Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells

Citation: Muñoz-García, R.; Sánchez-Hidalgo, M.; Montoya, T.; Alcarranza, M.; Ortega-Vidal, J.; Altarejos, J.; Alarcón-de-la-Lastra, C. Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells. Pharmaceuticals 2022,15, 1338. https://doi.org/ 10.3390/ph15111338 Academic Editor: Carlos Vaamonde-García Received: 8 October 2022 Accepted: 26 October 2022 Published: 28 October 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceuticals Article Effects of Oleacein, a New Epinutraceutical Bioproduct from Extra Virgin Olive Oil, in LPS-Activated Murine Immune Cells Rocío Muñoz-García1,2, Marina Sánchez-Hidalgo 1,2 , Tatiana Montoya 1,2 , Manuel Alcarranza 1,2 , Juan Ortega-Vidal 3, Joaquín Altarejos 3and Catalina Alarcón-de-la-Lastra 1,2,* 1Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, 41012 Sevilla, Spain 2Instituto de Biomedicina de Sevilla, IBiS, Universidad de Sevilla, HUVR, Junta de Andalucía, CSIC, 41013 Seville, Spain 3Department of Inorganic and Organic Chemistry, Faculty of Experimental Sciences, Campus de Excelencia Internacional Agroalimentario (ceiA3), Universidad de Jaén, 23071 Jaén, Spain *Correspondence: calar[email protected]; Tel.: +34954559877 Abstract: The present study was designed to evaluate the immunomodulatory effects of the secoiridoid from extra virgin olive oil, oleacein (OLA), deepening into the possible signaling pathways involved in LPS-activated murine peritoneal macrophages. Moreover, we have explored OLA-induced epigenetic changes in histone markers and related cytokine production in murine LPS-stimulated murine splenocytes. Murine cells were treated with OLA in the presence or absence of LPS (5 µ g/mL) for 18 or 24 h. OLA modulated the oxidative stress and the inflammatory response produced by LPS stimulation in murine peritoneal macrophages, by the inhibition of pro-inflammatory cytokines (TNF- α , IL-6, IL-1 β , IFN- γ , IL-17 and IL-18) and ROS production and the expression of pro-inflammatory enzymes such as iNOS, COX-2 and m-PGES1. These protective effects could be due to the activation of the Nrf-2/HO-1 axis and the inhibition of JAK/STAT, ERK and P38 MAPKs and inflammasome canonical and non-canonical signaling pathways. Moreover, OLA modulated epigenetic modifications throughout histone methylation deacetylation (H3K18ac) and (H3K9me3 and H3K27me) in LPS-activated spleen cells. In conclusion, our data present OLA as an interesting anti-inflammatory and antioxidant natural compound that is able to regulate histone epigenetic markers. Nevertheless, additional in vivo studies are required to further investigate the beneficial effects of this EVOO secoiridoid, which might be a promising epinutraceutical bioproduct for the management of immune-related inflammatory diseases. Keywords: epigenetic; histone; inflammation; immunity; macrophages; oleacein; olive oil; spleen cells 1. Introduction Macrophages are one of the most important components of the immune system and play a critical role in the inflammatory process. However, abnormal macrophage activation is involved in the pathogenesis of immune-inflammatory diseases. Specifically, an imbalance in the polarization of M1/M2 macrophages has been described as a key factor in the development of systemic lupus erythematosus [ 1 ]. Furthermore, macrophages are implicated in rheumatic diseases such as rheumatoid arthritis and osteoarthritis promoting synovitis and the destruction of cartilage and bone, characteristics of these diseases [2,3]. In activated macrophages, the Toll-like receptor 4 (TLR4) recognizes the pathogenassociated molecular pattern, bacterial lipopolysaccharide (LPS), leading macrophages to an inflammatory state and generating reactive oxygen species (ROS), Th1 and Th17 cytokines and chemokines. Furthermore, the expression of inflammatory-related enzymes, in particular, cyclooxygenase (COX)-2, microsomal prostaglandin E synthase (mPGES)-1, and inducible nitric oxide synthase (iNOS), which synthesize prostaglandin (PG)E2 and nitric oxide (NO), respectively, has been widely reported in LPS-stimulated macrophages [4,5] . The process of gene expression of these pro-inflammatory mediators involves multiple signaling Pharmaceuticals 2022,15, 1338. https://doi.org/10.3390/ph15111338 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2022,15, 1338 2 of 20 cascades that culminate in the activation of transcription factors, mainly mitogen-activated protein kinases (MAPKs), nuclear transcription factor kappa B (NF- κ B), mitogen-activated protein kinases (MAPKs), Janus kinase/signal transducer and transcription activator of transcription (JAK/STAT) or inflammasome activation [6]. In addition, the nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) is a redox-sensitive transcription factor that regulates the expression of antioxidant enzymes such as heme oxygenase-1 (HO-1) [ 7 ]. This signaling pathway is repressed during the induction of activated macrophages through a regulative function on the activation of ROS, MAPKs, and inflammasome signaling pathways [8]. Dietary nutrients could regulate pathological processes through critical epigenetic mechanisms, modulating gene expression without altering the genetic code. In particular, extra virgin olive oil (EVOO) has exhibited anti-inflammatory activities in human macrophages by epigenetic mechanisms [ 9 ]. The beneficial effects of EVOO have been attributed to the high content of monounsaturated fatty acids, specifically oleic acid, and also a combination of minor phytochemicals, including polyphenols such as secoiridoids. Secoiridoids mainly represent the major group of phenolic compounds (>90%), particularly ligustroside and oleuropein aglycons and their derivatives oleocanthal and oleacein (OLA), respectively. In fact, during the EVOO extraction process, oleuropein is hydrolyzed by endogenous glycosylases to generate OLA [10]. Several studies have demonstrated the protective effects of OLA on the metabolic alterations presented in a high-fat diet-fed mice model, such as body weight gain, obesity, steatohepatitis, and insulin resistance [ 11 , 12 ]. In addition, OLA inhibited the formation of foam cells in human monocyte-derived macrophages [ 13 ] and prevented the destabilization of atherosclerotic plaque from hypertensive patients [ 14 ]. Additionally, this phenolic compound has shown anticancerous activity [ 15 ]. Singularly, OLA has exhibited anti-proliferative and anti-metastatic effects in myeloma multiple and SH-SY5Y human neuroblastoma cell lines [ 16 , 17 ]. Interestingly, OLA presented anti-inflammatory and antioxidant effects in a murine model of encephalomyelitis, improving the disease progression [ 18 ]. Moreover, OLA has been shown to be a potent antioxidant even more than its precursor oleuropein [19]. On the other hand, epigenetic changes such as histone modifications, DNA methylation, and non-coding RNA profiling have been described to play a significant role in the pathogenesis of autoimmune diseases. Immune system tolerance is regulated by histone modifications, preventing damage from an exacerbated inflammatory response. In this context, post-translational histone modifications could hold great therapeutic potential [ 20 , 21 ]. In fact, Lauterbach et al. have shown that macrophages redirect metabolic fluxes to foster histone acetylation in response to TLR4-LPS ligation in a time frame that is relevant for gene induction [22]. We have previously reported that LPS can promote the production of inflammatory cytokines in murine splenocytes and peritoneal macrophages suggesting that LPS stimulation disrupted H3 acetylation or methylation resulting in sustained IL-1 β , IL-6, and IL-17 production [ 23 – 25 ]. Consequently, it could be considered that the use of both types of immune cells could provide a more complete view for a better comprehensive understanding of the potential effects of OLA on the immune-inflammatory response. In this context, prior studies by our research group have explored drug-induced epigenetic changes in histone markers (H3K9me3, H3K27me3, and H3K18ac) and cytokine-correlated production compared in spleen cells after LPS induction with relevant results production [25]. Given this background, the present study was designed to assess the antioxidant and anti-inflammatory effects of OLA in LPS-stimulated murine peritoneal macrophages. We attempted to characterize the action mechanisms underlying the possible beneficial effects. In addition, to explore the epigenetic mechanisms involved in the anti-inflammatory effects of this EVOO secoiridoid, we evaluated OLA-induced epigenetic changes in histone markers (H3K9me3, H3K27me3, and H3K18ac) and related cytokine levels in murine LPS-stimulated splenocytes. Pharmaceuticals 2022,15, 1338 3 of 20 2. Results 2.1. Isolation of OLA from Olive Oil The amount of OLA required to carry out this work was obtained from a sample of olive oil of the cultivar Picual elaborated in the province of Jaen, Spain. The phenolic fraction was obtained by extraction of the oil with a mixture of methanol–water according to the International Olive Council protocol. The resulting dry extract (0.4% yield, with respect to oil) was chromatographed by the fast centrifugal partition chromatography (FCPC) technique yielding several fractions, one of them containing crude OLA, which was further chromatographed by semi-preparative high-performance liquid chromatography (HPLC) to give pure OLA (2.1% yield, with respect to phenolic fraction). The structure of this final sample of OLA was properly characterized by nuclear magnetic resonance (NMR) and polarimetry as (–)-oleacein as described in Section 4.1. 2.2. Antioxidant Activity of OLA At first, the antioxidant effect of OLA was evaluated by inhibition of the 2,20-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)+ radical. OLA presented a value of IC50 for ABTS radicals of 23.92 ± 3.96 µ M. OLA showed similar antioxidant activity to the standard TROLOX, which presented IC50 values of 20.8 ±3.81 µM. 2.3. Effects of OLA on Cell Viability We determined the effect of OLA on the viability of murine peritoneal macrophages by the sulforhodamine B (SRB) assay. After 18 h of OLA treatment, at concentrations of 200–1.56 µM , the viability of peritoneal macrophages was not significantly affected (Figure 1). In all doses the survival % was higher than 80%, taking as a reference the untreated control cells. In the same way, the vehicle dimethylsulfoxide (DMSO) did not alter cell viability. Pharmaceuticals 2022, 15, x FOR PEER REVIEW 3 of 20 Given this background, the present study was designed to assess the antioxidant and anti-inflammatory effects of OLA in LPS-stimulated murine peritoneal macrophages. We attempted to characterize the action mechanisms underlying the possible beneficial effects. In addition, to explore the epigenetic mechanisms involved in the anti-inflammatory effects of this EVOO secoiridoid, we evaluated OLA-induced epigenetic changes in histone markers (H3K9me3, H3K27me3, and H3K18ac) and related cytokine levels in murine LPS-stimulated splenocytes. 2. Results 2.1. Isolation of OLA from Olive Oil The amount of OLA required to carry out this work was obtained from a sample of olive oil of the cultivar Picual elaborated in the province of Jaen, Spain. The phenolic fraction was obtained by extraction of the oil with a mixture of methanol–water according to the International Olive Council protocol. The resulting dry extract (0.4% yield, with respect to oil) was chromatographed by the fast centrifugal partition chromatography (FCPC) technique yielding several fractions, one of them containing crude OLA, which was further chromatographed by semi-preparative high-performance liquid chromatography (HPLC) to give pure OLA (2.1% yield, with respect to phenolic fraction). The structure of this final sample of OLA was properly characterized by nuclear magnetic resonance (NMR) and polarimetry as (–)-oleacein as described in Section 4.1. 2.2. Antioxidant Activity of OLA At first, the antioxidant effect of OLA was evaluated by inhibition of the 2,20-Azino- Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS)+ radical. OLA presented a value of IC50 for ABTS radicals of 23.92 ± 3.96 μM. OLA showed similar antioxidant activity to the standard TROLOX, which presented IC50 values of 20.8 ± 3.81 μM. 2.3. Effects of OLA on Cell Viability We determined the effect of OLA on the viability of murine peritoneal macrophages by the sulforhodamine B (SRB) assay. After 18 h of OLA treatment, at concentrations of 200–1.56 μM, the viability of peritoneal macrophages was not significantly affected (Figure 1). In all doses the survival % was higher than 80%, taking as a reference the untreated control cells. In the same way, the vehicle dimethylsulfoxide (DMSO) did not alter cell viability. Figure 1. Effect of OLA on cell viability. Cells were treated with OLA (200–1.6 μM) for 18 h. Cell survival was expressed as the percentage of viability with respect to control untreated cells (100%). Data are represented as means ± SEM (n = 6). Figure 1. Effect of OLA on cell viability. Cells were treated with OLA (200–1.6 µ M) for 18 h. Cell survival was expressed as the percentage of viability with respect to control untreated cells (100%). Data are represented as means ±SEM (n= 6). 2.4. Intracellular ROS, Nitrite Production, and iNOs Overexpression Are Inhibited by OLA in LPS-Stimulated Murine Peritoneal Macrophages To know the effect of OLA on the inflammatory and oxidative response induced by LPS, we measured intracellular ROS, NO levels, and iNOS expression using 20,70–dichlorofluorescein diacetate (DCFDA), Griess, and Western blot assays, respectively, in LPS-activated murine peritoneal macrophages. The DCFA fluorescence signal was increased by LPS stimulation (+++ p< 0.001 vs. unstimulated cells). However, pretreatment with OLA was able to reduce ROS production (* p< 0.05, *** p < 0.001 vs. LPS-DMSO cells) (Figure 2B). This proves that OLA could have antioxidant activity. Pharmaceuticals 2022,15, 1338 4 of 20 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 4 of 20 2.4. Intracellular ROS, Nitrite Production, and iNOs Overexpression Are Inhibited by OLA in LPS-Stimulated Murine Peritoneal Macrophages To know the effect of OLA on the inflammatory and oxidative response induced by LPS, we measured intracellular ROS, NO levels, and iNOS expression using 2′,7′–dichlorofluorescein diacetate (DCFDA), Griess, and Western blot assays, respectively, in LPS- activated murine peritoneal macrophages. The DCFA fluorescence signal was increased by LPS stimulation (+++ p < 0.001 vs. unstimulated cells). However, pretreatment with OLA was able to reduce ROS production (* p< 0.05, *** p < 0.001 vs. LPS-DMSO cells) (Figure 2B). This proves that OLA could have antioxidant activity. Nitrites measurement was used as an indicator of NO production. Like the DCFDA signal, nitrites generation increased in the presence of LPS (+++ p < 0.001 vs. unstimulated cells). Nonetheless, pretreatment with OLA displayed a significant reduction of NO production in treated cells (*** p < 0.001 vs. stimulated cells). These data were related to the reduction in iNOS protein expression after OLA treatment (*** p < 0.001 vs. stimulated cells) (Figure 2A,C, respectively). Figure 2. OLA downregulated ROS production, nitrites production, and iNOS protein expression in murine peritoneal macrophages. Cells were pretreated with different concentrations of OLA (25 and 12.5 μM) and then stimulated with LPS for 18 h. (A) Nitrite production; (B) ROS production; (C) Western blot analysis of iNOS protein expression. Densitometry was performed following normalization to the control (β-actin housekeeping gene). Data are represented as means ± SEM (n = 6). (+++) p < 0.001 vs. control cells (no stimulated). *** p < 0.001 vs. LPS-MSO-treated cells. 2.5. OLA Decreased LPS-Induced Secretion of Pro-Inflammatory Cytokines To assess whether OLA could inhibit the secretion of pro-inflammatory cytokines, specific ELISA assays were used to measure interleukin (IL)-1β, necrosis tumoral factor (TNF)-α, IL-17, IL-6 and interferon (IFN)-γ secretion levels in macrophages culture supernatant. After LPS stimulation for 18 h, the levels of IL-1β, TNF-α, IL-17, IL-6, and IFN-γ were significantly augmented (+++ p < 0.001 vs. unstimulated control cells). Nevertheless, exposure to OLA significantly decreased the secretion of all these cytokines compared to DMSO-LPS cells (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. stimulated cells) (Figure 3A–E). Figure 2. OLA downregulated ROS production, nitrites production, and iNOS protein expression in murine peritoneal macrophages. Cells were pretreated with different concentrations of OLA (25 and 12.5 µ M) and then stimulated with LPS for 18 h. ( A ) Nitrite production; ( B ) ROS production; ( C ) Western blot analysis of iNOS protein expression. Densitometry was performed following normalization to the control ( β -actin housekeeping gene). Data are represented as means ± SEM (n= 6). (+++) p< 0.001 vs. control cells (no stimulated). *** p< 0.001 vs. LPS-MSO-treated cells. Nitrites measurement was used as an indicator of NO production. Like the DCFDA signal, nitrites generation increased in the presence of LPS (+++ p < 0.001 vs. unstimulated cells). Nonetheless, pretreatment with OLA displayed a significant reduction of NO production in treated cells (*** p < 0.001 vs. stimulated cells). These data were related to the reduction in iNOS protein expression after OLA treatment (*** p < 0.001 vs. stimulated cells) (Figure 2A,C, respectively). 2.5. OLA Decreased LPS-Induced Secretion of Pro-Inflammatory Cytokines To assess whether OLA could inhibit the secretion of pro-inflammatory cytokines, specific ELISA assays were used to measure interleukin (IL)-1 β , necrosis tumoral factor (TNF)- α , IL-17, IL-6 and interferon (IFN)- γ secretion levels in macrophages culture supernatant. After LPS stimulation for 18 h, the levels of IL-1 β , TNF- α , IL-17, IL-6, and IFN- γ were significantly augmented (+++ p< 0.001 vs. unstimulated control cells). Nevertheless, exposure to OLA significantly decreased the secretion of all these cytokines compared to DMSO-LPS cells (* p< 0.05, ** p< 0.01, *** p< 0.001 vs. stimulated cells) (Figure 3A–E). 2.6. Inhibitory Effect of OLA on COX-2 and mPGES-1 Overexpression Induced by LPS Next, in order to elucidate the anti-inflammatory activity of OLA, we analyzed the effects of OLA on the COX-2 and mPGES-1 markers. As shown in Figure 4, LPS stimulation increased COX-2 and mPGES-1 expression (+++ p< 0.001 vs. no stimulated cells). On the contrary, OLA pretreatment (25 and 12.5 µ M) significantly decreased both COX-2 and mPGES-1protein expressions compared to LPS-DMSO cells (*** p< 0.001 vs. LPS-DMSO control cells) (Figure 4A,B). Pharmaceuticals 2022,15, 1338 5 of 20 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 5 of 20 Figure 3. Effects of OLA on IL-1β (A), TNF-α (B), IL-17(C), IL-6 (D), and IFN-γ (E) levels. Cells were pretreated for 30 min(min) with OLA and LPS-stimulated (5 μg/mL) for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Data are expressed as means ± SEM (n = 7). +++ p < 0.001 vs. control cells (no stimulated); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO control-treated cells. 2.6. Inhibitory Effect of OLA on COX-2 and mPGES-1 Overexpression Induced by LPS Next, in order to elucidate the anti-inflammatory activity of OLA, we analyzed the effects of OLA on the COX-2 and mPGES-1 markers. As shown in Figure 4, LPS stimulation increased COX-2 and mPGES-1 expression (+++ p < 0.001 vs. no stimulated cells). On the contrary, OLA pretreatment (25 and 12.5 μM) significantly decreased both COX-2 and mPGES-1protein expressions compared to LPS-DMSO cells (*** p < 0.001 vs. LPS-DMSO control cells) (Figure 4A,B). Figure 4. OLA reduced COX-2 and mPGES-1 overexpression in LPS-stimulated murine peritoneal macrophages. (A) Western blot analysis of COX-2 expression; (B) Western blot analysis of mPGES- 1 expression. Cells were pretreated with OLA (25 and 12.5 μM) and incubated in absence or presence of LPS for 18 h. Densitometry was performed after normalization to the control (β-actin housekeeping gene). Data are represented as mean ± SEM (n = 6). +++ p < 0.001 vs. control cells (no stimulated); *** p < 0.001 vs. LPS−DMSO cells. Figure 3. Effects of OLA on IL-1 β ( A ), TNF- α ( B ), IL-17( C ), IL-6 ( D ), and IFN- γ ( E ) levels. Cells were pretreated for 30 min(min) with OLA and LPS-stimulated (5 µ g/mL) for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Data are expressed as means ±SEM ( n= 7 ). +++ p< 0.001 vs. control cells (no stimulated); * p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS−DMSO control-treated cells. Pharmaceuticals 2022, 15, x FOR PEER REVIEW 5 of 20 Figure 3. Effects of OLA on IL-1β (A), TNF-α (B), IL-17(C), IL-6 (D), and IFN-γ (E) levels. Cells were pretreated for 30 min(min) with OLA and LPS-stimulated (5 μg/mL) for 18 h. Control cells were also treated with DMSO (solvent control) and LPS or nontreated. Data are expressed as means ± SEM (n = 7). +++ p < 0.001 vs. control cells (no stimulated); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO control-treated cells. 2.6. Inhibitory Effect of OLA on COX-2 and mPGES-1 Overexpression Induced by LPS Next, in order to elucidate the anti-inflammatory activity of OLA, we analyzed the effects of OLA on the COX-2 and mPGES-1 markers. As shown in Figure 4, LPS stimulation increased COX-2 and mPGES-1 expression (+++ p < 0.001 vs. no stimulated cells). On the contrary, OLA pretreatment (25 and 12.5 μM) significantly decreased both COX-2 and mPGES-1protein expressions compared to LPS-DMSO cells (*** p < 0.001 vs. LPS-DMSO control cells) (Figure 4A,B). Figure 4. OLA reduced COX-2 and mPGES-1 overexpression in LPS-stimulated murine peritoneal macrophages. (A) Western blot analysis of COX-2 expression; (B) Western blot analysis of mPGES- 1 expression. Cells were pretreated with OLA (25 and 12.5 μM) and incubated in absence or presence of LPS for 18 h. Densitometry was performed after normalization to the control (β-actin housekeeping gene). Data are represented as mean ± SEM (n = 6). +++ p < 0.001 vs. control cells (no stimulated); *** p < 0.001 vs. LPS−DMSO cells. Figure 4. OLA reduced COX-2 and mPGES-1 overexpression in LPS-stimulated murine peritoneal macrophages. ( A ) Western blot analysis of COX-2 expression; ( B ) Western blot analysis of mPGES-1 expression. Cells were pretreated with OLA (25 and 12.5 µ M) and incubated in absence or presence of LPS for 18 h. Densitometry was performed after normalization to the control ( β -actin housekeeping gene). Data are represented as mean ± SEM (n= 6). +++ p< 0.001 vs. control cells (no stimulated); *** p< 0.001 vs. LPS−DMSO cells. 2.7. OLA Upregulated Nrf-2/HO-1 Pathway Protein Expression To evaluate whether OLA could regulate the antioxidant signaling pathway Nrf-2/HO- 1, we determined the expression of Nrf-2 and HO-1 protein by Western blotting. Nrf-2 Pharmaceuticals 2022,15, 1338 6 of 20 is a key transcription factor in the response to oxidative stress, which can regulate the expressions of antioxidant enzymes such as HO-1 [8]. Treatment with OLA caused a significant increase in Nrf-2 and HO-1 expression compared to LPS-DMSO cells (* p< 0.05, ** p< 0.01 vs. LPS − DMSO-treated cells) (Figure 5). These results, along with ROS production downregulation could prove the potential role of OLA in the antioxidant response. Pharmaceuticals 2022, 15, x FOR PEER REVIEW 6 of 20 2.7. OLA Upregulated Nrf-2/HO-1 Pathway Protein Expression To evaluate whether OLA could regulate the antioxidant signaling pathway Nrf- 2/HO-1, we determined the expression of Nrf-2 and HO-1 protein by Western blotting. Nrf-2 is a key transcription factor in the response to oxidative stress, which can regulate the expressions of antioxidant enzymes such as HO-1 [8]. Treatment with OLA caused a significant increase in Nrf-2 and HO-1 expression compared to LPS-DMSO cells (* p< 0.05, ** p < 0.01 vs. LPS−DMSO-treated cells) (Figure 5). These results, along with ROS production downregulation could prove the potential role of OLA in the antioxidant response. Figure 5. OLA upregulated Nrf-2/HO-1 signaling pathway in LPS-activated peritoneal macrophages. Murine cells were pretreated with OLA for 30 min, followed by LPS stimulation for 18 h. Nrf-2 and HO-1 protein levels were detected by Western blot. Densitometry was performed following normalization to the control (β-actin housekeeping gene). Data are represented as the means ± SEM (n = 6). * p < 0.05, ** p < 0.01 vs. LPS−DMSO-treated cells. 2.8. Effects of OLA on MAPKs Phosphorylation In addition, we assessed the role of OLA in MAPKs activation by Western blotting. Our results showed that OLA inhibited the phosphorylation of P38 and ERK induced by LPS stimulation (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO control cells). Despite this fact, OLA was unable to significantly reduce JNK MAPKs phosphorylation after LPS treatment, as shown in Figure 6. Figure 5. OLA upregulated Nrf-2/HO-1 signaling pathway in LPS-activated peritoneal macrophages. Murine cells were pretreated with OLA for 30 min, followed by LPS stimulation for 18 h. Nrf-2 and HO-1 protein levels were detected by Western blot. Densitometry was performed following normalization to the control ( β -actin housekeeping gene). Data are represented as the means ± SEM (n= 6). * p< 0.05, ** p< 0.01 vs. LPS−DMSO-treated cells. 2.8. Effects of OLA on MAPKs Phosphorylation In addition, we assessed the role of OLA in MAPKs activation by Western blotting. Our results showed that OLA inhibited the phosphorylation of P38 and ERK induced by LPS stimulation (* p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS − DMSO control cells). Despite this fact, OLA was unable to significantly reduce JNK MAPKs phosphorylation after LPS treatment, as shown in Figure 6. 2.9. LPS-Induced JAK/STAT Pathway Activity Is Downregulated by OLA in Murine Peritoneal Macrophages To go deeper into the possible molecular mechanism of OLA, we explored its potential role in JAK/STAT phosphorylation. The JAK/STAT signaling pathway participates in the inflammatory process to mediate immune responses. Usually, JAK2 is in the cytoplasm. Under cellular stress, it is phosphorylated and activated. Subsequently, STAT-3 is phosphorylated on tyrosine residues and translocated to the nucleus to bind DNA and moderate gene transcription [5,6]. We observed that LPS treatment significantly increased JAK2 and STAT-3 phosphorylation in peritoneal macrophages (+ p< 0.05, ++ p< 0.01, +++ p< 0.001 vs. non-stimulated cells) while in cells treated with OLA, JAK2 and STAT-3 phosphorylation was significantly downregulated (** p< 0.01, *** p< 0.001 vs. cells treated with LPS-DMSO) (Figure 7). Pharmaceuticals 2022,15, 1338 7 of 20 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 7 of 20 Figure 6. Effects of OLA on MAPKs activation in murine peritoneal macrophages. Cells were pretreated with OLA (25 and 12.5 μM) for 30 min and then incubated in presence or absence of LPS for 18 h. As controls, cells were also treated with DMSO (solvent control). Densitometry was performed after normalization to the control (P38, JNK, ERK housekeeping gene). Results are represented as means ± SEM (n = 6). + p < 0.05, +++ p < 0.001 vs. control cells (no stimulated); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells. 2.9. LPS-Induced JAK/STAT Pathway Activity Is Downregulated by OLA in Murine Peritoneal Macrophages To go deeper into the possible molecular mechanism of OLA, we explored its potential role in JAK/STAT phosphorylation. The JAK/STAT signaling pathway participates in the inflammatory process to mediate immune responses. Usually, JAK2 is in the cytoplasm. Under cellular stress, it is phosphorylated and activated. Subsequently, STAT-3 is phosphorylated on tyrosine residues and translocated to the nucleus to bind DNA and moderate gene transcription [5,6]. We observed that LPS treatment significantly increased JAK2 and STAT-3 phosphorylation in peritoneal macrophages (+ p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. non-stimulated cells) while in cells treated with OLA, JAK2 and STAT-3 phosphorylation was significantly downregulated (** p < 0.01, *** p < 0.001 vs. cells treated with LPS-DMSO) (Figure 7). Figure 7. OLA treatment modulated JAK/STAT signaling pathway in LPS-stimulated murine peritoneal macrophages. Cells were untreated or treated with OLA (25 and 12.5 μM) for 18 h in presence or absence of LPS. Data shown are means ± SEM (n = 6). + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. control cells (no stimulated); ** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells. Figure 6. Effects of OLA on MAPKs activation in murine peritoneal macrophages. Cells were pretreated with OLA (25 and 12.5 µ M) for 30 min and then incubated in presence or absence of LPS for 18 h. As controls, cells were also treated with DMSO (solvent control). Densitometry was performed after normalization to the control (P38, JNK, ERK housekeeping gene). Results are represented as means ± SEM (n= 6). + p< 0.05, +++ p< 0.001 vs. control cells (no stimulated); *p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS−DMSO-treated cells. 2.10. Effect of OLA in the Canonical and Non-Canonical Inflammasome Activation in LPS-Activated Murine Peritoneal Macrophages “NOD-like” receptor (NLRP)-3 activation triggers an increase in the oligomerization of the adapter protein caspase recruitment domain (ASC). Consequently, ASC activates pro-caspase-1 in caspase-1, which is involved in the maturation of IL-1- β and IL-18 [ 26 ]. Hence, we analyzed the effects of OLA on canonical and non-canonical inflammasome activation using Western blot. Our results showed that after LPS-stimulation there was an increase in NLRP3 and ASC protein expression compared to unstimulated cells, also, we observed parallel results in both forms of caspase-1 (+ p< 0.05, ++ p< 0.01, +++ p< 0.001 vs. no stimulated cells). However, in those cells treated with OLA, the expression of these proteins was negatively regulated (* p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS-DMSO treated cells). According to these results, we found that OLA reduced both IL-1 β and IL-18 levels, in comparison with LPS-DMSO induced cells (** p< 0.01, *** p< 0.001 vs. LPS − DMSO-treated cells) (Figures 3A and 8C, respectively). On the other hand, we evaluated the impact of OLA on noncanonical inflammasome activation in LPS-induced murine peritoneal macrophages (Figure 8D). As expected, pro-, partially cleaved and cleaved-caspase-11 were upregulated after LPS stimulus (+ p< 0.05, ++ p< 0.01 vs. control cells). Nevertheless, OLA treatment could mitigate this activation (* p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS−DMSO-treated cells). Pharmaceuticals 2022,15, 1338 8 of 20 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 7 of 20 Figure 6. Effects of OLA on MAPKs activation in murine peritoneal macrophages. Cells were pretreated with OLA (25 and 12.5 μM) for 30 min and then incubated in presence or absence of LPS for 18 h. As controls, cells were also treated with DMSO (solvent control). Densitometry was performed after normalization to the control (P38, JNK, ERK housekeeping gene). Results are represented as means ± SEM (n = 6). + p < 0.05, +++ p < 0.001 vs. control cells (no stimulated); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells. 2.9. LPS-Induced JAK/STAT Pathway Activity Is Downregulated by OLA in Murine Peritoneal Macrophages To go deeper into the possible molecular mechanism of OLA, we explored its potential role in JAK/STAT phosphorylation. The JAK/STAT signaling pathway participates in the inflammatory process to mediate immune responses. Usually, JAK2 is in the cytoplasm. Under cellular stress, it is phosphorylated and activated. Subsequently, STAT-3 is phosphorylated on tyrosine residues and translocated to the nucleus to bind DNA and moderate gene transcription [5,6]. We observed that LPS treatment significantly increased JAK2 and STAT-3 phosphorylation in peritoneal macrophages (+ p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. non-stimulated cells) while in cells treated with OLA, JAK2 and STAT-3 phosphorylation was significantly downregulated (** p < 0.01, *** p < 0.001 vs. cells treated with LPS-DMSO) (Figure 7). Figure 7. OLA treatment modulated JAK/STAT signaling pathway in LPS-stimulated murine peritoneal macrophages. Cells were untreated or treated with OLA (25 and 12.5 μM) for 18 h in presence or absence of LPS. Data shown are means ± SEM (n = 6). + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. control cells (no stimulated); ** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells. Figure 7. OLA treatment modulated JAK/STAT signaling pathway in LPS-stimulated murine peritoneal macrophages. Cells were untreated or treated with OLA (25 and 12.5 µ M) for 18 h in presence or absence of LPS. Data shown are means ± SEM (n= 6). + p< 0.05, ++ p< 0.01, +++ p< 0.001 vs. control cells (no stimulated); ** p< 0.01, *** p< 0.001 vs. LPS−DMSO-treated cells. 2.11. Effects of OLA on LPS-Induced IκB-αDegradation in Peritoneal Macrophages In addition, we evaluated the role of OLA in I κ B- α degradation in cytosolic fractions of murine peritoneal cells. After cellular stimulation, I κ B- α is phosphorylated and degraded by the proteasome (+ p < 0.05 vs. no stimulated cells), leading to NF- κ B signaling pathway activation [ 27 ]. Pretreatment with OLA at 25 µ M prevented I κ B- α degradation (* p< 0.05 vs. LPS-DMSO cells). However, at the dose of 12.5 µ M this effect was not observed (Figure 9). 2.12. Epigenetic Histone Modifications by OLA in Spleen Cells Finally, in order to explain the role of OLA in epigenetic changes, we studied whether this secoiridoid could induce post-translational modification in histone H3 and its correlated cytokines production. After LPS stimulation, H3K18ac levels were increased (+++ p< 0.001 vs. control cells). In cells treated with OLA (25 µ M), we could appreciate a marked decrease in the acetylation level of Histone H3 (*** p< 0.001 vs. LPS-DMSO cells) (Figure 10A). However, we noticed the opposite effect on histone methylation levels. In OLA treated cells, we found a higher expression of methylated H3K9 and H3K27 while in the LPS cells the methylation of H3 was downregulated (Figure 10B,C) (+ p< 0.05, ++ p< 0.01, vs. control cells (no stimulated); *p< 0.05, ** p< 0.01, LPS−DMSO-treated cells). Based on these results, we measured the production of cytokines that were associated with these epigenetic marks in their promoter regions. As expected, OLA reduced the LPS-induced overproduction of pro-inflammatory cytokines such as IL-1 β , IL-6, and IL-17 in spleen cells (* p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS−DMSO-treated cells). Pharmaceuticals 2022,15, 1338 9 of 20 Pharmaceuticals 2022, 15, x FOR PEER REVIEW 8 of 20 2.10. Effect of OLA in the Canonical and Non-Canonical Inflammasome Activation in LPS-Activated Murine Peritoneal Macrophages “NOD-like” receptor (NLRP)-3 activation triggers an increase in the oligomerization of the adapter protein caspase recruitment domain (ASC). Consequently, ASC activates pro-caspase-1 in caspase-1, which is involved in the maturation of IL-1-β and IL-18 [26]. Hence, we analyzed the effects of OLA on canonical and non-canonical inflammasome activation using Western blot. Our results showed that after LPS-stimulation there was an increase in NLRP3 and ASC protein expression compared to unstimulated cells, also, we observed parallel results in both forms of caspase-1 (+ p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. no stimulated cells). However, in those cells treated with OLA, the expression of these proteins was negatively regulated (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS-DMSO treated cells). According to these results, we found that OLA reduced both IL-1β and IL-18 levels, in comparison with LPS-DMSO induced cells (** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells) (Figures 3A and 8C, respectively). On the other hand, we evaluated the impact of OLA on noncanonical inflammasome activation in LPS-induced murine peritoneal macrophages (Figure 8D). As expected, pro- , partially cleaved and cleaved-caspase-11 were upregulated after LPS stimulus (+ p < 0.05, ++ p < 0.01 vs. control cells). Nevertheless, OLA treatment could mitigate this activation (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO-treated cells). Figure 8. Inflammasome signaling pathway was downregulated by OLA in murine peritoneal macrophages. (A) NLRP3, (B) ASC, (C) IL-18, (D) pro-caspase-11, partially cleaved and cleaved, (E) procaspase-1 and cleaved-caspase-1. Protein expressions were analyzed by immunoblot. Murine peritoneal macrophages were pretreated for 30 min with OLA (25 and 12.5μM) followed by stimulation with 5 μg/mL LPS for 18 h. Densitometry was performed following normalization to the control (βactin housekeeping gene). Data are represented as the means ± SEM (n = 6). + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. control cells (no stimulated); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS−DMSO- treated cells. Figure 8. Inflammasome signaling pathway was downregulated by OLA in murine peritoneal macrophages. ( A ) NLRP3, ( B ) ASC, ( C ) IL-18, ( D ) pro-caspase-11, partially cleaved and cleaved, ( E ) pro-caspase-1 and cleaved-caspase-1. Protein expressions were analyzed by immunoblot. Murine peritoneal macrophages were pretreated for 30 min with OLA (25 and 12.5 µ M) followed by stimulation with 5 µ g/mL LPS for 18 h. Densitometry was performed following normalization to the control ( β -actin housekeeping gene). Data are represented as the means ± SEM (n= 6). + p< 0.05, ++ p < 0.01, +++ p< 0.001 vs. control cells (no stimulated); * p< 0.05, ** p< 0.01, *** p< 0.001 vs. LPS−DMSO-treated cells. Pharmaceuticals 2022,15, 1338 16 of 20 4.6. Measurement of Nitrite Production Cells were seeded in 24-well plates (1 × 10 6 cells/mL) and treated with different concentrations of OLA (25 and 12.5 µ M). After 30 min, peritoneal macrophages were stimulated with LPS for 18 h. Nitrites levels were determined by Griess reaction (Sigma- Aldrich®, St. Louis, MO, USA) through extrapolation from a standard curve with sodium nitrite 22. Absorbance at 540 nm was measured using an ELISA reader (BioTek ® , Bad Friedrichshall, Germany) and represented as a percentage compared with LPS-DMSO cells. 4.7. DCFDA Cellular Reactive Oxygen Species Detection Intracellular ROS were detected using a DCFDA assay kit (Abcam ® , Cambridge, UK) according to the manufacturer’s instructions. The 2.5 × 10 4 cells/well were seeded on a 96-well black plate and LPS-stimulated in presence or absence of OLA pretreatment (25 and 12.5 µ M). DCFDA (25 µ M) was added to each well at 37 ◦ C for 45 min. The excitation and emission wavelengths (485 and 535 nm, respectively), were read using a fluorescence microplate reader (Biotek ® , Bad Friedrichshall, Germany). We used H 2 O 2 (Sigma-Aldrich ® , Barcelona, Spain) as a positive pro-oxidant control (100% intracellular ROS production). 4.8. Determination of Pro-Inflammatory Cytokines by Enzyme-Linked ImmunoSorbent Assay Pro-inflammatory cytokines levels were measured on culture supernatants using IL-1 β , TNF- α (R&D System ® , Minneapolis, MN, USA), IL-17 (Peprotech ® , London, UK), IL-6, and interferon (IFN)- γ (Diaclone ® , Besancon Cedex, France) ELISA kits, according to manufacturer’s instructions. 4.9. Histone Extraction Histones were extracted using an acid extraction protocol [ 58 ] with modifications. Spleen cells were washed twice with PBS, resuspended in lysis buffer (10 mM Tris pH 6.5, 50 mM sodium bisulfite, 10 nM MgCl2, 8.6% sucrose, 1% Triton X-100) and incubated for 15 min at 4 ◦ C. Then, cells were centrifuged at 3500 × grpm for 10 min at 4 ◦ C and rewashed with lysis buffer. The supernatant was discarded and Tris-EDTA buffer (10 mM Tris pH 7.4 and 13 mM EDTA) was added to the samples. The pelleted nuclei were resuspended with acid sulfuric 0.2M. After, at least, 1 h of incubation, samples were centrifuged at 15,000×g rpm for 1 h at 4 ◦ C. Supernatants were collected and incubated overnight at − 20 ◦ C with acetone. The samples were centrifuged at 15,000 × grpm for 10 min at 4 ◦ C, pellets were diluted with H2O and the protein content was measured according to Bradford’s method [59]. 4.10. Isolation of Proteins and Western Blot Analysis Cells were lysed in ice-cold PBS containing a cocktail of protease and phosphatase inhibitors to isolate proteins. Protein concentration was determined according to the Bradford method [59] using γ-globulin as a standard. Aliquots of 15 µ g of protein were separated on 10% or 15% acrylamide gel by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated overnight at 4 ◦ C with specific primary antibodies against mPGES-1, IL-18 (Abcam ® , Cambridge, UK), COX-2, iNOS, pJNK, JNK, P38, pP38, pERK1/2, ERK 1 2 , Nrf-2, pSTAT-3, pJAK2, NLRP3, ASC, H3K18ac, H3K27me3, H3K9me3, H3 (Cell Signaling ® , Danvers, MA, USA), HO-1 (Henzo ® , Madrid, Spain), caspase-1 and caspase-11 (Novus Biologicals ® , Littleton, CO, USA). Nitrocellulose membranes were incubated for 2 h in a blocking solution with antirabbit horseradish peroxidase-labeled (HRP) secondary antibody (Cell Signaling ® , Danvers, MA, USA) or antimouse HRP secondary antibody (Dako ® , Atlanta, GA, USA). β -Actin primary antibody (Abcam ® , Cambridge, UK) was employed to demonstrate equal loading. The immunosignals were captured using Amersham Imager 600 from GE Healthcare ® (Buckinghamshire, UK) and analyzed and quantified by Image Processing and Analysis in Java (ImageJ ® , Softonic) and expressed comparing with the DMSO-LPS treated cells. Pharmaceuticals 2022,15, 1338 17 of 20 4.11. Statistical Analysis All values in the figures and text are expressed as arithmetic means ± standard error (SEM). Data were evaluated with GraphPad Prism_Version 5.01 software. The statistical significance of any difference in each parameter among the groups was evaluated by oneway analysis of variance (ANOVA), using Tukey–Kramer multiple comparisons test as post hoc test. pvalues of <0.05 were considered statistically significant. 5. Conclusions In conclusion, our study proves, for the first time, that OLA was able to modulate oxidative stress and the acute inflammatory response produced by LPS stimulation in murine peritoneal macrophages, by inhibiting the production of ROS, pro-inflammatory cytokines (TNF- α , IL-6, IL-1 β , IFN- γ , IL-17, and IL-18) and pro-inflammatory enzymes such as iNOS, COX-2, and mPGES-1. These protective effects could be due to the activation of the Nrf-2/HO- 1 axis and inhibition of JAK/STAT, ERK, and P38 MAPKs and inflammasome canonical and non-canonical signaling pathways. Moreover, OLA modulated epigenetic modifications through histone methylation (H3K9me3 adnH3K27me) and deacetylation (H3K18ac) in LPS-activated spleen cells. Despite these promising results, further in vivo studies would be necessary to explore the immunomodulatory effects of OLA, which could be a novel epinutraceutical bioproduct, useful in the management of immuno-inflammatory pathologies. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/ph15111338/s1, Figure S1: (–)-Oleacein chemical structure; Figure S2: 1 H NMR of (–)-oleacein in deuterated chloroform; Figure S3: 13 C NMR (bottom) and Dept-135 (superior) of (–)-oleacein in deuterated chloroform. Author Contributions: Conceptualization, C.A.-d.-l.-L. and M.S.-H.; methodology, R.M.-G.; product extractions and chemical synthesis, J.A. and J.O.-V.; software, R.M.-G.; validation, R.M.-G., M.A., and T.M.; formal analysis, R.M.-G.; investigation, R.M.-G.; resources, C.A.-d.-l.-L. and J.A.; data curation, R.M.-G.; writing—original draft preparation, R.M.-G. and J.O-V.; writing—review and editing, C.A.-d.-l.-L., M.S.-H., T.M. and J.A.; visualization, R.M.-G.; supervision, C.A.-d.-l.-L. and M.S.-H.; project administration, C.A.-d.-l.-L., M.S.-H. and J.A; funding acquisition, C.A.-d.-l.-L. and J.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD OF SPAIN, grant number AG-2017-89342-P and JUNTA DE ANDALUCÍA (CTS-259 and FQM-182). Institutional Review Board Statement: The study was conducted according to the Guidelines of the European Union regarding animal experimentation (Directive of the European Counsel 2012/707/EU) and following a protocol approved by the Animal Ethics Committee of the University of Seville (CEEA-US 2018-11/2 and CEEA-US 2019-6) and by the Consejeria de Agricultura, Pesca y Desarrollo (Junta de Andalucía, 23/07/2018/119), according to the RD 53/1 February 2013. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Acknowledgments: The authors gratefully acknowledge the assistance of the Center for Technology and Innovation Research, University of Seville (CITIUS), the Technical Scientific Instrumentation Center (CICT) R. Muñoz-García gratefully acknowledges support from a Postgraduate National Program of FPU fellowship and financial sponsorship from the Spanish Ministerio de Universidades; M. Alcarranza is thankful for the FPU fellowship and financial sponsorship from the Spanish Ministerio de Universidades; T. Montoya thanks support from a Postgraduate Program of PIF fellowship and financial sponsorship from VI Plan Propio de Investigación y Transferencia at the University of Seville. Conflicts of Interest: The authors declare no conflict of interest. Pharmaceuticals 2022,15, 1338 18 of 20 References 1. Funes, S.C.; Rios, M.; Escobar-Vera, J.; Kalergis, A.M. Implications of Macrophage Polarization in Autoimmunity. Immunology 2018,154, 186–195. [CrossRef] [PubMed] 2. 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