Anti-inflammatory mechanisms of neovestitol from Brazilian red propolis in LPS-activated macrophages

Journal of Functional Foods 36 (2017) 440–447

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Anti-inflammatory mechanisms of neovestitol from Brazilian red propolis in LPS-activated macrophages Bruno Bueno-Silva a,1, Pedro L. Rosalen b, Severino M. Alencar c, Marcia P.A. Mayer a,⇑ a

Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Zip-code: 05508-900, São Paulo, SP, Brazil Piracicaba Dental School, University of Campinas – UNICAMP, Department of Physiologic Science, C.P. 52, Zip-code: 13414-903, Piracicaba, SP, Brazil c College of Agriculture ‘‘Luiz de Queiroz” (ESALQ), University of São Paulo, C.P. 9, Zip-code: 13418-900, Piracicaba, SP, Brazil b

a r t i c l e

i n f o

Article history: Received 27 March 2017 Received in revised form 14 June 2017 Accepted 10 July 2017

Chemical compounds studied in this article: Neovestitol (PubChem CID44257510) Keywords: Natural products Propolis Cytokines Inflammation Nutraceutical

a b s t r a c t Neovestitol is considered one of the main bioactive components of Brazilian red propolis. Neovestitol’s antimicrobial and antioxidant effects have already been demonstrated. Here, neovestitol immune modulatory effects were investigated on LPS activated macrophages. RAW264.7 murine macrophages activated with LPS were treated with neovestitol and NO production, cell viability and cytokines profile were determined. Activation of inflammatory signaling pathways and macrophage polarization were determined by RT-qPCR and Western blot. Neovestitol at 0.22 mM inhibited NO production by 60% without affecting cell viability and reduced GM-CSF, IFN-c, IL-1b, IL-4, TNF-a and IL-6 levels, whereas increased IL-10 production. These cytokines profile changes were associated with the downregulation of transcription of genes involved in nitric oxide production, NF-jB, IL-1b, and TNF-a signaling pathways. NF-jB and MAPK signaling pathways inhibition and decreased levels of TIRAP were further confirmed by Western blot. Neovestitol, as a nutraceutical, is a potential candidate to modulate chronic inflammation in humans. Ó 2017 Published by Elsevier Ltd.

1. Introduction Brazilian red propolis (BRP) is a resinous natural product collected by Apis mellifera bees in Alagoas state (Northeast region, Brazil) (Silva et al., 2008). Natural products are proven rich sources of bioactive compounds with distinct therapeutic properties (Cragg & Newman, 2013). These agents can be used as nutraceuticals as long as they lack toxic effects and provide physiological benefits or protection against chronic diseases (Massaro, Scoditti, Carluccio, & De Caterina, 2010; Nicoletti, 2012). BRP presents pharmacological properties such as antimicrobial (Bueno-Silva, Marsola, Ikegaki, Alencar, & Rosalen, 2017; Silva et al., 2008), antibiofilm, anticaries (Bueno-Silva, Koo, et al., 2013), antioxidant (Oldoni et al., 2011) and anti-inflammatory Abbreviations: BRP, Brazilian red propolis; DAMP, danger-associated molecular patterns; MAMP, microbe-associate molecular patterns; TIRAP, Toll-Interleukin 1 Receptor; TIR, Domain Containing Adaptor Protein. ⇑ Corresponding author at: Lineu Prestes, 1374 Cidade Universitária, São Paulo, SP, Brazil. E-mail address: [email protected] (M.P.A. Mayer). 1 Present address: Dental Research Division, Guarulhos University, Guarulhos, Zip-code: 07023-070, São Paulo, SP, Brazil. http://dx.doi.org/10.1016/j.jff.2017.07.029 1756-4646/Ó 2017 Published by Elsevier Ltd.

(Bueno-Silva, Alencar, et al., 2013; Bueno-Silva et al., 2016). We have recently shown that BRP is able to inhibit lipopolysaccharide (LPS) signaling in macrophages (Bueno-Silva et al., 2015), although the bioactive component associated with this activity was not determined. Neovestitol is an isoflavonoid shown to be a major bioactive component (Bueno-Silva, Alencar, et al., 2013; Bueno-Silva, Koo, et al., 2013; Inui et al., 2014; Righi et al., 2011) of BRP. It was isolated for the first time in 1976 from fungus-infected stems of Cajanuscajan, an African leguminosae plant (Ingham, 1976) and remained unnoticed until few years ago, when chemical studies revealed the presence of neovestitol in the Cuban red propolis (Campo Fernandez et al., 2008; Cuesta-Rubio et al., 2007). Our group has demonstrated that BRP from the Northeast region is a very unique propolis with isoflavonoids in its chemical profile, such as neovestitol, a major bioactive constituent. Some of the BRP therapeutic properties are shared by neovestitol, indicating its potential use in new drugs or alimentary formulations (Bueno-Silva, Alencar, et al., 2013; Bueno-Silva, Koo, et al., 2013; Franchin et al., 2016). Exacerbated inflammatory responses are common features in several human diseases, such as diabetes, cardiovascular diseases

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and cancer, and new therapeutic agents targeting different inflammatory pathways are needed (Dinarello, Simon, & van der Meer, 2012). Previous data reported the anti-inflammatory properties of neovestitol due to its ability to reduce neutrophil migration in an animal model (Bueno-Silva, Alencar, et al., 2013). Thus, in the present study, we demonstrated that neovestitol inhibits LPS induced response by macrophages and determined the molecular bases for this inhibition by gene expression analysis and evaluation of inflammatory pathways activation.

2. Material and methods 2.1. Bio-guided fractionation of Brazilian red propolis to obtain neovestitol Neovestitol was obtained from BRP according to bio-guided fractionation method (Bueno-Silva, Alencar, et al., 2013) and diluted in DMSO (1:500) in concentrations ranging from 0.11 to 0.38 mM.

2.2. Eukaryotic cells The monocytic cells line Raw 264.7, derived from murine tumors (leukemia) induced with Abelson leukemia virus (Raschke, Baird, Ralph, & Nakoinz, 1978), were cultured and maintained in DMEM (Cultilab, Campinas, Brazil) supplemented with 10% fetal bovine serum, 1% antibiotic solution [1000 U/ml penicillin G (ICN Biomedicals, Irvine, CA, USA) and 100U/ml streptomycin sulfate (Calbiochem, Darmstadt, Germany)].

2.3. Raw 264.7 cells treatment, cell viability and nitric oxide (NO) production 500 ng/mL LPS from E. coli serotype O111:B4 (Sigma, St. Louis, MI, USA) were used to activate Raw 264.7 cells (1  105 cells/well) in 96-well plate. At the same time, aliquots of neovestitol ranging from 0.11 to 0.38 mM (30–100 mg/ml) were added to each well. Plates were then incubated for 48 h at 37 °C in 5% CO2. Wells containing cells plus vehicle (DMSO) with and without LPS were used as controls. NO production was determined by measuring nitrite in cell culture supernatants with the use of the Griess reagent (Sigma, St. Louis, MI, USA). Results were expressed as mM of NO2. Cells viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-dip henyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MI, USA) method. Results were expressed as% of viable cells and viability of cells plus vehicle (DMSO) was considered as 100% (AndoSuguimoto et al., 2014). The lower neovestitol concentrations which inhibited nitric oxide production without affecting cell viability in LPS activated macrophages were used to determine cytokines profiles (0.18 and 0.22 mM), gene expression and signaling pathways activation (0.22 mM), and compared with negative control (cells with the addition of vehicle).

2.4. Cytokines production Levels of IL-12, Granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-c, IL-1b, IL-4, IL-10, IL-13, Transforming growth factor-beta (TGF-b), tumor necrosis factor-alpha (TNF-a) and IL-6 in cell supernatants were determined by ELISA, by using commercial kits (Becton-Dickinson, San Diego, CA, USA), and data were expressed in qg/ml.

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2.5. Gene expression Total RNA was extracted with the aid of RNA extraction kit (Qiagen, Hilden, Germany). RNA was obtained from 3 wells of each experiment. cDNA was generated by reverse transcription with 1 lg of RNA using RT2 First Strand Kit (Qiagen). Gene expression was determined by real time PCR using arrays for mouse common cytokines (PAMM-021CZ), mouse Signal Transduction Pathway (PAMM 014CZ), mouse PI3K-AKT Signaling Pathway (PAMM058CZ) and nitric oxide signaling pathway (PAMM-062CZ) (Qiagen), totalizing 360 evaluated genes. Differences in target genes transcripts were determined relative to the mean cycle threshold (CT) values of five different calibrator genes (gusb, hprt, hsp90ab1, gapdh and actb) using the DDCT method (Huggett, Dheda, Bustin, & Zumla, 2005). mRNA levels of arg1, mrc1, cd80 and cd86, relative to gapdH levels, were determined, using the comparative method of DDCT (Pfaffl, 2001) and indicated macrophage polarization at M1 or M2. 2.6. Activation of signaling pathways associated with LPS response Raw 264.7 macrophages were lysed by re-suspending in loading dye (BioRad, Hercules, CA, USA) and heated. Proteins were resolved on 12% SDS Bis-acrylamide -Tris gel, blotted into nitrocellulose membrane (Life Technologies). Western Blot was performed with primary antibodies to the phosphorylated Factor nuclear kappa B [(pNF-jB p65) (Ser536) (93H1)], pC-Fos (Ser 32) (5348), pp42/44 (phospho Mitogen Activated Protein Kinases (MAPK) – p42/44 (Thr180/Tyr182)-4631S) and Toll-Interleukin 1 Receptor (TIR) Domain Containing Adaptor Protein (TIRAP) (Invivogen 482300, San Diego, CA, USA) at 1: 1000 dilution with Tris-buffered saline, 0.1% Tween 20 plus 5% skin milk, according to manufacturer guidelines. Anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2118) (Sigma-Aldrich, St. Louis, MI, USA) was used as control. Anti-rabbit IgG (Sigma-Aldrich) was diluted at 1: 2000 and used as the secondary antibody. The autoradiograms were produced with the aid of Lumax X-ray Cassete – IBF (São Paulo, Brazil), photographed and bands intensity compared visually. 2.7. Statistical analysis Differences in cell viability, NO and cytokines levels among the groups were determined by variance test analysis (One-Way ANOVA), with the aid of Biostat Software. Differences in gene expression were determined by using the tools at the SABiosciences Technical Core website (SABiosciences/Qiagen Corp., Frederick, MD, USA), and considered significant when p < 0.05. Student’s t-test was used to assess differences in gene transcription profiles between control and experimental groups.

3. Results 3.1. NO quantification and cells viability Neovestitol decreased cells viability at concentrations ranging from 0.26 to 0.38 mM, as shown in Fig. 1B and D for cells not activated and activated with LPS, respectively. The lower concentrations of neovestitol (0.15 to 0.22 mM) were able to decrease NO production in LPS activated cells without affecting cell viability (Fig. 1A and C). Neovestitol at 0.18 and 0.22 mM had no effect on NO release and cell viability in non-LPS activated cells. However, higher concentrations of neovestitol (0.26 to 0.38 mM) promoted a reduction in cell viability and induced NO production in nonLPS activated cells.

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Fig. 1. Quantification of NO production and cell viability of Raw 264.7 cells treated with neovestitol during 48 h. A: NO quantification of non-activated cells; B: cell viability of non-activated cells; (C) NO quantification of activated cells and (D) activated cells viability. Results are expressed as Means followed by Standard Deviation of three experiments performed in triplicate. (*) Indicates significance statistically difference compared to control group by One–way ANOVA analysis (p < 0.05).

3.2. Cytokines in cell supernatant

4. Discussion

Since neovestitol at 0.18 and 0.22 mM were the lowest concentrations to promote a reduction in NO release without any effect on viability of LPS activated cells, these concentrations were selected for the determination of the effect of neovestitol on cytokines profile of LPS activated cells. The treatment of LPS activated cells with neovestitol at 0.18 mM promoted a significant reduction in IL-1b, TNF-a, IFN-c, IL-4 levels (Fig. 2). Furthermore, the treatment of these cells with neovestitol at 0.22 mM inhibited the production of IL-1b, TNF-a, IFN-c, IL-4, IL6 and GM-CSF, and induced the production of IL-10 (Fig. 2), whereas levels of IL-13, IL-12 and TGF-b were not affected (data not shown).

Inflammation is a human host-defense mechanism against internal or external danger signs. It can be activated by different stimulus such as physical injuries and bacterial products, which release danger-associated molecular patterns (DAMP) or microbe-associate molecular patterns (MAMP) that are sensed by resident tissue macrophages, and differentiate to mount an inflammatory response. Inflammation is mediated by conventional inflammatory cytokines and aims to eliminate the invading microorganisms or damaged cells, but also promotes tissue repair and regeneration (Karin & Clevers, 2016). However, uncontrolled inflammation leads to massive macrophage activation resulting in self-inflicted death, which subsequently triggers extensive neutrophil recruitment, thereby causing severe immunopathologies (Zhong, Sanchez-Lopez, & Karin, 2016). Macrophages polarize in response to diverse microbial and environmental signals into various subpopulations with distinct effector functions. Toll-like receptor ligands such as LPS or proinflammatory cytokines polarize macrophages to M1 aiming to eliminate their primary triggers which are disrupting homeostasis. This phenotype is associated with high levels of NO and proinflammatory cytokines, and enhanced phagocytic activity. Macrophages polarized at M2 produce high levels of anti-inflammatory factors such as IL-4, IL-10, IL-13 and TGF-B, and lead to healing and regeneration. M2 macrophages are also characterized by production of arginase 1 (arg1) and expression of the mannose receptor C type 1 (Mrc1) (Italiani & Boraschi, 2014). Thus, the modulation of macrophage activation is a major step in controlling inflammation. Our data clearly indicated that, despite LPS activation, macrophages treated with neovestitol produced increased levels of IL-10 (Fig. 2). Furthermore, neovestitol attenuated LPSinduced production of pro-inflammatory mediators such as IL-6,

3.3. Gene expression Gene expression analysis was performed using neovestitol at 0.22 mM in LPS activated cells, since this concentration promoted the most intense effect on cytokines profile. The relative expression rate and function of genes significantly regulated by 0.22 mM neovestitol in LPS- activated cells when compared to control are shown in Table 1. 3.4. Signaling pathways activation and TLRs expression The treatment of LPS- activated Raw 267.4 cells with neovestitol at 0.22 mM decreased the amount of the phosphorylated proteins: pNF-jB, pC-FOS and pMAPK p42/44 in relation to control, when the bands intensities were compared to the control protein GAPDH. Moreover, TIRAP levels were also reduced by the neovestitol treatment (Fig. 3).

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Fig. 2. Neovestitol effect on levels of IL-1b, TNF-a, IFN-c, IL-4, IL-6, GM-CSF and IL-10 in cell supernatant of LPS-activated Raw 267. cells. Results are expressed as means pg/ml of three experiments performed in triplicate. (*) Indicates statistically difference compared to DMSO control group (ANOVA) using the Tukey-Kramer HSD (p < 0.05).

TNF-a, GM-CSF, IFN-c and IL-1b. IL-1b and TNFa are the most potent inflammatory cytokines and gene transcription analysis have further indicated that neovestitol was able to inhibit the transcription of genes involved in IL-pathway, members of the IL-1 and TNF families and GM-CSF. IL-1a and IL-1b bind to the receptor IL-1RI, and trigger a cascade that lead to activation of the transcription nuclear factor kappa B (NF-jB p50/p65) (Guha & Mackman, 2001; Janssens, Burns, Tschopp, & Beyaert, 2002); expression of inducible nitric oxide synthase (iNOS) (Hewett, Corbett, McDaniel, & Choi, 1993), proinflammatory cytokines such as IL-6 (Sironi et al., 1989), and TNF-a (Ikejima, Okusawa, Ghezzi, van der Meer, & Dinarello, 1990). Con-

sequently, IL-1 inhibition is noteworthy for neovestitol antiinflammatory properties (Dinarello et al., 2012). The reduced IL-1 levels promoted by neovestitol were also demonstrated by gene expression analysis. Neovestitol treatment was associated with reduced levels of mRNA of capns1, il1b and il1f6. Capns1 encodes the small subunit of calpain I which is required for the conversion of pro-IL-1-a into its active form, IL1a (Dinarello, 1997). IL1-a leads to systemic inflammation by activation of TNF-a and induction of IL-6 secretion (Xie et al., 2013) and also acts as an alarm signal to initiate inflammation in response to a tissue injury (Ramadas, Ewart, Iwakura, Medoff, & LeVine, 2012). Transcription of il-1b and consequent release of IL-

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Table 1 Genes regulated by neovestitol 0.22 mM treatment in Raw 264.7 cells activated with LPS. The data were compared with those of LPS-activated control cells (treated with DMSO) (Student’s t-test, p < 0.05). Gene

Fold-changes

Function

Nox1 Ifnb1 Capns1 Il1b Egr1 Calm1 Elk1 Chuk Tnfsf13b Cd70 Il19 Fos Il18 Csf2 Il1f6 Cd86 Tnfsf13 Tnfsf18 Cd80 Mrc1

25.0 21.4 19.8 16.9 10.8 9.3 6.9 5.3 4.8 4.5 4.4 4.1 3.7 3.4 3.4 2.5 2.3 2.2 2.1 2.2

NF-jB pathway (O’Leary et al., 2012) Host-defense (Wang et al., 2011) IL-1 pathway (Dinarello, 1997) IL-1 pathway (Dinarello, 2011) Early growth response protein 1, a transcription factor involved in inflammatory response (Diaz-Munoz et al., 2010) Nitric oxide pathway (Kim et al., 2014) ETS domain-containing protein, transcription factor involved in TLR response, MAPK and NF-jB pathway (Al-Sadi et al., 2013) NF-jB pathway (Karin & Delhase, 2000) TNF superfamily (Jeong et al., 2010; Lee et al., 2011) TNF superfamily (Denoeud & Moser, 2011) IL-10 superfamily; function not clear (Ellison et al., 2013) Transcription factor involved in TLR response (Srivastava et al., 2016) IL1 family (Jitprasertwong, Jaedicke, Nile, Preshaw, & Taylor, 2014) GM-CSF IL1 family, also known as IL36a (Frey et al., 2013) M1 Macrophage polarization marker (Italiani & Boraschi, 2014; Martinez, Sica, Mantovani, & Locati, 2008) TNF superfamily (Jeong et al., 2010; Lee et al., 2011) TNF superfamily and cancer development (Bae et al., 2008) Macrophage polarization marker (Italiani & Boraschi, 2014; Martinez et al., 2008) Macrophage polarization marker (Italiani & Boraschi, 2014; Martinez et al., 2008)

Fig. 3. Western blotting image showing the levels of phosphorylated proteins: pNFjB, pc-FOS, pMAPK p42/44, TIRAP and GAPDH (control) in control and neovestitol treated LPS –activated Raw cells.

1b is induced upon NF-jB activation caused by PAMPs or DAMPs binding to Toll-like receptors (TLR) or engagement of TNF receptors (Karin & Clevers, 2016). Il1f6 encodes IL-36a, a member of the IL-1 family, is involved in IL-1 independent inflammatory response by activation of NF-jB and MAPK pathways with the consequent induction of IL-6 and IL-8 production. Its expression is upregulated in psoriatic and, rheumatoid arthritis (Frey et al., 2013; Ramadas et al., 2012). Neovestitol has also promoted the down-regulation of il18, encoding IL-18, a member of IL-1 family produced by T-cells, neutrophils and macrophages after LPS-activation. Interestingly IL-18 is encountered in diverse inflammatory and autoimmune diseases (Sedimbi, Hagglof, & Karlsson, 2013) and also induced/produced by inflamassome activation (Karin & Clevers, 2016). The observation that neovestitol treatment has also downregulated the mRNA levels of the transcription factors Egr-1 and

Elk-1, both inductors of TNF expression, is in accordance with the data indicating that neovestitol reduced TNF-a production and down-regulated the transcription of tnfsf13b (BAFF), tnfsf13 (APRIL), tnfsf18 (GITRL) and tnfsf7 (CD70), encoding members of super-family TNF. All these features are of interest in therapies aiming to modulate inflammation. BAFF and APRIL can induce inflammatory activation of macrophages via MAPK activation, which leads to subsequent activation of NF-jB (Jeong et al., 2010; Lee, Kim, Suk, & Lee, 2011). GITRL induces NF-jBdependent expression of matrix metalloproteinase (MMP)-9, TNF, IL-6 and other pro-inflammatory cytokines in macrophages (Bae et al., 2008). Down regulation of transcription tnfsf7, encoding CD70, is also involved in NF-jB pathway activation and consequently in inflammatory diseases (Denoeud & Moser, 2011). Therapies aiming to control CD70, using an anti-CD70, attenuated the development of arthritis and inflammatory bowel diseases (Denoeud & Moser, 2011). Despite the reduction of pro-inflammatory cytokines promoted by neovestitol, it has also reduced the levels of IFN-c, reduced the transcription of the gene encoding IL-19, a member of IL-10 family, but increased the levels of IL-10. IL-19 plays a double role in inflammation. It presents antiinflammatory properties by decreasing production of TNF-a, IL-6 and IL-12 (Azuma et al., 2010; Azuma, Nakajima, & Takeuchi, 2011; Ellison et al., 2013); but also participates in proinflammatory activities in psoriasis and rheumatoid arthritis, inducing IL-6 release (Leng, Pan, Tao, & Ye, 2011; Sakurai et al., 2008). IFN-b was shown to exhibit anti-inflammatory properties (Wang et al., 2011) by inducing the release of IL-10 in LPSstimulated macrophages (Chang, Guo, Doyle, & Cheng, 2007) and by inhibiting IL-12 p40 production at the transcriptional level in human monocytes (Byrnes et al., 2001). On the other hand, autoimmune diseases such as systemic lupus erythematosus have been associated with excessive IFN production (Noppert, Fitzgerald, & Hertzog, 2007). It also has been shown that IFN-b knockout mice are more resistant to high-dose LPS challenge than wild-type mice (Sheikh, Dickensheets, Gamero, Vogel, & Donnelly, 2014). Thus, it seems that neovestitol activity in reducing relevant proinflammatory cytokines but concomitantly reducing mediators associated with autoimmune diseases such as IFN-b and IL-19 could have a therapeutic value in several damaging conditions and diseases.

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The control of inflammation mediated by neovestitol is also shown by its ability to increase IL-10 production. IL-10 is produced by several distinct immune cells after inflammatory stimulus and plays a crucial role in regulating both innate and adaptive immune responses in order to keep the homeostasis. IL-10 macrophagederived is produced after DAMPs recognition by macrophagesPRRs such as TLRs and NODs receptors (Ma et al., 2015)and it acts through Jak/Stat pathways, downregulating NF-jB pathway with consequently reduction of IL-1b, TNF-a, IL-8, IL-12, monocyte chemotactic protein 1 (MCP-1), Intercellular Adhesion Molecule 1 (ICAM 1) and iNOS levels (Han & Boisvert, 2015). IL-10 orchestrated LPS-activated intestinal macrophages avidly remove DAMPs and MAMPs and do not lead to tissue injury but are associated with tissue regeneration. (Simon et al., 2016). Therefore, IL-10 high levels, as promoted by neovestitol-treatment, may prime macrophages into a ‘‘regenerative-tissue inducer phenotype”. LPS activated macrophages produce not only proinflammatory cytokines, and chemokines but also reactive oxygen species (ROS) and nitric oxide (NO) (Hayden, West, & Ghosh, 2006). High concentrations of neovestitol (0.26–0.38 mM) seemed to induce the production of NO and decrease macrophages viability. It is already demonstrated in the literature that macrophage apoptosis may be nitric oxide dependent trough enhancement of Bax/Bcl-2 ratio and augmented expression of caspase-3 (Guo et al., 2016). In this way, the neovestitol 0.38 mM was the unique concentration to reduce more than 50% of macrophage viability. Since the viability test used here is elementary and used for initial analysis regarding citoxicity, future studies should further evaluate possible neovestitol toxic effects associated with concentrations higher than 0.38 mM. On the other hand, macrophages treated with neovestitol at 0.22 mM did not alter macrophage viability and decreased NO production, which is in accordance to the transcription profile of NO pathway. The neovestitol 0.22 treatment led to down regulation of nox1, encoding NADPH oxidase 1 which is one of the major sources of ROS in response to inflammatory mediators (O’Leary et al., 2012), and of calm1which encodes a protein that maintains iNOS stability and activation (Kim et al., 2014). We then investigated whether the altered cytokines and gene expression profiles promoted by neovestitol in LPS activated macrophages were the result of altered macrophages polarization to M2. Neovestitol treated macrophages revealed up-regulation of transcription of M2 markers mrc1 and arg1 (although the last was not significant) and down-regulation of M1 markers Cd80 and Cd86 (Table 1), suggesting polarization in the M2 antiinflammatory phenotype. Moreover, the low IL-4/high IL-10 production phenotype of neovestitol treated LPS-activated macrophages indicates polarization into M2c, macrophages with antiinflammatory, phagocytosis and tissue remodeling functions (Jiang & Zhu, 2016). The macrophage polarization to M2 was not typical, since TNFa production, considered an M1 macrophage marker leading to pro-inflammatory, pathogen clearance and tissue damage function (Jiang & Zhu, 2016) was reduced but not eliminated in neovestitol LPS-activated macrophages. In fact, macrophages populations do not polarize to only one of the two conditions, but, depending on the environment, differentiate into a spectrum of different phenotypes, where M1 and M2 represent the extreme ends (Mosser & Edwards, 2008). As shown in subsequent analysis, the phenotype of the macrophages induced by neovestitol may be of relevance in modulation of inflammation, since TNFa drives inflammation to a regenerative process that was demonstrated to occur during liver, epithelial and intestinal mucosa regeneration after tissue injury (Karin & Clevers, 2016). In order to elucidate the mechanisms underlying the macrophage phenotype induced by neovestitol in the presence of LPS, a PAMP recognized to induce the M1 phenotype, we then investi-

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gated whether neovestitol would interfere with TLR signaling. In general, inflammation starts with TLR activation that promotes initial changes in cytokines gene transcription, inflammasome initiation and matrix metalloproteinases (MMPs) induction (Karin & Clevers, 2016). Protein analysis revealed that the expression of TIRAP, an adaptor protein of toll-interleukin receptor with a crucial role at early stages of TLR1, TLR2, TLR4 and TLR 6 (Bonham et al., 2014; Takeda & Akira, 2004), was decreased after neovestitol treatment in LPS-activated macrophages. Therefore, neovestitol effect on TIRAP (Fig. 3), either by inhibition of expression or degradation, would account to neovestitol anti-inflammatory properties by decreasing LPS recognition at an early stage of the inflammatory process. Furthermore, the neovestitol treated macrophages exhibited a phenotype compatible with TIRAP decreased expression since TIRAP deficiency leads to reduced release of inflammatory cytokines (Kim, Jin, Choi, & Park, 2011). Interestingly, the natural product-derived compound resveratrol has also TIRAP-inhibition as a central role for its well-recognized beneficial pharmacological properties (S. Kim et al., 2011). Furthermore, p110d isoform of the kinase PI3K may be responsible for calpain-mediated degradation of TIRAP (Aksoy et al., 2012). Therefore, future studies to elucidate neovestitol TIRAP-reduction effect should evaluate whether neovestitol affects p110d expression. We then investigated the activation of different inflammatory pathways in the neovestitol treated LPS- activated macrophages. The low levels of phosphorylated MAPK, Extracellular Signalregulated Kinase-1 (ERKp) and Jun N-terminal kinase (JNKp), NFjBp p65 sub-unit are indicative of mitogen-activated protein (MAP) kinases, ERK 1 and 2, p38, JNK and NF-jB reduced activation in LPS- activated macrophages after neovestitol treatment. This decreased activation is in accordance to the phenotype observed in TIRAP deficient macrophages, when compared to wild-type macrophages (Yamamoto et al., 2002). Elk1 is a substrate of MAP kinases pathways (ERK1/2) and plays a crucial role in inflammatory response mediated by TNF-a (Al-Sadi, Guo, Ye, & Ma, 2013). Elk1 is activated by Toll-like response, particularly TLR4 (Hodgkinson, Patel, & Ye, 2008) and TLR9 (Thapa, Kim, Kwon, & Kim, 2012) and thus is involved in the activation of the NF-jB, and production of TNF-a, IL-6 and MCP-1 (Hodgkinson et al., 2008). Early growth response protein 1 (EGR-1) belongs to a group of early response genes induced by different stimuli such as LPS, cytokines, growth factors and hypoxia, being implicated in chronic inflammatory diseases such as atherosclerosis and arthritis rheumatoid. In addition, EGR-1 interacts with NF-jB in order to stimulate macrophages-Prostaglandin E2 synthesis that is related to several physiological and pathological processes such as inflammation, pain, vascular regulation, neuronal functions, female reproduction, gastric mucosal protection, and kidney function (Diaz-Munoz, Osma-Garcia, Cacheiro-Llaguno, Fresno, & Iniguez, 2010).

5. Conclusions Taken all the data together, neovestitol reduced TIRAP levels in LPS activated macrophages, and drove macrophages to the M2c phenotype, which is associated with tissue regeneration. The inhibition of NF-jB activation promoted by neovestitol resulted in the inhibition of several other downstream pathways and modulated multiple signaling such as those promoted by TNF-a and IL-1b, leading to reduced levels of oxygen reactive products, and proinflammatory cytokines, but induced the production of high levels of IL-10. Thus, due to its immunemodulatory effects, neovestitol therapeutic and nutraceutical properties should be investigated in diseases associated with exacerbated inflammation or as functional food.

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Acknowledgments The authors are grateful to Mr. Alessandro Esteves (in memoriam) for providing the Brazilian red propolis samples and to Dr. Toshihisa Kawai (Forsyth Institute, Boston, MA) for providing RAW 264.7 cells. This work was supported by São Paulo Research Foundation - FAPESP grants 2012/14323-3 and 2012/01500-4.

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