Immunomodulatory Effects of (R)-Sulforaphane on LPS-Activated Murine Immune Cells: Molecular Signaling Pathways and Epigenetic Changes in Histone Markers

The aim of this study was to explore the immunomodulatory effects of the natural enantiomer (R)-Sulforaphane (SFN) and the possible signaling pathways involved in an ex vivo model of LPS-stimulated murine peritoneal macrophages. Furthermore, we studied the epigenetic changes induced by (R)-SFN as well as the post-translational modifications of histone H3 (H3K9me3 and H3K18ac) in relation to the production of cytokines in murine splenocytes after LPS stimulation. (R)-SFN was able to modulate the inflammatory response and oxidative stress induced by LPS stimulation in murine peritoneal macrophages through the inhibition of reactive oxygen species (ROS), nitric oxide (NO) and cytokine (IL-1β, IL-6, IL-17, IL-18 and TNF-α) production by down-regulating the expression of pro-inflammatory enzymes (iNOS, COX-2 and mPGES-1). We also found that activation of the Nrf-2/HO-1 axis and inhibition of the JAK2/STAT-3, MAPK, canonical and non-canonical inflammasome signaling pathways could have been responsible for the immunomodulatory effects of (R)-SFN. Furthermore, (R)-SFN modulated epigenetic modifications through histone methylation (H3K9me3) and deacetylation (H3K18ac) in LPS-activated spleen cells. Collectively, our results suggest that (R)-SFN could be a promising epinutraceutical compound for the management of immunoinflammatory diseases.


Introduction
Specific functional food supplements, such as sulforaphane (SFN)-an isothiocyanate compound-(1-isothiocyanate-(4R)-(methylsulfinyl)butane), are currently attracting a great amount of interest due to their high biological activity. Several vegetables of the cruciferous family, such as broccoli and cauliflower, among others, present SFN in the form of glucosinolate (glucoraphanin), which is hydrolyzed in part by the action of the enzyme myrosinase during cooking or chewing and by the gut microbiota after ingestion [1].
Available data indicate that the biological activities of natural isothiocyanates are defined by their structural characteristics, as it can be observed that small changes can induce important modifications in their effects. Another feature to take into account is the oxidation state of the sulfur atom, as this can modify the potency of the compound; for example, the sulfoxide derivative (sulforaphane) is more active than the thioether I and the sulfone II derivative ( Figure S1) [2][3][4].
Even though two enantiomers are possible, the natural SFN exists as a single enantiomer with an R S absolute configuration ( Figure S1) [5].
SFN is considered a natural antioxidant and free radical scavenger and is also a potent activator of the nuclear factor erythroid 2 (Nrf-2) signaling pathway [6]. In addition, SFN Nutritional compounds are capable of modulating pathological processes, such as inflammation, by regulating gene expression without changing the genetic code, through key epigenetic mechanisms, e.g., histone acetylation/deacetylation, small non-coding RNA action and DNA methylation, as reported with recent data. Their ability to change epigenetic patterns depends directly on the interaction with enzymes that add or suppress epigenetic markers, or indirectly on the normalization of proteins encoded by genes involved in the epigenetic system [37].
Recent studies suggest that epigenetic changes, in particular histone modifications, play an important role in immune system tolerance, preventing damage by excessive inflammatory response. Indeed, Lauterbach et al. have shown that macrophages adopt their metabolism upon LPS activation by promoting histone acetylation [12].
It has been documented that rac-SFN positively influences the acetylation/deacetylation balance of histones in chromatin. The epigenetic control of histone deacetylase (HDAC) activity has clearly explained the molecular mode of action of this isothiocyanate. In fact, rac-SFN has been identified as an inhibitor of HDAC not only in cancer cells, but also in dendritic cells, cortical neurons and neural crest cells [38]. Moreover, this isothiocyanate has also been considered as a potential regulator of DNA methylation in the development of cancer and hypertension [39,40]. In addition, rac-SFN was also reported to modulate miRNA expression in colorectal cells [41]. However, the role of the natural enantiomer (R)-SFN as an epigenetic modulator in the inflammatory response generated by LPS is unknown.
Although the bioactivity of the rac-SFN compound has been previously reported in a wide variety of experimental models, as discussed above, the pharmacological effects of the natural isothiocyanate have not yet been investigated. In this context, the main objective of the present study was to evaluate the anti-inflammatory and antioxidant activity of (R)-SFN in an ex vivo model of inflammation using LPS-stimulated murine peritoneal macrophages in order to initially validate its future use as a nutraceutical compound. In particular, we aimed to characterize the mechanism of action and signaling pathways possibly involved in the potentially beneficial effects of (R)-SFN. Finally, to explore the (R)-SFN-induced epigenetic changes involved, we studied histone H3 post-translational modifications (H3K9me3, H3K27me3 and H3K18ac) and related cytokine production in spleen cells after LPS stimulation.

Effect of (R)-SFN on the Viability of Murine Peritoneal Macrophages
The cytotoxic effects of (R)-SFN on peritoneal macrophages was determined using the SRB (sulforhodamine B) assay. Cells were treated with various concentrations of (R)- Nutritional compounds are capable of modulating pathological processes, such as inflammation, by regulating gene expression without changing the genetic code, through key epigenetic mechanisms, e.g., histone acetylation/deacetylation, small non-coding RNA action and DNA methylation, as reported with recent data. Their ability to change epigenetic patterns depends directly on the interaction with enzymes that add or suppress epigenetic markers, or indirectly on the normalization of proteins encoded by genes involved in the epigenetic system [37].
Recent studies suggest that epigenetic changes, in particular histone modifications, play an important role in immune system tolerance, preventing damage by excessive inflammatory response. Indeed, Lauterbach et al. have shown that macrophages adopt their metabolism upon LPS activation by promoting histone acetylation [12].
It has been documented that rac-SFN positively influences the acetylation/deacetylation balance of histones in chromatin. The epigenetic control of histone deacetylase (HDAC) activity has clearly explained the molecular mode of action of this isothiocyanate. In fact, rac-SFN has been identified as an inhibitor of HDAC not only in cancer cells, but also in dendritic cells, cortical neurons and neural crest cells [38]. Moreover, this isothiocyanate has also been considered as a potential regulator of DNA methylation in the development of cancer and hypertension [39,40]. In addition, rac-SFN was also reported to modulate miRNA expression in colorectal cells [41]. However, the role of the natural enantiomer (R)-SFN as an epigenetic modulator in the inflammatory response generated by LPS is unknown.
Although the bioactivity of the rac-SFN compound has been previously reported in a wide variety of experimental models, as discussed above, the pharmacological effects of the natural isothiocyanate have not yet been investigated. In this context, the main objective of the present study was to evaluate the anti-inflammatory and antioxidant activity of (R)-SFN in an ex vivo model of inflammation using LPS-stimulated murine peritoneal macrophages in order to initially validate its future use as a nutraceutical compound. In particular, we aimed to characterize the mechanism of action and signaling pathways possibly involved in the potentially beneficial effects of (R)-SFN. Finally, to explore the (R)-SFN-induced epigenetic changes involved, we studied histone H3 post-translational modifications (H3K9me3, H3K27me3 and H3K18ac) and related cytokine production in spleen cells after LPS stimulation.

Effect of (R)-SFN on the Viability of Murine Peritoneal Macrophages
The cytotoxic effects of (R)-SFN on peritoneal macrophages was determined using the SRB (sulforhodamine B) assay. Cells were treated with various concentrations of (R)-SFN (200-1.6 µM) for 18 hours (h). (R)-SFN did not compromise cell viability (≥80%) at doses

(R)-SFN Down-Regulated iNOS, Nitrite Production and Intracellular ROS in LPS-Stimulated Murine Peritoneal Macrophages
To gain insight into the effects of (R)-SFN on the LPS-induced oxidative response, intracellular NO and ROS levels, as well as iNOS expression, were determined using the Griess assay, a 2′,7′-dichlorofluorescin diacetate (DCFDA) kit and Western blotting, respectively. Figure 3 shows a significant increase in the DCFDA fluorescence signal in LPS-DMSO-induced cells as a positive control (+++ p < 0.001 vs. unstimulated control cells). However, pretreatment with (R)-SFN was able to significantly attenuate ROS production, demonstrating potent antioxidant activity (*** p < 0.001 vs. LPS-DMSO cells) (Figure 3). When murine peritoneal macrophages are exposed to LPS, NO is synthesized and released into the extracellular milieu via iNOS protein expression. Due to the low half-life of NO, nitrites were measured as an indicator of NO synthesis levels. LPS activation resulted in a statistically significant increase in NO production levels compared to the unstimulated control group (+++ p < 0.001 vs. control cells (unstimulated)). As shown in Figure 3, pretreatment with 12.5 and 6.25 μM of (R)-SFN significantly reduced NO generation, which was confirmed by a reduction in iNOS expression (*** p < 0.001 vs. LPS-DMSOtreated cells).

(R)-SFN Down-Regulated iNOS, Nitrite Production and Intracellular ROS in LPS-Stimulated Murine Peritoneal Macrophages
To gain insight into the effects of (R)-SFN on the LPS-induced oxidative response, intracellular NO and ROS levels, as well as iNOS expression, were determined using the Griess assay, a 2 ,7 -dichlorofluorescin diacetate (DCFDA) kit and Western blotting, respectively. Figure 3 shows a significant increase in the DCFDA fluorescence signal in LPS-DMSO-induced cells as a positive control (+++ p < 0.001 vs. unstimulated control cells). However, pretreatment with (R)-SFN was able to significantly attenuate ROS production, demonstrating potent antioxidant activity (*** p < 0.001 vs. LPS-DMSO cells) ( Figure 3). When murine peritoneal macrophages are exposed to LPS, NO is synthesized and released into the extracellular milieu via iNOS protein expression. Due to the low half-life of NO, nitrites were measured as an indicator of NO synthesis levels. LPS activation resulted in a statistically significant increase in NO production levels compared to the unstimulated control group (+++ p < 0.001 vs. control cells (unstimulated)). As shown in Figure 3, pretreatment with 12.5 and 6.25 µM of (R)-SFN significantly reduced NO generation, which was confirmed by a reduction in iNOS expression (*** p < 0.001 vs. LPS-DMSO-treated cells).

(R)-SFN Down-Regulated iNOS, Nitrite Production and Intracellular ROS in LPS-Stimulated Murine Peritoneal Macrophages
To gain insight into the effects of (R)-SFN on the LPS-induced oxidative response, intracellular NO and ROS levels, as well as iNOS expression, were determined using the Griess assay, a 2′,7′-dichlorofluorescin diacetate (DCFDA) kit and Western blotting, respectively. Figure 3 shows a significant increase in the DCFDA fluorescence signal in LPS-DMSO-induced cells as a positive control (+++ p < 0.001 vs. unstimulated control cells). However, pretreatment with (R)-SFN was able to significantly attenuate ROS production, demonstrating potent antioxidant activity (*** p < 0.001 vs. LPS-DMSO cells) ( Figure 3). When murine peritoneal macrophages are exposed to LPS, NO is synthesized and released into the extracellular milieu via iNOS protein expression. Due to the low half-life of NO, nitrites were measured as an indicator of NO synthesis levels. LPS activation resulted in a statistically significant increase in NO production levels compared to the unstimulated control group (+++ p < 0.001 vs. control cells (unstimulated)). As shown in Figure 3, pretreatment with 12.5 and 6.25 μM of (R)-SFN significantly reduced NO generation, which was confirmed by a reduction in iNOS expression (*** p < 0.001 vs. LPS-DMSOtreated cells).

(R)-SFN Up-Regulated Nrf-2/HO-1 Axis Protein Expression in LPS-Stimulated Murine Peritoneal Macrophages
Western blotting was used to determine whether (R)-SFN could regulate the antioxidant Nrf-2/HO-1 signaling pathway. Treatment with (R)-SFN produced a significant upregulation in the expression of both proteins in comparison to the control LPS-DMSO cells (** p < 0.01; *** p < 0.001 vs. LPS-DMSO-treated cells) ( Figure 6). These results, together with the decrease in ROS production, could demonstrate the significant antioxidant activity of (R)-SFN in this preclinical model.

(R)-SFN Up-Regulated Nrf-2/HO-1 Axis Protein Expression in LPS-Stimulated Murine Peritoneal Macrophages
Western blotting was used to determine whether (R)-SFN could regulate the antioxidant Nrf-2/HO-1 signaling pathway. Treatment with (R)-SFN produced a significant up-regulation in the expression of both proteins in comparison to the control LPS-DMSO cells (** p < 0.01; *** p < 0.001 vs. LPS-DMSO-treated cells) ( Figure 6). These results, together with the decrease in ROS production, could demonstrate the significant antioxidant activity of (R)-SFN in this preclinical model.

Effects of (R)-SFN on the JAK2/STAT-3 Signaling Pathway in LPS-Activated Peritoneal Macrophages
The JAK/STAT signaling pathway plays an important role in the inflammatory response. Under conditions of cellular stress, phosphorylation of JAK2 is induced, which in turn allows phosphorylation of STAT, which is released from the receptor-binding complex and translocated to the cell nucleus to regulate the transcription of pro-inflammatory genes, including inducible enzymes or cytokines [42].

Effects of (R)-SFN on the JAK2/STAT-3 Signaling Pathway in LPS-Activated Peritoneal Macrophages
The JAK/STAT signaling pathway plays an important role in the inflammatory re sponse. Under conditions of cellular stress, phosphorylation of JAK2 is induced, which in turn allows phosphorylation of STAT, which is released from the receptor-binding com plex and translocated to the cell nucleus to regulate the transcription of pro-inflammatory genes, including inducible enzymes or cytokines [42].

(R)-SFN Inhibited the Canonical and Non-Canonical Inflammasome in LPS-Activated Murine Peritoneal Macrophages
The domain-like receptor protein 3 (NLRP3) inflammasome is a multiprotein com-plex composed of a NLRP3 sensor molecule, an adaptor caspase recruitment protein (ASC) and pro-caspase 1. In the presence of inflammatory stimuli, such as LPS in murine peritoneal macrophages, activation of this complex occurs, producing the maturation of pro-caspase 1 to caspase 1, leading to the generation of IL-1β and IL-18 due to the activation of pro-IL-1β and pro-IL-18 cytokine precursors. In addition, the non-canonical pathway is also activated by LPS, which triggers the activation of pro-caspase 11 to caspase 11 and, consequently, the generation of IL-1β and IL-18 maturation, as already mentioned [43]. Accordingly, the effects of (R)-SFN on canonical and non-canonical activation of the inflammasome were determined by Western blotting.

(R)-SFN Produced Epigenetic Histone Modifications in Spleen Cells
Finally, in order to explain the role of (R)-SFN in post-translational modification in H3K27me3, H3K18ac and H3k9me3 histones and its correlated cytokine production, we evaluated epigenetic modifications in spleen cells by Western blotting.

(R)-SFN Produced Epigenetic Histone Modifications in Spleen Cells
Finally, in order to explain the role of (R)-SFN in post-translational modification in H3K27me3, H3K18ac and H3k9me3 histones and its correlated cytokine production, we evaluated epigenetic modifications in spleen cells by Western blotting.
In addition, the opposite behaviour was observed in histone methylation. In fact, the levels of H3K9me3 and H3k27me3 were significantly reduced when the spleen cells were stimulated with LPS (++ p < 0.01; +++ p < 0.001 vs. unstimulated control cells) ( Figure  10E,F); however, the treatment with (R)-SFN only statistically raised the H3k9me3 expression (* p < 0.05 vs. LPS-DMSO-treated cells), while the H3k27me3 immunosignal remained unchanged.

Discussion
The results of our study confirmed that the natural enantiomer (R)-SFN was able to induce immunomodulatory effects in LPS-activated murine peritoneal macrophages. These data are in agreement with the only study we have located in the literature that showed immunomodulatory activities of (R)-SFN in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from healthy adults [44].
LPS-stimulated macrophages alter the intracellular redox balance, leading to the production of ROS. This plays a critical role in the pathogenesis of oxidative stress-related diseases. LPS induces ROS expression through several mechanisms, such as activation of NADPH oxidase and inhibition of antioxidant enzymes. Upon LPS stimulation, iNOS expression increases and high levels of NO are produced, which acts as an intracellular messenger modulating ROS production [45]. Interestingly, our results showed that (R)-SFN treatment was not only able to decrease these oxidation-related mediators, but that it also reduced intracellular ROS generation. Ranaweera et al. obtained similar results with rac-SFN by inhibiting LPS-induced ROS, NO and iNOS production in RAW 264.7 cells [23].
LPS-mediated activation is closely related to an imbalance of the cytokine network leading to the production of pro-inflammatory Th1 and Th17 cytokines, including IL-6, IL-1β, TNF-α and IL-17, among others [46,47]. Consistent with these observations, our data showed a marked increase in pro-inflammatory cytokine expression following LPS stimulation. However, treatment with (R)-SFN significantly decreased the secretion of the cytokines IL-1β, IL-6, TNF-α, IL-18 and IL-17. Similar data have been reported after treatment with the racemic compound in LPS-stimulated RAW 264.7 macrophages [21,23] and after treatment with (R)-SFN in LPS-activated PBMC populations in healthy adult volunteers [44].

Discussion
The results of our study confirmed that the natural enantiomer (R)-SFN was able to induce immunomodulatory effects in LPS-activated murine peritoneal macrophages. These data are in agreement with the only study we have located in the literature that showed immunomodulatory activities of (R)-SFN in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from healthy adults [44].
LPS-stimulated macrophages alter the intracellular redox balance, leading to the production of ROS. This plays a critical role in the pathogenesis of oxidative stress-related diseases. LPS induces ROS expression through several mechanisms, such as activation of NADPH oxidase and inhibition of antioxidant enzymes. Upon LPS stimulation, iNOS expression increases and high levels of NO are produced, which acts as an intracellular messenger modulating ROS production [45]. Interestingly, our results showed that (R)-SFN treatment was not only able to decrease these oxidation-related mediators, but that it also reduced intracellular ROS generation. Ranaweera et al. obtained similar results with rac-SFN by inhibiting LPS-induced ROS, NO and iNOS production in RAW 264.7 cells [23].
LPS-mediated activation is closely related to an imbalance of the cytokine network leading to the production of pro-inflammatory Th1 and Th17 cytokines, including IL-6, IL-1β, TNF-α and IL-17, among others [46,47]. Consistent with these observations, our data showed a marked increase in pro-inflammatory cytokine expression following LPS stimulation. However, treatment with (R)-SFN significantly decreased the secretion of the cytokines IL-1β, IL-6, TNF-α, IL-18 and IL-17. Similar data have been reported after treatment with the racemic compound in LPS-stimulated RAW 264.7 macrophages [21,23] and after treatment with (R)-SFN in LPS-activated PBMC populations in healthy adult volunteers [44].
Exposure of peritoneal macrophages to LPS induces overexpression of the inducible COX isoform (COX-2) and mPGES-1, which are involved in the overproduction of PGE2 responsible for increased chemotaxis, blood flow and subsequent tissue dysfunction during inflammation through activation of the EP4 receptor [48]. In agreement with our previous reports by Montoya et al., LPS stimulation significantly increased protein expression of COX-2 and mPGES-1 [49,50]. However, (R)-SFN decreased the immunosignal of both proteins. In this regard, other studies have shown that rac-SFN was able to inhibit COX-2 production in LPS-stimulated RAW 264.7 macrophages [23,51].
In addition, in order to further investigate the mechanism of action involved in (R)-SFN immunomodulatory effects, we studied several intracellular signaling pathways. Nrf-2 is an important modulator of cellular detoxification responses and redox status and is able to respond by translocating to the nucleus and interacting with the specific antioxidant responsive element (ARE) present in the promoter gene encoding detoxifying and antioxidant proteins, such as HO-1. Indeed, this enzyme plays a cytoprotective role against oxidative stress and inflammation in LPS-activated macrophages. In line with previous studies, we found that the Nrf-2/HO-1 signaling pathway was down-regulated after LPS treatment [49,50].
SFN is a known activator of Nrf-2 that directly induces Nrf-2 translocation and accumulation in the nucleus and may be phosphorylated by the activation of multiple kinases that stimulate the expression of detoxification enzymes, such as HO-1 [52]. Our results indicated that (R)-SFN treatment significantly increased the expression of both Nrf-2/HO-1 proteins. Although HO-1 also influences the anti-inflammatory action of SFN independently of the Nrf-2 pathway [53], our findings suggest that activation of the Nrf-2/HO-1 signaling pathway could be partly responsible for the anti-inflammatory activity of (R)-SFN by reducing the expression of pro-inflammatory enzymes, in particular COX-2 and iNOS, and other pro-inflammatory mediators, including TNF-α, IL-6, IL-1β, PGE2 and NO. Similar results were obtained with rac-SFN in a murine model of LPS-induced acute lung injury inflammation [24] and in LPS-activated microglia [54].
Furthermore, we proceeded to explore other effector signals, such as the JAK2/STAT-3 and MAPK signaling pathways.
JAK2 is a cytoplasmic tyrosine kinase responsible for the activation of STAT-3 prior to its phosphorylation and dimerization. Phosphorylated STAT-3 is then translocated to the nucleus to initiate the transcription of target genes. The JAK/STAT axis is known to play an important role in cytokine receptor signaling [16]. Therefore, we studied the possible involvement of the JAK2/STAT-3 signaling pathway in the immunomodulatory effects of (R)-SFN. Consistent with our previous studies, incubation with LPS promoted the phosphorylation of STAT-3 proteins in murine peritoneal macrophages [49,55]. In contrast, (R)-SFN was able to reduce this phosphorylation. Thus, our results consistently demonstrated that the natural isothiocyanate could down-regulate inflammatory cytokine expression by inhibiting the JAK2/STAT-3 signaling pathway in LPS-activated cells.
As mentioned above, MAPKs play a key role in macrophage activation by LPS. They are a family of serin-threonine kinases, including c-Jun N-terminal kinases (JNKs), p38 and extracellular signal-regulated kinases (ERKs-1 and -2) [56]. In response to inflammatory and oxidative stress stimuli, MAPKs can modulate the JAK/STAT pathway by inducing the expression of multiple genes that together regulate the expression of inflammatory chemokines and cytokines [57]. Our results in agreement with those of our recent reports indicated that after the incubation of macrophages with LPS, phosphorylation of JNK, p38 and ERK was statistically increased [55]. However, (R)-SFN treatment counteracted the effects of LPS by significantly decreasing the activation of JNK, ERK and p38 MAPKs. These data suggest that inactivation of MAPKs by (R)-SFN may be partly responsible for the down-regulation of the Th1-and Th17-mediated inflammatory response. Similar results have been obtained with rac-SFN in other experimental models, such as primary mouse bone-marrow-derived macrophages (BMDMs), human THP-1-derived macrophages and primary human peripheral blood mononuclear cell-derived macrophages [57].
A key role in pathogen defense and cytosolic innate immune sensing is played by the inflammasomes-intracellular complexes that have been extensively studied. Regarding the NLR family, the pyrin domain-containing protein 3 (NLRP3) inflammasome comprises three elements: a sensor (NLRP3), an adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and a zymogen (pro-caspase 1). "Canonical inflammasome" is a multimeric complex formed by the activation of NLRP3, allowing the recruitment of ASC, which couples with pro-caspase 1, leading to its activated form, caspase 1. This caspase is able to contribute to the maturation and secretion of IL-18 and IL-1β, inducing pyroptosis and inflammatory cell death [58]. The non-canonical pathway is another activation mechanism for NLRP3 complex formation. In murine macrophages, in the presence of LPS, caspase 11 matures, resulting in potassium efflux leading to NLRP3 activation and pyroptosis. Thus, mature caspase 11 cooperates with the canonical NLRP3 inflammasome [59].
Our previous reports have shown that LPS-induced inflammation leads to activation of the NLRP3 inflammasome in macrophages [49,55]. Accordingly, in our research, inflammation induced by LPS led to the activation of canonical and non-canonical inflammasomes, as characterized by an increased expression of NLRP3, ASC, caspase 1 and caspase 11 protein expression, which was accompanied by an enhancement of IL-18 and IL-1β production.
Surprisingly, we show for the first time in this investigation that (R)-SFN treatment suppressed the activation of both canonical and non-canonical inflammasome signaling pathways after LPS activation in murine peritoneal macrophages. Thus, we provide evidence confirming that (R)-SFN modulates inflammatory responses by inhibiting the NLRP3 inflammasome via caspase 1 and caspase 11.
Epigenetic aberrations, including DNA methylation, histone modifications and miRNA deregulation, play a pivotal role in the development of inflammation. Recent studies have suggested that H3 post-translational modifications could influence pro-inflammatory cytokine production. In particular, histone acetylation is generally associated with increased gene expression due to decreased histone-DNA interaction; consequently, chromatin opens up and DNA accessibility increases, inducing gene transcription. In fact, Zhao et al. have suggested that NLRP3 inflammasome activation depends on the level of histone H3 acetylation [71]. On the other hand, methylation of H3K9me3 and H3K27me3 generally leads to suppressed gene expression by closing chromatin [72]. Furthermore, interesting studies have shown that histone methyltransferases contribute synergistically to inflammasome activation, collaborating in the inflammasome-induced inflammatory response [73].
In agreement with our results, increased H3K18ac and decreased H3K9me3 and H3K27me3 could be involved in LPS-induction of pro-inflammatory cytokine production in spleen cells. Nonetheless, the treatment with (R)-SFN regulated both H3K18ac and H3K9me3 expression and subsequently controlled IL-6, IL-1β and IL-17 production.
To summarize, (R)-SFN was able to modulate the inflammatory response and oxidative stress induced by LPS stimulation in murine peritoneal macrophages through the activation of the Nrf-2/HO-1 axis and inhibition of JAK2/STAT-3, ERK, JNK, p38 MAPKs and canonical and non-canonical inflammasome signaling pathways. Finally, the treatment with (R)-SFN down-regulated H3K18ac and up-regulated H3K9me3 expression and subsequently reduced IL-6, IL-1β and IL-17 production in spleen cells.

Reagents and Instruments
Oven-dried glassware and dried solvents were used for the reactions, which were run under an atmosphere of dry argon. Chemicals were purchased from commercial sources and were used without further purification. Compounds were detected by thin-layer chromatography (TLC), which analyses were carried out on silica gel GF254 (Merck) and charred with phosphomolybdic acid/EtOH. Merck 230-400 mesh silica gel was used for flash chromatography. Positive pressure of air was used to elute chromatographic columns, and eluents are given as volume-to-volume ratios (v/v). Optical rotations were determined with a PerkinElmer 341 polarimeter. Nuclear magnetic resonance (NMR) spectra were recorded with Bruker Avance 500 MHz spectrometers. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. High-resolution mass spectra (HRMS) were recorded at the Centro de Investigación, Tecnología e Innovación of the University of Seville with a Kratos MS-80RFA 241-MC apparatus.

Synthesis of 4-Azidobutyl-1-thioacetate (3)
To a solution of 4-azidobutyl methanesulfonate 2 (30.26 g, 156.61 mmol) in dry DMF (500 mL) under argon atmosphere, potassium thioacetate (23.3 g, 203.59 mmol) was added at room temperature. The reaction was left stirring overnight. Then, water was used to wash the mixture and EtOAc was used to extract it three times. NaHCO 3 aqueous solution and brine were used to wash the combined organic phases. Afterwards, the product was dried over anhydrous Na 2 SO 4 and evaporated to obtain 24.7 g (142.51 mmol, 91% yield) of 3 as a brown oil, which was used without further purification [33,35].

Synthesis of 4-Azidobutane-1-sulfinyl chloride (4)
To a solution of thioacetate 3 (13.3 g, 76.84 mmol) in methylene chloride (67 mL) at −20 • C, acetic anhydride (7.3 mL, 7.84 mmol) and sulfuryl chloride (12.4 mL, 153.67 mmol) were added. The resulting mixture was stirred for 1 h at −5 • C, then the solvent was evaporated and the residue was dried under vacuum to give 14.0 g (76.82 mmol, quantitative yield) of 4 as a black low-melting point solid. The crude sulfinyl chloride, which was kept under argon, was used in the following reaction without further purification, in order to preparate sulfinate esters [33,35]. Hydrogen-1 NMR (300 MHz, CDCl 3  60 mmol) was added, while the reaction mixture was vigorously stirred. An hour later, the reaction mixture was treated with 1 M HCl aqueous solution and extracted with CH 2 Cl 2 . Saturated NaHCO 3 aqueous solution and brine were used to successively wash the combined organic layers. Then, the product was dried over anhydrous Na 2 SO 4 and evaporated to obtain S sulfinate as the major diastereomer with a 94% diastereomeric excess. The crude was purified by column chromatography (hexane/2-propanol 20:1) to give 1.5 g (3.74 mmol, 87% yield) of diastereomerically pure 5-(S) as a yellow oil [33,35].  (15 mL), at 0 • C. Saturated NH 4 Cl aqueous solution was added after stirring for 1 h at 0 • C. CH 2 Cl 2 was used to extract the aqueous layer and then the organic layers obtained were combined, dried on anhydrous Na 2 SO 4 and concentrated. The crude product was purified by column chromatography (EtOAc/MeOH 12:1) to give 127 mg of 6-(R) (0.79 mmol, 93% yield) as a colourless liquid [33,35]. Triphenylphosfine (343 mg, 1.31 mmol) was added to a solution of azide 6-(R) (140 mg, 0.69 mmol) in Et 2 O (5 mL), followed by refluxing for an hour. Carbon disulfide (3 mL) was added after removing the solvent at vacuum, leaving the reaction reflux for 3 h. Finally, the solvent was removed under vacuum and the crude product was purified by column chromatography (EtOAc/MeOH 9:1) to give 149 mg of (R)-sulforaphane (0.68 mmol, 98% yield) as a colourless liquid [33,35].

Isolation and In Vitro Culture of Murine Peritoneal Macrophages and Spleen Cells
The method used was as described by Aparicio-Soto et al. [74]. After a week of acclimatization of the animals, 1 mL of sodium thioglycolate solution (3.8% w/v) (BD Difco, Le Pont de Claix, France) was administered to the mice, leaving food and drink ad libitum. After 72 h, the mice were sacrificed by CO 2 exposure and the peritoneal cavities were washed with cold sterile phosphate-buffered saline solution (PBS) to obtain cellular exudates. Once the cells were centrifuged, they were resuspended in RPMI1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in the presence of 100 mg/mL streptomycin and 100 U/mL penicillin and seeded onto plates (1 × 10 6 cells/mL) for 2 h at 37 • C in a humidified atmosphere of 5% CO 2 . After 2 h, non-adherent cells were eliminated by washing with PBS, and fresh RPMI 1640 without FCS containing 6.25 or 12.5 µM of (R)-SFN was added. Thirty minutes later, the cells were stimulated with 5 µg/mL of LPS from Escherichia coli (Sigma-Aldrich, St. Louis, MO, USA) for 18 h in the presence or absence of the study compound. Ultimately, the supernatants were collected.
The spleens were extracted, crushed and filtered using a nylon cell filter (70 µm) (BD ® Biosciences, Franklin Lakes, NJ, USA) to prepare a cell suspension. Splenocytes were cultivated in supplemented RPMI1640 culture medium (10% FCS, 100 mg/mL streptomycin and 100 U/mL penicillin, 2 mM L-glutamine, 50 µM 2-mercaptoethanol and 1 mM sodium pyruvate). The cell suspension was centrifuged and resuspended in red cell lysis buffer to remove erythrocytes (BD ® Biosciences, Franklin Lakes, NJ, USA). Subsequently, the cells were washed with PBS. Finally, the cells were centrifuged and resuspended in RPMI 1640 supplemented medium to obtain a cell suspension of 3 × 10 6 cells/mL and were cultured in 24-well plates at 37 • C in humidified air with 5% CO 2 for 24 h in the presence or absence of (R)-SFN (12.5 or 6.25 µM) and LPS (5 µg/mL). Once the culture time had passed, the cells were centrifuged and the cell pellets and supernatants were collected and stored at −80 • C until the ELISA assays and histone extractions were conducted.

Cell Viability Assay
Cellular viability was assessed by the SRB assay [75]. Cells were cultured at 1 × 10 5 cells/well and incubated in the presence or absence of (R)-SFN (200 µM-1.6 µM) for 18 h at 37 • C in a 5% CO 2 humidified atmosphere. After the incubation time, adherent cells were fixed in situ with 50 µL of cold trichloroacetic acid (Sigma-Aldrich ® , St. Louis, MO, USA) solution (50% w/v) and incubated for 1 h at 4 • C. The supernatant was discarded, and the plates were washed with deionized water 5 times and allowed to air dry. Then, 100 µL of the SRB (Sigma-Aldrich ® , St. Louis, MO, USA) solution (0.4% w/v) was added to each well, followed by incubation in the dark and at room temperature for 30 min. The supernatant was discarded and washed 5 times with 1% acetic acid (Panreac ® , Barcelona, Spain), allowing it to air dry. Finally, 100 µL per well of 10 mmol/L Tris base (pH 10.5) (Sigma-Aldrich ® , St. Louis, MO, USA) was added. The cell viability was quantified by optical density at 492 nm using a multi-well plate reader spectrophotometer (BioTek ® , Bad Friedrichshall, Germany). Cell survival was measured as a percentage of absorbance by comparing treated cells with control cells (untreated, 100% cell survival).
In each experiment, the viability was always ≥80%. (R)-SFN stock solutions were prepared in DMSO (Sigma-Aldrich ® , St. Louis, MO, USA) and diluted to achieve the desired concentrations in the culture medium. The DMSO concentration in the culture medium was ≤1% in all experiments, which had no significant effects on cells.

Measurement of Nitrite Production
To a 96-well plate together with a standard curve of sodium nitrite, 100 µL of cell supernatants were transferred and subsequently mixed with Griess reagent (Sigma-Aldrich ® , St. Louis, MO, USA). The plate was then incubated at room temperature for 15 min. Finally, the absorbance at 540 nm was measured using an ELISA reader (BioTek ® , Bad Friedrichshall, Germany). The amount of nitrite was determined as the NO generation index and obtained by extrapolating a standard curve with sodium nitrite. The results were expressed as the percentage of nitrite production compared to DMSO-LPS cells (stimulated untreated cells).

Detection of DCFDA Cell-Reactive Oxygen Species
The quantification of the intracellular ROS concentration was performed using a DCFDA assay kit (Abcam ® , Cambridge, UK), according to the manufacturer's instructions. For this, 2.5 × 10 4 cells/well were seeded in a black 96-well plate pretreated with (R)-SFN (12.5 and 6.25 µM). Thirty minutes later, both treated and untreated cells were stimulated with LPS. Next, DCFDA (25 µM) was added to each of the wells and the cells were incubated at 37 • C for 45 min. Excitation and emission wavelengths (485 and 535 nm, respectively) were read using a fluorescence microplate reader (SynergyTM HTX BioTek ® , Bad Friedrichshall, Germany). H 2 O 2 (Sigma-Aldrich ® , Cambridge, UK) was used as a positive pro-oxidant control (100% intracellular ROS production).

Determination of Pro-Inflammatory Cytokines
The supernatants obtained from the murine peritoneal macrophage and murine splenocyte culture were collected 18 or 24 h after LPS stimulation. Subsequently, they were processed by ELISA kits to determine levels of IL-1β (BD OptEIA ® , San Jose, CA, USA), IL-6 (Diaclone ® , Besacon Cedex, France), IL-17 and TNF-α (Peprotech ® , London, UK), according to the manufacturer's instructions. Results were expressed in pg/mL.

Histone Extraction
The method used was as described by Hajji et al., based on acid extraction of histones, with slight modifications [76]. Splenocytes were washed twice with cold PBS. Cells were centrifuged 900× g for 5 min at 4 • C. Then, the pellet was resuspended with 1 mL of Lysis buffer (10 mM Tris pH 6.5, 50 mM sodium bisulfite, 10 nM MgCl 2 , 8.6% sucrose, 1% Triton X-100), incubated on ice for 15 min. Once the time had elapsed, it was centrifuged at 3500 rpm for 10 min at 4 • C, after which the supernatant was discarded and 1 mL of Tris-EDTA buffer (10 mM Tris pH 7.4 and 12 mM EDTA) was added to the sample. The pellet was resuspended with sulfuric acid 0.2 M. Subsequently, with at least 1h of incubation, the samples were centrifuged at 15,000 rpm for 1 h at 4 • C. The supernatants were collected and incubated with acetone overnight at −20 • C. Finally, the samples were centrifuged at 15,000 rpm for 10 min at 4 • C, the supernatant was discarded and the pellet was resuspended in H 2 O milli-Q, after which the protein content was determined following the method described by Bradford et al. [77].
Immune signals were captured using the Amersham TM Imager 600 from GE Healthcare ® (Buckinghamshire, UK), and densitometric data were studied after normalization to the housekeeping loading control. The signals were analyzed and quantified by Image Processing and Analysis in Java (Image J ® , Bethesda, MD, USA) and expressed in relation to the DMSO-LPS treated cells.

Statistical Evaluation
Data were evaluated with GraphPad Prism version 5.01 software (San Diego, CA, USA). All values in the figures and text are expressed as arithmetic means ± SEMs. Oneway analysis of variance (ANOVA) was used to evaluate the statistical significance of any difference in each parameter between groups, and after that the Tukey-Kramer multiple comparison test was used as a post hoc test. p-values < 0.05 were considered statistically significant. Experiments were carried out in triplicate, and specifically, in the densitometry assays, the figures shown are representative of at least six different experiments performed on different days.

Conclusions
Taken together, these findings demonstrate, for the first time, that (R)-SFN was able to modulate the inflammatory response and oxidative stress induced by LPS stimulation in murine peritoneal macrophages, reducing pro-inflammatory enzyme expression (iNOS, COX-2 and mPGES-1) and cytokine production (IL-1β, IL-6, IL-17, IL-18 and TNF-α) via inhibition of MAPK, JAK2/STAT-3 and canonical and non-canonical inflammasome signaling pathways. Additionally, this natural isothiocyanate could reduce NO and ROS levels and up-regulate the Nrf-2/HO-1 axis. Lastly, in spleen cells activated by LPS, (R)-SFN modulated epigenetic changes through histone methylation (H3K9me3) and deacetylation (H3K18ac). Consequently, (R)-SFN could be a new epinutraceutical compound useful for the management of several immunoinflammatory diseases.