Resveratrol differentially modulates immune responses in human THP-1 monocytes and macrophages

Journal Pre-proof Resveratrol differentially modulates immune responses in human THP-1 monocytes and macrophages

Li Feng, Rumana Yasmeen, Norberta W. Schoene, K. Y. Lei, Thomas T. Y. Wang PII:

S0271-5317(19)30396-3

DOI:

https://doi.org/10.1016/j.nutres.2019.10.003

Reference:

NTR 8054

To appear in:

Nutrition Research

Received date:

15 April 2019

Revised date:

9 September 2019

Accepted date:

2 October 2019

Please cite this article as: L. Feng, R. Yasmeen, N.W. Schoene, et al., Resveratrol differentially modulates immune responses in human THP-1 monocytes and macrophages, Nutrition Research(2019), https://doi.org/10.1016/j.nutres.2019.10.003

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© 2019 Published by Elsevier.

Journal Pre-proof RESVERATROL DIFFERENTIALLY MODULATES IMMUNE RESPONSES IN HUMAN THP-1 MONOCYTES AND MACROPHAGES Li Fenga, Rumana Yasmeenb, Norberta W. Schoeneb, K. Y. Leia, Thomas T. Y. Wang b,*

a

Department of Nutrition and Food Science, University of Maryland, 0112 Skinner Building,

Diet, Genomics and Immunology Lab, Beltsville Human Nutrition Research Center, ARS,

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b

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College Park, MD 20742, USA; [email protected] (L.F.); [email protected] (K.Y.L.).

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USDA, Beltsville, MD 20705, USA; [email protected] (R.Y.);

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[email protected] (N.S.); [email protected] (T.T.Y.W.) .

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*Corresponding Author

Bldg 307C, BARC-EAST

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Beltsville, MD20705-2350

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10300 Baltimore Avenue

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Phone: (301) 504-8459 ext. 239

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Journal Pre-proof Abbreviations Resveratrol

D-THP-1;

Differentiated THP-1

AHR ;

Aryl Hydrocarbon Receptor

LPS ;

Lipopolysaccharide

PAM ;

PAM3CSK4

TLR ;

Toll-like Receptor

IL-1β ;

Interleukin-1β

IL-6 ;

Interleukin-6

IL-8 ;

Interleukin-8

Ptes ;

Pterostilbene

Gen ;

Genistein

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Res ;

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Journal Pre-proof Abstract

Resveratrol (Res), a natural polyphenol compound found in grapes and red wine has been shown to exhibit anti-inflammatory, anti-oxidant, and anti-carcinogenic effects. However, proinflammatory/tumor-promoting properties of Res have also been reported, rendering the polyphenol’s reported therapeutic benefits less convincing and controversial. In order to evaluate

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the underlying plausible factors contributing to the differential immunomodulatory effects

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imparted by Res, herein, we investigated, at both physiological and pharmacological doses, the in vitro effects of Res on cell survival/proliferation, inflammatory genes and cytokine production

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in human monocytic cell line (THP-1) and phorbol 12-myristate 13-acetate (PMA) differentiated

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human THP-1-derived (D-THP-1) macrophages. We hypothesized that the differential effects

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observed in monocytes and macrophages may largely depend on dietary vs. pharmacological doses of Res, duration of treatment and the target cells it acts upon. Our data showed that Res, at

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physiological concentrations, inhibited proliferation of THP-1 monocytes with S phase arrest. On

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the other hand, at pharmacological concentrations, Res induced cell apoptosis and caused G0/G1

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phase arrest. Additionally, Res showed differential effects on pro-inflammatory cytokine expression and production measured by RT-PCR and ELISA, respectively, in THP-1 monocytes versus macrophages: promoting inflammation in monocytes, while exhibiting anti-inflammatory effects in macrophages. Comparative analysis on Res, and two other phytochemicals, pterostilbene and genistein revealed that the immunomodulatory effects of Res were consistent with those observed in pterostilbene and not genistein. Our results reveal a pleiotropic immunomodulatory property of Res that is dose-time-target cell-dependent and thus serve as a caution for the use of Res in the treatment of inflammatory diseases.

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Journal Pre-proof Keywords: Resveratrol; inflammation; monocytes; macrophages; stilbene structure

1. Introduction

Monocytes and macrophages are important cell types in the immune system that play central roles in an individual’s inflammatory processes [1,2]. Following an infection or tissue injury,

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monocytes are rapidly recruited to inflamed tissues, and differentiate into tissue macrophages

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and dendritic cells [3,4]. Additionally, monocytes play key roles in several biological processes,

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such as wound healing, chemotaxis, tissue homeostasis and even cancer progression [5,6].

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Macrophages, through their ability to clear pathogens and instruct other immune cells (such as T cells), protect the host and resolve inflammation. The capacity of macrophages to rapidly release

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a cascade of pro-inflammatory cytokines, such as interleukins (i.e. IL-1β, IL-6, IL-8, TNF-α),

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reactive oxygen species, complement factors and proteolytic enzymes upon exposure to various stimuli render them as critical contributors to the early inflammatory response following an

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injury [7]. Heightened immune responses elicited by dysregulated monocytes/macrophages and

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other immune cells could lead to persistent inflammation/injury and the development of acute and chronic systemic diseases [8,9]. Therefore, preventive/therapeutic strategies that reduce inflammation through modulation of these immune cells may hold promise for diseases associated with severe or chronic inflammation. Currently, the practiced line of treatment for inflammatory diseases resorts to the non-steroidal anti-inflammatory drugs (NSAID). However, long-term use of NSAID lends to serious health issues, such as cardiovascular and gastrointestinal complications [10-12]. Thus, the need for the identification of safer alternative anti-inflammatory preventive/therapeutics is a priority.

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Journal Pre-proof Numerous studies have shown that dietary nutrients can impart profound beneficial effect on human health that span from reducing potential risk factors as well as preventing/delaying onset of chronic diseases. At present, foods rich in polyphenolic compounds are being considered as a promising safer alternative for the prevention and treatment of inflammatory diseases [13-17]. Resveratrol (Res), a polyphenol, abundant in the skin of grapes, berries and other plants has been widely studied for its anti-inflammatory, anti-oxidant, anti-proliferative and chemopreventive

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properties [18-20]. Res has been shown to inhibit biological processes associated with the

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initiation, promotion and progression of tumors in vitro [21, 22]. Moreover, Res has been

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reported to inhibit the proliferation of oral, liver, colon, breast and prostate cancer cell lines in

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vitro in a dose-and time-dependent manner [23-33]. Studies reporting cell cycle arrest or apoptosis induced by higher doses of Res however may not be a desirable physiological outcome

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as upregulation of cyclin-dependent kinase inhibitor 1 A (CDKN1A/p21) or B (CDKN1B/p27) -

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often used as indicators of growth inhibition, may also indicate cellular stress/DNA damage [34] and thus require careful interpretation. Several in vitro macrophage studies have also shown that

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Res pretreatment can modify the macrophage inflammatory and oxidative response to

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lipopolysaccharide, an inflammatory stimulus and a toll-like receptor (TLR) 4 ligand. A recent study [35] demonstrated that pretreatment of murine RAW 264.7 macrophages and human differentiated THP-1 with Res (≥25 μM), followed by LPS stimulation blunted iNOS,PGE2, cytokines (IL-1β, IL-6, TNF-α) and chemokines (CCL2/MCP1, CCL4/MIP-1β, CCL5/RANTES, CXCL10/IP-10). In accordance with these findings [35-37], U-937 and RAW 264.7 macrophages, showed that 0.1 to 10 mM Res inhibited LPS-stimulated release of TNF-α and gene expression of TNF-α, IL-1β, IL-6. However, most of these in vitro studies use

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Journal Pre-proof supraphysiological doses of Res and often fail to reproduce or contradict the purported beneficial health effects in vivo. Previously, we have reported that Res exposure can promote tumor angiogenesis and inhibit apoptosis in a xenograft mouse model of prostate cancer [38]. Several studies [39, 40], in addition to our own, have reported detrimental and/or tumor-promoting effects of Res. Andreani C. et al. [39] showed that Res treatment (0.0001% in drinking water; daily intake of 4µg/mouse)

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shortened tumor latency and enhanced tumor multiplicity in Δ16HER2 mice, a HER2+/ERα+

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breast cancer model. Another study [40] reported promotion of mammary tumor growth and

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metastasis by Res at all concentrations (0.5, 5, 50 mg/kg body weight) tested in tumors derived

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from the low metastatic estrogen receptor (ER)α(-), ERβ(+) MDA-MB-231 and the highly

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metastatic ER(-) MDA-MB-435 cancer cell lines, using immunocompromised mice. These results lead to questioning the actual health benefits of Res. Moreover, apart from selection of

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effective doses of Res supplementation, concerns regarding bioavailability of Res and attainment

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of effective plasma concentrations serve as potential drawbacks to achieving desired therapeutic outcome. Res, following oral administration, is rapidly metabolized by phase II enzymes such as

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glucuronides and sulfatases [41, 42]. We have previously reported highest achievable plasma Res concentrations consuming 100 mg Res /kg diet to be 5.25µM in a xenograft model for prostate cancer [38]. Therefore, feeding an enormous amount of Res to mimic observed in vitro health benefits in vivo may not be realistic. Moreover, Res has been reported to bind to aryl hydrocarbon receptor (AHR), a critical transcription factor regulating xenobiotic mechanisms that either activate or repress its trans-activating ability [43, 44]. The AHR-dependent P450 (CYP) cytochromes, CYP1A1 and B1 are highly expressed in a diversity of tumor cells. Studies have reported that exposure to AHR-activating toxicants contributes to the development of a 6

Journal Pre-proof myriad of immune disorders, including rheumatoid arthritis, asthma, multiple sclerosis and allergic responses [45-46]. Of note, activation of differentiating monocytes or macrophages is associated with increased levels of AHR [47]. Given that Res is reported to act through multiple pathways including that of AHR-mediated pathways [43, 44], study of the concentrationdependent biological effects exerted by Res in monocytes vs. macrophages may offer valuable insights into understanding how Res modulates immune responses. Despite well-documented

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beneficial properties of Res, to date, there has been little research investigating the

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immunomodulatory effects of Res, at physiologically attainable concentrations, in human

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immune cells. Moreover, the existing literature leans heavily toward illustrating the effects on

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macrophages and relatively less information on monocytes. Given that monocytes are also immune responsive and play a critical role in the immune system [1-5], understanding the effects

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of Res or any other bioactive on monocytes is important in providing a better view of overall

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effects of these compounds on the immune system. The objective of the present study was to evaluate how Res impacts the immune system,

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primarily, monocytes and macrophages. We hypothesized that Res, at dietary and

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pharmacological achievable concentrations, imparts differential biological and immunomodulatory effects in monocytes and macrophages. We also asked whether AHR activation can influence the observed biological effects. To test our hypothesis, we evaluated and compared Res’ effects on cell cycle, development of cytotoxicity, inflammatory markers, cytokine production using the human THP-1 cell line. The THP-1 cells can exist as monocytes and differentiated macrophages (D-THP-1), and thus a suitable model to use to compare and elucidate the effects of Res at both physiological and pharmacological doses in monocytes and macrophages.

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Journal Pre-proof 2. Methods and materials

2.1 Materials and reagents Cell culture media, DMEM (Cat #11,995) and RPMI 1,640 (Cat #11,875), were purchased from GIBCO (Grand Island, NY, USA). Lipopolysaccharides (LPS) (derived from Escherichia coli 0111:B4), resveratrol (Res), pterostilbene (Ptes), genistein (Gen) and Phorbol 12-myristate 13-

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acetate (PMA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). PAM3CSK4 (PAM)

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was purchased from InvivoGen (San Diego, CA, USA). Human IL-6 ELISA kit II (Cat #550799)

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was purchased from BD Biosciences (San Jose, CA, USA).

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2.2 Cell Culture and Treatment

Human THP-1 cells were obtained from American Type Culture Collection (ATCC, Manassas,

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VA, USA) and were cultured in RPMI-1640 Medium, supplemented with 10% fetal bovine

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serum (FBS; Invitrogen) and 100 U/mL penicillin, 100 mg/mL streptomycin (ThermoFisher Scientific, Grand Island, NY, USA), hereinafter referred as the culture medium. Cells were

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incubated at 37 °C in the presence of 5% CO2. THP-1 monocytes (5×105 cells/mL, 2mL) were

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seeded in 6-well plate (Costar; Corning Incorporated, Corning, NY, USA) overnight, followed by treatment with different concentrations of resveratrol. For generation of THP-1 macrophages, THP-1 monocytes (5×105 cells/mL, 2mL) seeded in 6-well plate were differentiated with PMA (25ng/mL) for 48 hours, which were incubated at 37 °C with 5% CO 2 in ambient air. PMA is a differentiating agent that stimulates differentiation of non-adherent monocytic THP-1 into adherent D-THP-1[35]. After washing three times with PBS, attached cells were grown in fresh culture media and ready for treatments. Res concentrations used in this study was based on our

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Journal Pre-proof previously observed in vitro and in vivo physiological (5µM) and pharmacological (>10µM) effects of Res [38]. To assess the effect of Res on monocyte and macrophage function, we examined effects of Res on Toll-like receptor (TLR) -mediated induction of inflammatory cytokines. TLRs are pattern recognition receptors responsible for defense against bacterial infection [1-5]. We used the TLR4 ligand bacterial cell wall lipopolysaccharide (LPS) and the synthetic TLR2 ligand

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PAM3CSK4 (PAM) for our experiments. THP-1 or D-THP-1 cells were treated with Res for 48

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hours with daily changes of media. For gene expression experiments, following Res treatment,

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cells were stimulated with TLR ligands for 2 hours and harvested for RT-PCR as described

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below. For detection of cytokine secretion, after Res treatment, cells were stimulated with TLR

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ligands for 4 hours and secreted cytokine determined using ELISA as described below. 2.3 Evaluation of cell viability by Trypan Blue staining in THP-1 cells

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THP-1 monocytes (1×105 cells/mL, 2mL) were plated in 6-well plates for 24 hours and then

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treated with (1, 5, 10, 25 μM) or without resveratrol (control, DMSO) for 0, 24, 48 hours. Total

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cell count for each sample was measured with Trypan Blue staining [48]. 2.4 Cell Viability Assay for D-THP-1 cells Cell viability of D-THP-1 was measured using the sulforhodamine B (SRB) assay [49]. Following differentiation of THP-1 with 25 ng/mL PMA in 6-well plates for 48 hours, D-THP-1 cells were treated with (1, 5, 10, 25 μM) or without Res (control, DMSO) for another 48 hours. Cells were harvested and cell viability was measured using the SRB assay. Briefly, cells were first fixed with trichloroacetic acid (TCA, 10%) for one hour at 4 °C, and then stained for 20 minutes with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. Unbound dye was removed by five washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris 9

Journal Pre-proof base (pH 10.5) for determination of optical density in a computer-interfaced, 96-well microtiter plate reader (530 nm) [49]. 2.5 Cell cycle analysis/FACS analysis THP-1 monocytes (1×105 cells/mL, 10mL) were plated in 75-cm2 flasks overnight and then treated with (1, 5, 10, 25 μM) or without Res (control, DMSO) for 48 hours. Harvested cells, treated or not as described, were transferred to tubes (50 mL polypropylene; BD Biosciences,

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Bedford, MA, USA) and recovered by centrifugation(1000×g). Pellets were then washed in PBS

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(no Ca or Mg) and pelleted again. These pellets were then re-suspended in 1.5 mL PBS. To re-

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suspended cells, 15 mL of chilled 70% ethanol was added and the capped tubes vortexed gently

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and stored overnight at 40C. Followed by this storage, fixed cells were pelleted and washed one time in PBS with resuspension in the DNA PI staining solution (1 ug propidium iodide and 25

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mg ribonuclease A in 1 mL PBS) for 30 min at room temperature. Samples were then promptly

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analyzed for DNA content. Analyses were determined by flow cytometry using a FACScalibur cytometer (Becton Dickinson, San Jose, CA). Flow cytometric data files were collected and

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analyzed using the CELLQuest program (Becton Dickinson). A total of 10,000 cell events were

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collected for DNA analyses. Cell cycle distribution percentages of stained nuclei were calculated using Modfit LT software (Version 3.0, Verity Software House, Inc., Topsham, ME). Calibration standards (LinearFlow Green and DNA QC Particle Kit) for verification of instrument performance were purchased from Molecular Probes (Eugene, OR) and Becton Dickinson, respectively [50]. 2.6 Real-Time PCR Analysis Total RNA was isolated using TRIzol reagent (ThermoFisher Scientific, Grand Island, NY, USA) and cDNA was synthesized using AffinityScript Multiple Temperature cDNA Synthesis 10

Journal Pre-proof kit (Agilent Technologies, Santa Clara, CA, USA). Real-time PCR was carried out using a TaqMan Fast Universal PCR Master Mix on a 7900HT FAST Real-time PCR System (Applied Biosystems, Foster City, CA, USA). TaqMan gene expression assay (Life Technologies, Carlsbad, CA, USA) was used to quantify gene expression levels of human IL-1β (Hs01555410_m1), IL-6 (Hs00985639_m1), IL-8 (Hs00174103_m1), COX-2-PTGS2 (Hs00153133_m1), CYP1A1 (Hs01054797_g1), CYP1B1 (Hs02382916_s1), p21-CDKN1A

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(Hs00355782_m1), and p27-CDKN1B (Hs01597588_m1). Human glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH) (Hs02758991_g1) was used as a housekeeping gene for calculation of

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relative expression levels using the ΔΔCt method as previously described [38].

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2.7 CDKN1A/p21 protein determination by flow cytometry

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THP-1 cells were re-suspended in 300 mL of RPMI medium. Formaldehyde (16%) was added to obtain a final volume of 1.5% and cells were fixed for 15 min at room temperature followed by

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centrifugation. Pellets were re-suspended by vortexing in 50 mL RPMI media. Tubes were

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chilled and then 1 mL of ice-cold methanol was added and gently suspended to prevent cell clumping. Samples were capped tightly and stored at -20 °C until analysis by flow cytometry.

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Before staining, cells are centrifuged and re-suspended in PBS. Intracellular p21 protein was detected by combining steps suggested by the source of the antibody to p21 (F-5 fluorescein isothiocyanate labeled, Santa Cruz Biotechnology, Santa Cruz, CA) and those used in our laboratory for intracellular staining of proteins [51]. Control samples were stained using an appropriately labeled antibody isotype to show specificity for the presence of p21 protein. After staining for 30 min, cells were again centrifuged and resuspended in PBS prior to prompt analyses by flow cytometry (see Section 2.5 for details on instrument checks and software used).

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Journal Pre-proof Ten thousand events were analyzed per sample. Positive fluorescence intensities for p21 presence were calculated taking into account fluorescence of isotype control [51]. 2.8 IL-6 protein measurement by ELISA To determine IL-6 protein levels, THP-1 monocytes (5×105 cells/mL) were cultured in 6-well plates overnight, and treated with DMSO, Res (1, 5, 10, 25 μM), Ptes (10 μM), or Gen (10 μM) for 48 hours. Medium containing test compounds was replaced every 24 h. Forty-eight hours

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later, LPS (10 ng/mL) or Pam3CSK4 (PAM) (500 ng/mL) were added for another 4 hours. Then

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supernatant was collected for detection of IL-6 protein levels using commercially available

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ELISA kit (BD Biosciences, San Jose, CA, USA), while remaining cells were counted for

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normalization of quantity. Similar experiments were carried out using differentiated D-THP-1

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macrophages, supernatants were harvested for ELISA. Secreted cytokine levels were normalized

2.9 Statistical Analyses

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by total protein content (SRB assay) of D-THP-1 cells.

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Data were analyzed with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA) and are presented as means ± SD. A one-way ANOVA followed by post hoc test (Fisher’s LSD test)

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was used to analyze data. p values ≤ 0.05 were considered significant. Data are representative of three separate experiments. The inhibitory concentration (IC)/Effective concentration (EC) 50 was calculated using the dose response (nonlinear fit of stimulation or inhibition) function of Prism software. Sample size was calculated using G*power 3.1 [52] to provide at least 0.8 power to detect 0.8 effect size at p<0.05.

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Journal Pre-proof 3. Results 3.1 Differential effect of Res on the growth of THP-1 monocytes and D-THP-1 macrophages THP-1 monocytes: To evaluate the effects of various doses of Res on cell growth, we treated THP-1 monocytes and macrophages with 1, 10, and 25 μM Res. A dose-dependent inhibition of THP-1 monocyte

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growth was observed with higher doses of Res (Fig. 1A). The highest growth rate of THP-1

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monocytes was observed with the DMSO controls. A similar growth rate was observed with 1

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μM Res treated cells. Treatment with Res at 25 μM was associated with minimal cell growth at

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24 hours and cell death at 48 hours. Cells treated with Res 10 and 25 μM had respectively a 36.5% and 69.04% inhibition in growth compared to controls at 48 hours. Res at 25 μM was

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associated with a loss of 44.5% of cells from the initial plating. The half maximal inhibitory

D-THP-1 macrophages:

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concentration (IC50) of Res for THP-1 cells was 5 μM.

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Following 48 hours of incubation, Res treatment led to a dose-dependent effect on the growth of

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D-THP-1 cells. Res at 1 and 10 μM treated cells had a similar growth rate as the DMSO controls at 48 hours (Fig. 1B). Res at 25 μM was associated with a 14.3% reduction, in the growth of DTHP-1 cells at 48 hours compared to DMSO controls (p < 0.05) (Res 25 μM, Fig. 1B). The IC50 of Res for D-THP-1 cells was 76 μM.

3.2 Effect of Res on cell cycle in THP-1 monocytes Given that monocytic form of THP-1 was more sensitive to Res concentrations, we further characterized the inhibitory effects of resveratrol on monocytes. Cell cycle analysis was

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Journal Pre-proof performed to examine the influence of Res (1, 5, 10, 25 μM) on the progression of THP-1 cells through the cell cycle (Fig. 2A). Res at 1 μM did not have any effect on cell cycle as compared to control. Res at 5 and 10 μM caused a 1.2- and 2.0-fold increase, respectively, in % cells in S phase, compared to control, suggesting a block or reduced progression through the S phase. In contrast, Res at 25 μM increased the % cells in G0/G1 phase by 57.9%, indicating arrest in this phase. A 1.7-fold increase in sub G0/G1 was also observed with Res at 25 μM, indicating

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significant apoptosis at this high dosage.

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The cyclin inhibitors, p21 and p27, are genes commonly associated with cell cycle inhibition and

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apoptosis [53]. To further elucidate the molecular effects of Res, we examined the effects of Res on these genes. Res exerted a concentration-dependent effect on p21. Increases of 7.2-fold and

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25.8-fold in p21 mRNA expression were found in THP-1 cells treated with Res at 10 and 25 μM,

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respectively (Fig. 2B). In contrast, Res had no effect on p27 mRNA expression in THP-1 cells

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(Fig. 2C). Consistent with an effect on p21 mRNA, the expression of p21 protein (Fig. 2D) also increased in a dose-dependent fashion. Increased expression was first detected with Res 5 µM

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(3.1-fold). This expression was increased to 3.6- and 4.1-fold, respectively, at Res 10 and 25 µM.

3.3 Effect of Res on AHR-responsive gene expression in THP-1 monocytes and macrophages AHR plays a key role in xenobiotic metabolism as well as immune regulation (47). THP-1 and D- THP-1 cells were examined to determine if Res treatment may influence AHR -mediated pathway by assaying for the well-documented AHR-responsive genes, CYP1A1 and CYP1B1 mRNA expression. CYP1A1 mRNA expression was below detection limit under our experimental conditions. Additionally, Res treatment did not induce CYP1A mRNA. On the

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Journal Pre-proof other hand, CYP1B1 mRNA expression was inhibited in a Res concentration-dependent manner in both THP-1 (Fig. 3A) and D-THP-1 (Fig. 3B), with reductions at 34%-70% and 86%-90%, respectively. The IC50 for Res was 9.98 µM for THP-1 and 3 µM for D-THP-1.

3.4 Differential effect of Res on pro-inflammation-related cytokines/protein expression in TLR ligands-stimulated THP-1 monocytes and D-THP-1 macrophages.

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As monocytes and macrophages are known to regulate inflammatory mediators, we examined

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concentration-dependent (0-10 μM) effects of Res on inflammatory cytokine/protein expression

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in THP-1 and D-THP-1 cells. The cells were treated with the bacteria-related inflammatory

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stimulants TLR 4 and 1/2 ligands LPS and PAM3CSK4 (PAM) respectively to examine if the immunomodulatory effects of Res are TLR-4 or TLR1/2-mediated.

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THP-1 monocytes:

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IL-1β, IL-6, IL-8 and COX-2 mRNA expressions were all induced significantly by LPS and PAM as compared to control. (Fig. 4A and 4B). LPS stimulated cells pretreated with Res 1 μM

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showed no change in expression (Fig. 4A). A dose-dependent increase in expression of IL-1β

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was observed with Res 5 to 25 μM pretreatment. IL-6 expression level was similarly increased with Res 10 and 25 μM pretreatment. No increase in COX-2 expression was observed until a dose of Res 25 μM was reached. For PAM, enhanced mRNA expression of IL-1β (2.3-fold), IL6 (3.8-fold) and IL-8 (4.8-fold), was observed in THP-1 cells pretreated with Res 25 μM. In THP-1 monocytes, without LPS/PAM stimulations (Fig. 4C), THP-1 cells showed increased expression of IL-1β- IL-8 and COX-2 mRNA only at the pharmacological dose of 25 μM Res. Next, we sought to validate our expression data on inflammatory markers at protein level. We focused on IL-6, a well-documented inflammatory cytokine, secretion into the media following

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Journal Pre-proof Res pretreatment and subsequent stimulation of THP-1 monocytes and/or macrophages with LPS/PAM. LPS stimulation in THP-1 monocytes increased IL-6 protein level in a dosedependent manner with Res pretreatment at 5 to 25 μM, whereas, PAM stimulation in the same cell line increased IL-6 protein with Res pretreatment at 10 to 25 μM, as compared to controls (LPS/PAM 0 ng/mL) (Fig. 4D and 4E). D-THP-1 macrophages:

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LPS stimulation in D-THP-1 with no Res treatment (LPS 10 ng/mL, Res 0 μM) (Fig. 5A)

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markedly increased mRNA level of IL-1β, IL-6, IL-8 and COX-2, as compared to controls (LPS

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0 ng/mL, Res 0 μM). Resveratrol (1-25 μM) pretreatment exerted no additional increment in IL-

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1β and IL-8 mRNA levels in LPS stimulated D-THP-1 macrophages. In contrast, Res (5-25 μM) pretreatments markedly reduced IL-6 and COX-2 mRNA levels in LPS stimulated cells.

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PAM stimulation in these differentiated cells with no Res pretreatment (PAM 500 ng/mL, Res 0

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μM) (Fig. 5B) elevated IL-1β, IL-6 and IL-8 mRNA levels as compared to controls (PAM 0 ng/mL, Res 0 μM). In PAM stimulated D-THP-1 macrophages, Res (1-25 μM) pretreatment,

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mildly decreased IL-1β and IL-8 mRNA levels, however, exhibited a dose-dependent decrease in

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IL-6 mRNA levels with increasing Res pretreatment concentrations. In D-THP-1 macrophages, Res 1 to 25 μM pretreatments alone resulted in ~50% reductions in (Fig. 5C) IL-8 and COX-2 mRNA expression. IL-1β mRNA level was similarly decreased by Res (5 to 25 μM), LPS or PAM stimulation of D-THP-1 macrophages markedly elevated IL-6 protein level as compared to controls (LPS/PAM 0 ng/mL) (Fig. 5D and 5E). However, IL-6 protein level induction in LPS or PAM treated D-THP-1 cells (Fig. 5D and 5E) was attenuated by Res at 10 to 25 μM and 5 to 25 μM, respectively.

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Journal Pre-proof 3.5 Elucidation of structural-dependent effects of Res in THP-1 monocytes and D-THP-1 macrophages Next, we compared the effects of Res on a classic pro-inflammatory cytokine IL-6 to that of pterostilbene, another phytochemical with stilbene structure and genistein, which lacks the stilbene structure. In THP-1 monocytes (Fig. 6A), the bioactive compounds exerted little or no effect on expression of the inflammatory marker IL-6, compared to controls (LPS 0 ng/mL,

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DMSO 1 μM), except Res pretreatment. LPS stimulation in D-THP-1 macrophages (Fig. 6B,

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LPS 10 ng/mL, DMSO 1 μM), markedly elevated mRNA level of IL-6, as compared to controls

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(LPS 0 ng/mL, DMSO 1 μM). Most importantly, in D-THP-1 macrophages, LPS-induced IL-6 level was decreased by Res and Ptes pretreatments, both with inherent stilbene structure. On the

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contrary, Gen (10 μM) pretreatment elevated LPS-induced IL-6 mRNA level.

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When pretreated with the bioactive compounds, Res, Ptes and Gen, followed by LPS

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stimulation of THP-1 monocytes (Fig. 6C) or D-THP-1 macrophages (Fig. 6D), IL-6 protein level was markedly increased by Res or Ptes treatments in THP-1 monocytes, while mildly

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depressed in D-THP-1 macrophages. Gen pretreatment mildly induced IL-6 protein in THP-1

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monocytes (Fig. 6C), whereas no change was observed in D-THP-1 macrophages (Fig. 6D) compared to control.

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Journal Pre-proof 4. Discussion Res research has yielded controversial health effects both in vitro and in vivo. Given that monocytes and macrophages are involved in immune modulation, the objective of this study was to elucidate how Res at both physiological and pharmacological concentrations, impacts the immune system, primarily monocytes and macrophages. The results from our current study confirmed our hypothesis that Res may exert concentration-dependent effects on monocytes and

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macrophages.

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We chose the human THP-1 cell line as our model as monocytic THP-1 cells can differentiate

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into macrophages (D-THP-1). In the present study, we found Res to act differently on monocytes

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and macrophages. The most striking observation in this study was that Res enhanced inflammatory response in THP-1 monocytes, while it exerted anti-inflammatory effects in D-

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THP-1. Of note, Res enhanced LPS-induction of several pro-inflammatory cytokines’ gene

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expression in THP-1. Moreover, monocytes appeared to be much more sensitive to Res than macrophages. The IC50 related to growth for THP-1 monocytes treated with Res was ~5 μM

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while the IC50 of Res for D-THP-1 was ~ 80 μM. The effect of Res on monocytes appeared to

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be associated with impacting the cell cycle. At lower doses, 5 and 10 μM, Res inhibition of the proliferation of THP-1 appeared to be through S-phase arrest. However, Res at 25 μM appeared to induce G0/G1 phase arrest and apoptosis. Hence, our data support that Res exerted a concentration-dependent differential effect on cell proliferation and apoptosis depending on cell types, with monocytes being more sensitive toward Res than macrophages. At the molecular level, treatment of monocytes with Res was associated with activation of CDKN1A/p21. p21 is known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1. The p21 protein binds to and inhibits the activity of cyclin-CDK2 or -CDK1 complexes and functions as the p53-

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Journal Pre-proof dependent (tumor suppressor gene) regulator of cell cycle progression at G1 [53]. Moreover, the p21 protein can also interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair [54]. Up-regulation of p21 by Res would support the notion that exposure of THP-1 monocytes to Res would result in DNA damage/stress. The effect on p21 appeared to be specific, as CDKN1B/ p27 mRNA expression was not influenced by Res treatment. Thus, the induction of

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p21 by Res in THP-1 cells, together with cell cycle data, supported that the Res-mediated

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induction of p53-independent p21 gene expression may be responsible for concentration-

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dependent inhibition of cell-proliferation and induction of S phase arrest (Res at 5 and 10 μM) or

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G0/G1 arrest (Res at 25 μM) in monocytes.

One of our main goal in this study was to elucidate physiological vs. pharmacological effects

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of Res. Our previous work [38] indicates that circulating/tissue concentration of Res from dietary

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consumption of Res in a rodent model is at most ~ 5 μM. Taking this observation into account and our observed in vitro IC/EC50 of growth, inhibition of xenobiotics as well as inhibition of

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TLR-induced cytokines in the current study, we reason that at physiological level (i.e. from food)

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Res may act as an anti-inflammatory agent. Res, after entering circulation or tissues, can inhibit monocyte growth (IC50 = 5 μM), inhibit TLR-induced cytokine (IL-6) production by macrophages (IC50 = 3 μM). The promoting effects of Res on TLR-induction of cytokines in THP-1 monocytes are likely pharmacological as the effective concentration is above 5 μM (EC50>> 25 μM). However, it remains unclear if one can describe Res’ anti-inflammatory effects as beneficial to host. This property of Res may be a double-edged sword and may be dependent on the physiological state of a host. For example, it is not known whether Res would prevent or exacerbate an existing bacterial infection of the host. Further experiments using in

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Journal Pre-proof vivo inflammation models are necessary to elucidate under what condition Res may exert beneficial effects. The effects of Res on inflammatory cytokines in monocytes appeared relatively non-specific as all TLR inducible cytokines (IL-1, IL-6 and IL-8) appeared to be enhanced at similar concentrations. However, in D-THP-1 macrophages, IL-6 appeared to be the only one that was inhibited compared to IL-1 and IL-8. Moreover, The IC50 for Res inhibition of LPS induced IL-6 is within the physiological range achievable by diet which lends support to

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a physiological role for Res through the regulation of IL-6 -mediated inflammatory pathway.

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Additional studies are needed to validate the significance in-vivo.

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The AHR-dependent P450 (CYP) cytochromes, CYP1A1 and B1 are key members

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participating in the metabolism of xenobiotics and endobiotics [55] and are highly expressed in a diversity of tumor cells [55, 56]. In our study, CYP1A1 was not modulated by Res in THP-1 and

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D-THP-1 cells and was not detected in either type of cells at baseline. The observed differential

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effects of resveratrol on monocytes and macrophages may be due to the upregulation of expression of AHR/CYP1B1-related xenobiotic metabolism pathway. Figure 7 illustrates some

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possible scenarios based on our results and published literature. Previously, we reported that

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stimulation of monocytic THP-1 differentiation can lead to a 4-fold increase in AHR and an 85fold increase in CYP1B1 mRNA in D-THP-1 cells [47]. Our previous study also suggests that activation of AHR-dependent pathway by external AHR agonist can lead to up-regulation of CYP1B1 and inhibition of selective inflammatory cytokines induced by TLR ligands in THP-1 but not in D-THP-1 cells [47]. These results suggest that AHR agonist, often subjected to metabolism by xenobiotic metabolizing enzymes such as CYP1B1, acts as an inhibitor of TLRinduced inflammation. Baseline CYP1B1 expression levels may thus influence an agonist’s biological activity. Hence, it is possible that Res is less metabolized in THP-1, and therefore, can

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Journal Pre-proof exist in sufficient concentration to enhance TLR ligand-induced immune pathways (Figure 7, Scenario 1). According to our results, Res inhibits CYP1B1 expression in both THP-1 and DTHP-1. Hence, Res appeared to act as an antagonist of AHR in our system and thus expected to induce inflammatory cytokines. However, Res inhibited TLR-induced IL-6 in D-THP-1. We consider this to be related to the presence of endogenous AHR ligands generated via differentiation. It has been reported that enhanced AHR/CYP1B1 pathways may represent

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feedback from generation of endogenous AHR ligands such as 6-formylindolo[3,2-b]carbazole

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(FICZ) [57]. FICZ is also known to increase production of inflammatory cytokines [58]. The

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feedback upregulation of CYP1B1 would help terminate FICZ’s action and attenuate

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inflammatory responses. It is likely that in our study, initiation of differentiation to D-THP-1 cell resulted in an increase in endogenous AHR ligands which augmented AHR/CYP1B1 pathway.

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Res or its metabolites from CYP1B1 may compete with the endogenous AHR ligands and exert

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inhibitory effects on both AHR/CYP1B1 and TLR-induced IL-6 production in D-THP-1 cells (Figure 7, Scenario 2). Hence, the actions of Res on monocytes and macrophages may not be

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contradictory, but likely depend on multiple factors such as affinity of Res or Res metabolites to

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AHR compared to endogenous AHR ligands, the timing and extent of Res metabolism, inducibility of CYPs etc. Alternatively, Res or its metabolites may modulate AHR-independent pathway to influence inflammatory pathways. Additional studies are necessary to tease out and validate the complex interactions and our hypothesis. In this study, we also examined whether the effects exerted by Res was similar to other basic stilbene structure-containing polyphenols. According to our data, Ptes exerted similar proinflammatory effect as Res in monocytes, whereas the non-stilbene compound Gen showed no effects. Of note, our present study is limited to in vitro data. Further research on an in vivo

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Journal Pre-proof animal model of inflammatory disease and clinical trials would depict a well-rounded picture of Res’ immunomodulatory effects. Currently Res is commercially available as dietary supplements and promoted to exert antioxidant, anti-inflammatory, anti-carcinogenic effects, based on numerous in vitro studies [1416]. However, most of the beneficial effects reported in the literature often use supraphysiological doses of Res. Results from the present study confirmed our hypothesis that

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biological effects exerted by Res may be cell type-specific and concentration-dependent. Our

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data highlight pleiotropic biological effects of Res and caution against recommendation for

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human consumption without further clinical validation.

Acknowledgment

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declare no conflicts of interest.

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This work was supported by USDA appropriated fund #8040-51530-057-00-D. The authors

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Journal Pre-proof Figure Legends Figure 1. Effect of resveratrol on cell growth for THP-1 monocytes and D-THP-1 macrophages. THP-1 monocytes (1×105/mL) (A) and D-THP-1 macrophages (5×105/mL) (B) were plated in 6-well plates treated with (1, 5, 10, 25 μM) or without resveratrol (control, DMSO) for 48 hours. Total cell count of THP-1 cells was measured using Trypan Blue staining after 24 and 48 hours and value are expressed as mean cell number per ml ±SD (n=4). Growth of D-THP-1 was determined using the SRB method and

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values are expressed as the means ±SD (n=8) of OD530. * was used to indicate significantly different

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from vehicle control (p<0.05).

Figure 2. Effect of resveratrol on cell cycle in THP-1 monocytes. THP-1 monocytes (1×105/mL) were

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plated in 75-cm2 flasks for 24 hours and then treated with (1, 5, 10, 25 μM) or without resveratrol

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(control, DMSO) for 48 hours. Cell cycle distribution of collected cells was determined by using flow

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cytometry (A). Percentage of cells in each phase of the cell cycle (sub G0/G1, G0/G1, S, and G2/M) was calculated using CELL Quest. Values were expressed as the mean ± SD of 6 separate experiments. * was

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used to indicate significantly different from control at each phase of cell cycle (p<0.05). p21 (B) and p27 (C) mRNA expression was measured by quantitative RT-PCR. Values were expressed as means ± SD of

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3 separate experiments (n=3). Percentage of cells expressing p21 protein (D) was measured by using flow

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cytometry, according to manufacturer’s recommendations. Means with different letters signify significantly different (p<0.05). p21: cyclin-dependent kinase inhibitor 1A (CDKN1A). p27: cyclindependent kinase inhibitor 1B (CDKN1B). Figure 3. Effect of resveratrol on AHR-responsive gene expression in THP-1 monocytes and DTHP-1 macrophages, without LPS stimulation. THP-1 monocytes (A) and D-THP-1 macrophages (B) were treated with (1, 5, 10, 25 μM) or without resveratrol (control, DMSO) for 48 hours. CYP1B1 mRNA expression was then measured using quantitative RT-PCR. Values were expressed as the means ±SD of 3 separate experiments (n=3). Means with different letters were significantly different (p<0.05).

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Journal Pre-proof Figure 4. Effect of resveratrol on pro-inflammatory cytokine expression in THP-1 monocytes. THP1 monocytes (2.5×105/mL) were cultured for 24 hours and then treated with (1, 5, 10, 25 μM) or without resveratrol (control, DMSO) for 48 hours. (A) 10 ng/mL LPS was added to each well and incubated at 37 °C for 2 hours. (B) 500 ng/mL PAM was added to each well and incubated at 37 °C for 2 hours. (C) Cells were incubated at 37 °C for an additional 2 hours only with resveratrol and DMSO treatment. Cytokine mRNA expression was measured by quantitative RT-PCR. Values were presented as mean ± SD of 3

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separate experiments. Means with different letters were significantly different (p<0.05). * was used to indicate means are significantly different from control (p<0.01). (D, E) THP-1 monocytes were pretreated

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with resveratrol for 48 hours and then stimulated with LPS or PAM for 4 h. IL-6 secretion was measured

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by an ELISA. Data were normalized by cell count. Bars represent the means ± SD of three separate

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experiments (n=3). Means with different letters were significantly different (P < 0.05).

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Figure 5. Effect of resveratrol on pro-inflammatory cytokine expression in D-THP-1 macrophages. THP-1 monocytes (5×105/mL) were differentiated by PMA in the dark for 48 hours. D-THP-1

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macrophages were then treated with (1, 5, 10, 25 μM) or without resveratrol (control, DMSO) for 48 hours. (A) 10 ng/mL LPS was added to the final cell culture for 2 hours at 37 °C. (B) 500 ng/mL PAM

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was added to the final cell culture for 2 hours at 37 °C. (C) Cells were treated for additional 2 hours with

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only resveratrol and DMSO. Cytokine mRNA expression was measured by quantitative RT-PCR. Values were expressed as the means ±SD of 3 separate experiments. Means with different letters were significantly different (p<0.05). * was used to indicate significantly different from control (p<0.01). (D, E) D-THP-1 macrophages were pretreated with resveratrol for 48 hours and then stimulated with LPS or PAM for 5 hours. IL-6 secretion was measured by an ELISA. Data were normalized by cell count. Bars represent the means ± SD of three separate experiments (n=3). Means with different letters were significantly different (p < 0.05). Figure 6. Effect of resveratrol, pterostilbene and genistein on pro-inflammatory IL-6 expression in THP-1 monocytes and D-THP-1 macrophages. THP-1 monocytes (2.5×105/mL) and D-THP-1

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Journal Pre-proof macrophages (5×105/mL) were cultured overnight and then treated with DMSO (control), resveratrol (10 μM), pterolstilbene (10 μM) or genistein (10 μM) for 48 hours. Cells were then stimulated with 10 ng/mL LPS (A & B; THP-1; D-THP-1 respectively) for 2 hours for mRNA and 4h for IL-6 protein (C & D; THP-1; D-THP-1 respectively) secretion at 37 °C. Cytokine mRNA expression of treated cells was measured by quantitative RT-PCR. Values were expressed as the means ± SD of 3 separate experiments. Means with different letters were significantly different (p<0.05). * was used to indicate significantly

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different from control (p<0.05). IL-6 secretion was measured by an ELISA. Data were normalized by cell

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count. Bars represent the means ± SD of three separate experiments (n=3).

Figure 7. Hypothetical models of effects of Res on AHR and inflammatory pathways.

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A. Scenario 1: Effects of Res in low CYP1B1 THP-1 cells. Low baseline CYP1B1 minimizes metabolism

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of Res as well as external AHR agonist. This allow external ligands to activate AHR-dependent increase

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in CYP1B1 mRNA and inhibition of selected TLR-induced inflammatory cytokines. Higher Res levels enhance TLR-induced inflammatory cytokines production in THP-1 cells. B. Scenario B: Effects of Res

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in High CYP1B1 D-THP-1 cells. Differentiation leads to generation of endogenous AHR ligands that act through AHR to induce CYP1B1 and inhibit inflammatory cytokines production. Higher CYP1B1 would

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metabolize external AHR agonist and render it less effective. Resveratrol or its metabolite may compete

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with the endogenous AHR ligands and result in inhibition of CYP1B1 and TLR-induced inflammatory cytokines. Dashed red line represents hypothetical pathways.

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