. Our new data extend the previous in vitro studies with PUFAs, and emphasize their acute benefits on TRLs to a healthy population. In line with this notion, moDCs stimulated with DHA and EPA show a reduction in the expression of CD80 and CD86 and in the secretion of IL-12p70 [36]. To our best knowledge, for the first time, the current study demonstrates that oleic acid from olive oil decreases, even abrogates, the gene expression of DC maturation and activation gene markers and the pro-inflammatory cytokine release. However, functional assays with moDCs generated in the presence of different fatty acids and T cells could increase the knowledge of postprandial TRLs' effects on DC differentiation and function.

Finally, in line with previous data in human neutrophils [37], monocytes [15,38], and murine microglia [13], our study have showed that postprandial TRLs induced DC activation through ApoB48R upregulation in a FA-dependent manner. Dietary oleic acid, EPA, and DHA attenuated ApoB48R gene expression while triggering a depletion in intracellular TG accumulation compared to palmitic acid.

#### **5. Conclusions**

In conclusion, these findings suggest that dietary fatty acids play a relevant and interrelated role in protecting against DC postprandial differentiation. Our results open new opportunities for developing novel nutritional strategies with olive oil as the principal dietary source of MUFAs, notably oleic acid, to prevent development and progression of inflammatory- and autoimmune-related diseases.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/10/3139/s1, Table S1: Fatty acid composition of dietary fats, Table S2: Detailed information about primers' sequences used in this study.

**Author Contributions:** Conceptualization, C.V.-M., B.B., and S.M.d.l.P.; methodology, C.V.-M., E.G.-C., M.C.M.-L., and N.M.R.-M.; formal analysis, S.L.; investigation, M.E.M., G.A., and C.S.-M.; writing—original draft preparation, S.L. and S.M.-d.l.P.; project administration, S.M.-d.l.P.; funding acquisition, B.B. and S.M.-d.l.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the research Grant US-1263458 (Andalusian Ministry of Economy, Knowledge, Business, and University, Government of Andalusia, Spain) into the European Regional Development Fund Operational Programme 2014 to 2020.

**Conflicts of Interest:** The authors state no conflicts of interest.
