Secondary Logo

Share this article on:

Resveratrol Downregulates Biomarkers of Sepsis Via Inhibition of Proteasome's Proteases

Silswal, Neerupma*; Reddy, Nidhi S.; Qureshi, Asaf A.*; Qureshi, Nilofer*,†

doi: 10.1097/SHK.0000000000001080
Basic Science Aspects
Editor's Choice

ABSTRACT Lipopolysaccharide (LPS) is the main agonist of gram-negative bacteria and initiates inflammation. We recently reported that plasmas from sepsis patients revealed increased levels of following group of biomarkers; VCAM-1, ICAM1, CRP, resistin, and proteasome LMP subunits. Our objective here was to compare effects of resveratrol (shown to be a nonspecific proteasome inhibitor by us) and a known LMP7 inhibitor (ONX-0914, specific inhibitor) on proteasome's activities, as well as on inflammatory markers mentioned above in human blood monocytes. Using fluorescence-based assays on blood monocytes purified proteasomes, resveratrol (0–100 μM) inhibited all three protease activities, predominantly LMP7. Similarly, resveratrol inhibited all three protease activities using cell-based luminescence assay. In contrast, ONX-0914 was more selective and potent for LMP7 activity. Resveratrol and ONX-0914, both significantly inhibited expression of LPS-induced biomarkers mentioned above in CD14+ monocytes. Moreover, resveratrol itself, as well as in combination with LPS, accumulated pIκBα in CD14+ monocytes. Collectively, our data suggest that resveratrol is a less potent inhibitor of all three; CT-like (predominantly LMP7), T-like and PA protease activities and is less toxic to human monocytes than ONX-0914 (a selector inhibitor of only LMP7) as observed by an autophagy detection kit. Also, resveratrol reduces LPS-induced inflammatory cytokine expression by decreasing the translocation of NF-κB due to an increase in inhibitor pIκBα. Therefore, resveratrol can be used to curb inflammation in diseased states like sepsis and other disorders.

*Shock/Trauma Research Center, Department of Basic Medical Science, School of Medicine, University of Missouri Kansas City, Kansas City, Missouri

Departments of Pharmacology and Toxicology, School of Pharmacy, University of Missouri Kansas City, Kansas City, Missouri

Address reprint requests to Nilofer Qureshi, PhD, Department of Basic Medical Science, School of Medicine, Shock/Trauma Research Center, 2411 Holmes Street, University of Missouri Kansas City, Kansas City, MO 64108. E-mail: qureshin@umkc.edu

Received 7 June, 2017

Revised 23 June, 2017

Accepted 4 December, 2017

This study was supported in part by NIH grants GM102631 (NQ) and GM102631S1 (NQ).

The authors have no financial conflicts of interest.

Back to Top | Article Outline

INTRODUCTION

Lipopolysaccharide (LPS), a major constituent of gram-negative bacterial cell wall, triggers a dysregulated cascade of inflammation, which results in sepsis and ultimately septic shock. Septic shock affects about 400,000 patients per year in the US alone, of which 50% to 70% die due to complications with excessive amount of LPS triggered inflammation/cell death (1). Presently, despite the tremendous number of drugs that developed for septic shock, there is no effective cure for this disease. Increased bacterial load leads to elevated LPS, and activation of immune system resulting in systemic inflammation, followed by tolerance (2). Blood pressure drops due to increased nitric oxide (NO), there is lowered tissue perfusion that leads to cell death, and finally organ failure (3). Sepsis is more common than heart attacks and claims more lives than cancer.

We have used LPS and other agonists as prototypes for inflammation. We have demonstrated that proteasome inhibition reduces expression of cytokines and adhesion factors in the monocytes by inhibiting transcription factors, and other mediators in signal transduction pathways, which also greatly reduces mortality in the CLP mouse model treated with proteasome inhibitors and antibiotics. Recently, we have shown that customized treatment may be necessary for prevention and cure of septic shock depending on whether the patients are in the inflammatory mode or the tolerant mode, where the cells are refractory to further treatments to LPS (4). Thus novel strategies are crucially needed for prevention and cure for septic shock.

We have shown that the ubiquitin proteasome system (UPS) maintains the basic cellular processes of eukaryotic system and as part of UPS; proteasomes (P) controls a variety of physiological processes by regulating selective degradation of proteins modulated by LPS (5, 6). Multiple pro- and anti-inflammatory pathways triggered by release of LPS into the blood stream by the gram-negative bacteria. LPS is the prototype activator of human and murine tissue monocytes/macrophages and acts through both MyD88-dependent and MyD88-independent (TRIF/TRAM) pathways (7, 8). In cecal-ligation puncture model of sepsis, Chen et al. recently reported that LPS-induced expression of CD14 is via an activation of MyD88-dependent and TRIF-independent pathway (9). Human Monocytes differ from mouse macrophages and constitute an important part of our immune system: they have a dual role of providing immune defense as well as causing tissue damage during an infection.

Our previous work established that initial interaction of LPS and monocytes/macrophages induces all three proteasome activities, thus initiating a process of proteasome-mediated degradation of signaling mediators such as TLR4, IRAK-1, IRAKM, phosphorylated interferon regulatory factor 3 (P-IRF3) (10, 11). This leads to a net increase in ubiquitinated proteins and activation of transcription factors, such as NF-κB (proteasome degrades its inhibitor IκB) thus causing inflammation. The proteolytic active sites of proteasome reside on three important constitutively expressed subunits: X (β5), Y (β1), and Z (β2), which exhibit chymotrypsin-like (CT-like), post-acidic (PA) and trypsin-like (T-like) activities, respectively in murine macrophages (12). The constitutive subunits of proteasomes (P) can be induced to those containing immunoproteasome (IP) subunits LMP7 (β5i), LMP2 (β1i), and LMP10 (β2i) after priming with pro-inflammatory cytokines, or LPS with an increase in CT-like and T-like activities (13). Human monocytes primarily have LMP7, LMP2, and LMP10 catalytic proteasome subunits, which are encoded by PSMB8, PSMB9, and PSMB10 genes, respectively (12). The CT-like activity of X and LMP7 subunit containing proteasomes is most potent activity for modulating inflammation and controlling multiple signaling pathways in response to agonists such as LPS, CpG DNA, and peptidoglycan (5, 6, 14). Thus, proteasome inhibitors needed during the initial inflammatory mode of sepsis, where there may be excessive inflammation.

We had initially screened several compounds for inhibition of purified chymotrypsin-like activity from rabbit muscle, and showed that proteasome inhibitors like lovastatin and quercetin, in combination with antibiotic (Primaxin) prevents septic shock in a cecal-ligation and puncture mouse model of sepsis (15). We have also shown that resveratrol is a proteasome inhibitor of inflammation in mouse macrophages (16). Resveratrol (3,5,4′-trihydroxystilbene) is a plant based compound mainly found in grapes, berries, and peanuts. Resveratrol has various beneficial effects on human health due to its anti-inflammatory, antioxidant, anti-aging, and anti-tumor properties (17–19). Resveratrol inhibits the activity of cyclooxygenase and lipoxygenase enzymes that cause inflammation and ultimately leads to tumor formation by uncontrolled proliferation of cells (20). It also improves cardiovascular function by antihypertensive effects (21). In an endotoxic shock model, resveratrol prevents kidney and lung damage (22, 23). Many diseases like atherosclerosis, diabetes, obesity, sepsis, arthritis, neuro-degeneration, and cancer are directly based on inflammation, and proteasome inhibitors would be expected to slow down the progression of these diseases.

Resveratrol has been known to decrease various pro-inflammatory and anti-inflammatory cytokines like tumor necrosis factor (TNF-α), interleukin (IL)-1β, and IL-6 (17, 24). There are several synthetic proteasome inhibitors (PIs), such as bortezomib, carfilzomib, marizomib, ixazomib, delanzomib, oprozomib, PR-825, PR-924, and ONX-0914, which have been used in clinical trials for cancer and more recently for inhibiting inflammation (25, 26). PIs differ in their selectively to inhibit X, Y, Z, or LMP7, LMP2 and LMP10 subunits, for example PR-924 and ONX-0914 are LMP7-selective, while carfilzomib and oprozomib actively inhibit both X and LMP7 subunits (27, 28). However, selective proteasome inhibitors have not been tested along with resveratrol (a natural PI, not a specific inhibitor) to inhibit the LPS-induced inflammatory biomarkers of sepsis in CD14+ human monocytes.

In our recent communication (4), we have reported that biomarkers VCAM-1, ICAM1, CRP, and resistin, are upregulated in plasmas of septic patients. Moreover, during LPS-induced inflammatory phase, there is an increased expression of immunoproteasome subunits (10). Therefore, resveratrol (not a specific inhibitor) and LMP7-specific inhibitor ONX-0914 were tested for downregulating these LPS-induced biomarkers. Although both resveratrol and ONX-0914 are proteasome inhibitors, mechanisms by which resveratrol and ONX-0914 inhibit inflammation via the TLR4 signaling pathways, and inhibit markers of sepsis-induced inflammation in CD14+ monocytes are not well understood. In current study, we compared the effects of resveratrol and ONX-0914 on proteasome's activities using purified proteasomes as well as cell-based assays. Furthermore, we determined their effects on LPS-induced expression of cytokines and proteasome subunits using real-time PCR. We investigated their effects on LPS-induced NF-κB pathway using CD14+ human monocytes. Lastly, we established that inhibitory effects of resveratrol are not due to toxicity or autophagy (self-eating). Using high doses or potent proteasome inhibitors for at least 12 to 24 h can lead to induction of autophagy and cell death.

Back to Top | Article Outline

METHODS

Ethics statement

The use of human blood was approved by the Ethics Committee at UMKC, Kansas City, Mo.

Back to Top | Article Outline

Materials

RPMI-1640 medium and gentamycin were purchased from Lonza. Heat-inactivated low endotoxin fetal bovine serum (FBS) was from Omega scientific. Highly purified, deep rough chemotype LPS (Re-LPS) from Escherichia coli D31m4 was prepared as described previously (14). Proteasome-Glo reagents for determining the cellular proteasome's activities were purchased from Promega (Madison, Wis). Ac-WLA-AMC, Ac-ANW-AMC, and Ac-PAL-AMC fluorogenic substrates, as well as human proteasomes and immunoproteasomes were purchased from R&D systems. Trans-Resveratrol (later called resveratrol) was purchased from “Mega Resveratrol” (60 Newtown Toad # 32, Danbury, Conn). ONX-0914, specific inhibitor of LMP7 was purchased from UBPBio. MTT (Thiazolyl Blue Tetrazolium Bromide) for cell death assay and PMA (phorbol 12-myristate 13-acetate) for differentiation were purchased from Sigma-Aldrich. Cyto-ID Autophagy Detection Kit was purchased from Enzo Life Sciences Inc. (Farmingdale, NY). Proteasome-Glo Chymotrypsin-Like, Trypsin-Like and Caspase-Like Cell-Based Assays were purchased from Promega.

Back to Top | Article Outline

Human peripheral blood isolation and cell culture

Human mononuclear fractions of heparinized whole blood were obtained from volunteers in accordance with the policies of the University of Missouri Kansas City. Mononuclear cells were separated from blood by mixing 50 μL of monocyte enrichment cocktail and 25 μL of granulocyte depletion cocktail per ml of blood. After incubation for 20 min at room temperature, sample was diluted with equal volume of PBS+2% FBS and 1 mM EDTA. Diluted sample was layered on top of the Ficoll-Paque PLUS and centrifuged for 20 min at 1200 × g with the brake off. The enriched CD14+ cells were removed from plasma interface, washed and counted. Monocyte purity was >96%. Human blood monocytes were suspended in RPMI 1640 complete media. After each treatment, cells were pelleted and stored at −20oC.

Back to Top | Article Outline

THP-1 cell culture

Human monocyte cell line THP-1 was cultured in RPMI-1640 supplemented with 10% (v/v) heat-inactivated low endotoxin FBS and 1% (v/v) gentamycin in 5% CO2 at 37°C. The culture media was changed every 2 days. For differentiation into macrophages, THP-1 cells were treated with 10 ng/mL PMA and incubated at 37oC and 5% CO2 for 24 h.

Back to Top | Article Outline

Cell viability assays

Monocytes (2 × 104 cells/well) were cultured in a 96-well plate for 24 h and then incubated with different concentrations of resveratrol and ONX-0914. Stock solutions of resveratrol and ONX-0914 were prepared in dimethyl sulfoxide (DMSO) followed by dilution in RPMI1640 complete media containing 0.2% DMSO. After drug treatment, cells were first washed with 100 μl/well PBS and then, 100 μL MTT (1 mg/mL) stock solutions was added to each well. Cells were incubated for 2 h in the dark, washed and the reaction was terminated by addition of 100 μL of isopropanol. Plate was allowed to shake for 10 min at room temperature and absorbance was read at 570 nm using plate reader Cytation 3. We also performed lactate dehydrogenase assay (Promega) and observed similar results.

Back to Top | Article Outline

Autophagy detection assay

Blood monocytes were cultured on laminin-coated coverslips, followed by incubation with resveratrol (80 μM), rapamycin and chloramphenicol (+ve control), and vehicle for 4 h. While THP1 cells were first differentiated with PMA (10 ng/mL) in media for 24 h, and washed, followed by incubation with vehicle, rapamycin and chloramphenicol (as +ve control), resveratrol (80 μM), and ONX (0.25 μM) with or without LPS (10 ng/mL) for 4 h. Cells were washed twice with assay buffer followed by incubation with microscopy dual detection reagent (100 μL) for 30 min at 37oC in dark. The microscopy dual detection reagent was prepared following the protocol from the manufacturer. After incubation, monocytes were finally washed with assay buffer. Fluorescence measurements were performed under Cytation-3-fluorometric reader using DAPI and FITC filter sets.

Back to Top | Article Outline

Measurement of proteasome activities

Proteasome activity by purified human proteasome XYZ subunits and LMP proteasome subunits (Fluorescence assay). Purified human erythrocyte 20S proteasome and peripheral blood monocyte 20S immunoproteasomes (R&D Systems, Minneapolis, Minn) were assayed for different peptidase activities using fluorogenic tri- and tetra-peptide substrates coupled to AMC. For measurement of Ch-like, T-like and PA-specific activities, the fluorogenic substrate (Suc-LLVY-AMC), (Boc-LRR-AMC), and (Z-LLE-AMC) were used, respectively. Proteasome assay was performed in a reaction buffer (50 mM HEPES pH 8.0, 0.5 mM EDTA, 0.035% SDS) using flat bottom 96-well plate. Human proteasome or immunoproteasome enzymes (final concentration 2 nM) were added to the reaction buffer containing different concentrations of resveratrol and ONX-0914. Reaction mixture without peptide was incubated at 37°C for 20 min. Finally, 10 μM peptide pre-diluted in assay buffer was added to a final volume of 200 μL. Plate was read from top at Ex 345 nm/Em 445 nm in 10–20 sec interval in Cytation-3-fluorometric microplate reader for 60 min. All reactions were performed in triplicates and repeated at least 3 times. Percentage inhibition of proteasome and immunoproteasome activities was calculated relative to the vehicle controls.

Back to Top | Article Outline

Cell-based chymotrypsin-like, trypsin-like, and postacidic proteasome activity (luminescence assay)

THP1 cells 1 × 104 cells/100 μL were added in each well of 96-well white colored plates for luminescence studies. After the addition of medium and/or inhibitors (at various concentrations), plates were incubated in 37°C incubator at 5% CO2, for 30 min and were taken out of the incubator 20 min prior to the addition of the reagents to detect CT-like, T-like, and PA protease enzymatic activity. The relative luminescence units (RLUs) of assays were determined using a plate luminometer according to manufacturer's instructions in the linear dose ranges.

Back to Top | Article Outline

RNA isolation

CD14+ blood monocytes (2 × 106 cells /well) were seeded in six-well tissue culture plates in RPMI 1640 complete medium and incubated overnight. Cells were then pretreated with optimal doses of resveratrol (80 μM), ONX-0914 (0.25 μM), and DMSO for 1 h before stimulation with LPS (10 ng/mL) for 4 h in CO2 incubator depending on the treatment. Total RNA was isolated from cell lysates using Qiagen RNeasy Mini kit according to manufacturer's instructions. Quality of RNA was assessed by spectrophotometric measurements.

Back to Top | Article Outline

Real-time PCR

Real-time RT-PCR was performed on total RNA isolated from treated CD14+ monocytes. Primers-probes and One-Step qRT-PCR kit were obtained from Life Technologies (Foster City, Calif). All reactions were performed in triplicate using equal amount of mRNA per reaction. Real-time PCR assays were completed using a Step-one plus Real-time PCR system. Gene expression from cell cultures was normalized (2−ΔΔC T analysis) to GAPDH.

Back to Top | Article Outline

Western blot

Protein extracts were prepared from treated CD14+ monocytes using cell extraction buffer supplemented with a protease inhibitor cocktail, containing phenyl-methyl-sulfonyl fluoride, and phosphatase inhibitors. Protein concentrations were measured with BCA protein assay kits. Each well of SDS gel was loaded with 20 μg of protein and were electrophoresed at a constant 100 V in 1× Tris glycine buffer for 60 min. Proteins in gels were transferred to the PVDF membrane using wet transfer. After appropriate antibody treatments, the bands were visualized with SuperSignal West Pico enhanced chemiluminescence kit from Thermo Scientific (Rockford, Ill).

Back to Top | Article Outline

Immunocytochemistry

Freshly isolated CD14+ monocytes in RPMI-1640 complete media were added to poly-D-lysine coated 4-well culture slides (BD biocoat). After 24 h incubation at 37°C and 5% CO2, cells were treated with vehicle (DMSO), and 80 μM resveratrol for 1 h, followed by 10 ng/mL LPS for 30 min. After incubation, cells were immediately fixed in 4% paraformaldehyde for 15 min at room temperature followed by washing with PBS. Cells were permeabilized with ice-cold 100% methanol for 10 min at −20°C and blocked with blocking solution containing 5% normal goat serum for 1 h. Cells were then incubated with primary antibody (1:200) for overnight at 4°C. Next day, after 3× washing, cells were incubated with fluorochrome-conjugated secondary antibody (1:250) for 1 h at room temperature in dark. After washing, slides were cover slipped with Prolong Gold Antifade Reagent with DAPI, and immediately examined under fluorescence microscope.

Back to Top | Article Outline

Statistical analysis

All statistical procedures and graphs were performed with GraphPad Prism 5 (La Jolla, Calif). Data are presented as mean ± SEM. Data were compared using either a paired t-test or a one-way analysis of variance, and significance was set at the P < 0.05 level. One-way analysis of variance was followed with appropriate post hoc tests. A Bonferroni post hoc adjustment was used to correct for two to three comparisons to avoid type I error.

Back to Top | Article Outline

RESULTS

Resveratrol and ONX-0914 inhibit purified human proteasome (X, Y, Z) and immunoproteasome (LMP7, LMP2, and LMP10) activities

To investigate the proteasome inhibitory activity of resveratrol, we measured the activity of proteasomes in human monocyte cell line after incubation with increasing concentrations of resveratrol. There was a dose-dependent inhibition of PA, T-like, and CT-like proteasome activities after incubation with resveratrol, as shown in Figure 1. Resveratrol inhibited CT-like activity with an IC50 value of 1.895 μM, T-like activity with IC50 value of 4.999 μM, and PA activity with IC50 value of 1.370 μM. Therefore, at cellular level, resveratrol is a better inhibitor of CT-like and PA proteasome activity.

Fig. 1

Fig. 1

To evaluate preference of inhibition (constitutive or inducible proteasome) of resveratrol, we used purified human 20S proteasome and immunoproteasome in a cell-free system. Moreover, we compared resveratrol inhibitory effect with LMP7 specific inhibitor (ONX-0914). Resveratrol directly inhibited the CT-like activity of the constitutive proteasome (X, Y, and Z) and immunoproteasome (LMP7, LMP2, and LMP10) in a dose-dependent manner with IC50 59.44 μM and 39.77 μM, respectively, while ONX-0914 was active at nM dose (0.63 μM for X, Y, Z subunits and 0.17 μM for LMP subunits) (Fig. 2). In a similar manner, resveratrol inhibited PA activity, with IC50 for proteasomes 52.89 μM and for LMP subunits 40.75 μM, but only 30% inhibition of PA activity was observed with 50 μM dose of ONX-0914 (Fig. 2). We did not observe any inhibition of T-like activity even with highest dose of ONX-0914, while resveratrol inhibited it only 33% with 100 μM dose. These data suggest that in vitro, resveratrol mainly inhibits CT-like and PA activities with preference for LMP subunits.

Fig. 2

Fig. 2

Back to Top | Article Outline

Resveratrol and ONX-0914 and cell toxicity as observed by MTT, LDH, or autophagy assay

Most synthetic proteasome inhibitors are toxic to cells; therefore, we investigated the toxicity of drugs by quantifying the cell viability of monocytes by treating with different concentrations of resveratrol (20 μM–100 μM) and ONX-0914 (0.01 μM–0.5 μM) for 4 h. MTT and LDH cell death assay revealed that cells were >90% viable even after treating with highest dose of resveratrol (100 μM) (data not shown). Moreover, we also examined the effect of resveratrol on autophagy using human blood monocytes and THP1 cells. Resveratrol treatment did not induce autophagy in monocytes or THP1 cells after 4 h incubation (Figs. 3 and 4). However, rapamycin and chloroquine treatment (+ve control) caused increased green fluorescence, as evidence of autophagy vacuole accumulation (Figs. 3 and 4). On the other hand, ONX-0914, itself, at 0.25 μM concentration resulted in autophagy and vacuole accumulation, observed as bright green fluorescence. Also, there was no change in expression of Bax and Bcl2 genes at 80 μM dose of resveratrol. Taken together, these results suggest that resveratrol can safely be used at dose of 80 μM, as a proteasome inhibitor without inducing cell autophagy. We found that 80 μM dose of resveratrol was optimal for further cell culture studies.

Fig. 3

Fig. 3

Fig. 4

Fig. 4

Back to Top | Article Outline

Resveratrol and ONX-0914 inhibit expression of proteasome's subunits

We further analyzed resveratrol's effect on RNA expression of different proteasome subunits. The gene expression of X, Y, and Z proteasome subunits in CD14+ monocytes was much lower, as compared to LMP subunits, and we did not observe a significant change in proteasome subunits RNA (X, Y, Z) after incubation with resveratrol or ONX-0914 with LPS (Fig. 5). On the other hand, there was robust decrease in mRNA expression of LMP7 and LMP2 subunits after resveratrol and ONX-0914 pretreatment with LPS-induced CD14+ monocytes (Fig. 5). There was no significant change in expression of LMP10 subunit.

Fig. 5

Fig. 5

Back to Top | Article Outline

Resveratrol and ONX-0914 modulate LPS-induced expression of adhesion molecules, cytokines, and autophagy-linked genes

As reported by earlier published studies, resveratrol is anti-inflammatory in its action and at higher doses induces SIRT-1 (29). We studied anti-inflammatory effects of resveratrol on LPS-induced CD14+ monocytes and then compared with ONX-0914 in human CD14+ monocytes. Resveratrol and ONX-0914 both significantly downregulated expression of ICAM-1, TNF-α, IL-8, and IFNγ; however, there was no significant upregulation of Sirt-1 (Fig. 6). LPS-induced expression of resistin was also decreased by resveratrol and ONX-0914. We did not notice any effect on expression of Bcl2 and Bax responsible for cell death. We also investigated effect of resveratrol on expression of autophagy genes and detected that LPS-induced atg7 expression was downregulated by resveratrol but not by ONX-0914. In contrast, atg5 expression was significantly lower in all treatments compared to vehicle (Fig. 7). Similar to atg5, there was no significant change in expression of beclin1, eIF2α, p53, and hdac6 genes (Fig. 7).

Fig. 6

Fig. 6

Fig. 7

Fig. 7

Back to Top | Article Outline

Resveratrol and ONX-0914 accumulate pIκBα in LPS-treated CD14+ monocytes

LPS-induced activation of TNF-α, which is primarily controlled by nuclear factor NF-κB, largely depends on phosphorylation dependent ubiquitination and degradation of inhibitor of kappa B (IκB) proteins. Once the IκB protein is degraded then NF-κB is activated, binds to the promoter regions of genes and induces expression of cytokines. We detected that 60 min incubation of CD14+ monocytes with LPS alone, increased cytoplasmic pIκBα protein levels (Fig. 8). However, 60 min preincubation of LPS-induced monocytes with resveratrol inhibited degradation of pIκBα protein levels. Likewise, ONX-0914 also inhibited pIκBα protein degradation (Fig. 8). Similar to western blot results of pIκBα, we also observed increased red fluorescence in CD14+ monocytes after 30 min of LPS treatment that showed the accumulation of pIκBα (Fig. 9). Altogether, these results indicated that as a proteasome activity inhibitor, resveratrol accumulates LPS-induced pIκBα protein, thus inhibiting the degradation of ubiquitinated proteins such as IκB, activation of NF-κB for transcription, and secretion of various proteins.

Fig. 8

Fig. 8

Fig. 9

Fig. 9

Back to Top | Article Outline

DISCUSSION

In this study, we have demonstrated that although resveratrol and synthetic ONX-0914 are specific for different proteasome's proteases; yet similar results with respect to inhibition of LPS-induced gene expression of inflammatory biomarkers ICAM1, TNF-α and resistin (known marker of insulin resistance) were observed. However, in contrast, our data on autophagy detection suggests that ONX-0914 is far more active than resveratrol in inducing autophagy. Thus some data observed on inhibition of gene expression with LPS and ONX-0914 treatment may be due to autophagy of human monocytes. In our study, no cell death was observed at 80 μM dose of resveratrol in human monocytes using MTT, LDH or by autophagy tests. Therefore, our present study demonstrates that resveratrol is relatively nontoxic to human monocytes at effective concentrations used for decreasing gene expression of LPS-induced inflammatory cytokines in monocytes, without inducing autophagy.

Using purified human 20S proteasome and immunoproteasome in a cell-free system; resveratrol inhibited CT-like and PA activities, with a preference towards immunosubunits, LMP7, and LMP2. In a similar manner, resveratrol inhibited CT-like and PA activities using human monocytes. Mechanistically, once the proteasome's proteolytic activities are inhibited there is increased accumulation of ubiquitinated proteins in monocytes, because of reduced degradation. We detected increased accumulation of LPS-induced pIκBα protein in CD14+ monocytes as is normally observed with proteasome inhibitors. Altogether, our results indicate that natural and less toxic proteasome inhibitor, resveratrol, is at par with LMP7 specific inhibitor ONX-0914 in inhibiting biomarkers (described above) and those that are present during the inflammatory phase after LPS treatment and observed during shock (4). This inhibition of gene expression of these markers with resveratrol was not due to the toxicity of these compounds because of onset of autophagy.

We have identified that septic patient plasmas (4) have increased levels of circulating adhesion molecules like VCAM1 and ICAM1, as well as IL-8 (chemokine affecting neutrophil infiltration). In our monocyte preparations, resveratrol significantly downregulated LPS-induced expression of ICAM-1, TNF-α, and IL-8. We also detected increased expression of IFNγ in monocytes after LPS treatment, which is downregulated by resveratrol. IFNγ expression by monocytes is still debatable and it could be possible that our preparation was contaminated with T cells or natural killer cells, because monocytes do not readily adhere to the plastic. However, Kraaij et al. have also detected IFNγ expression in human monocytes on LPS stimulation supporting our findings (30). Thus, most of the LPS-induced inflammatory mediators (also observed during sepsis, Qureshi et al., unpublished data) could be inhibited by resveratrol in in vitro experiments with or without pretreatment.

The development of inflammatory and autoimmune diseases is dependent on UPS, because several intracellular regulatory proteins are degraded by proteasome are involved in transcription regulation, cytokine secretion, antigen presentation, cell cycle regulation and apoptosis (31). Recently, there is a lot of interest in developing proteasome inhibitors as a therapeutic intervention to control chronic inflammation, as of their ability to inhibit NF-κB signaling pathway. We have previously demonstrated that lactacystin and quercetin act as potent inhibitors of multiple proteases of proteasomes and also inhibit NO production by LPS-stimulated macrophages isolated from various strains of mice (6, 11, 32). Mechanistically, using mouse macrophages, our group has reported inhibition of NF-κB activation, NO and pro-inflammatory cytokines using trans-resveratrol (16).

Our group has also reported the cardiovascular benefits of a mixture of resveratrol (25 mg) on human subjects (33), and in other clinical trials, investigators have used higher doses of resveratrol up to 500 mg/d that were also well-tolerated (34). Importantly, our data using CD14+ monocytes displayed decreased levels of inflammatory markers in LPS-treated monocytes after resveratrol treatment. Therefore, resveratrol can be used as a potential drug for inhibiting pro-inflammatory cytokines, observed early on during sepsis or other inflammatory conditions.

Resveratrol has been tested in clinical trials for cancer, diabetes, metabolic, and cardiovascular disease without any adverse effects. In this study, we demonstrated that natural polyphenol resveratrol inhibited all three protease activities of proteasomes with varying IC50 values, with a preference towards LMP7, hence resulting in accumulation of polyubiquitinated proteins as well as pIκBα in CD14+ monocytes. Collectively, inhibiting LPS-induced p-IκBα degradation would prevent NF-κB translocation to the nucleus thus inhibiting inflammatory gene expression in CD14+ monocytes. In addition, by blocking proteasome activities, most of the other proteins degraded by this complex would also be expected to be stabilized and accumulate in the cell. Since the proteasome regulates both inflammation/tolerance in the cell (4, 10), overall, our study supports the use of resveratrol as a potential natural therapeutic drug for treating inflammatory diseases during the early stages of sepsis and other autoimmune diseases.

Back to Top | Article Outline

Acknowledgments

We thank Dr. Peter Silverstein for his help in obtaining blood samples.

Back to Top | Article Outline

REFERENCES

1. Biswal S, Remick DG. Sepsis: redox mechanisms and therapeutic opportunities. Antioxid Redox Signal 9 11:1959–1961, 2007.
2. Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol 30 10:475–487, 2009.
3. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315 8:801–810, 2016.
4. Qureshi N, Khan DA, Zuberi A, Vernon K, Kaja S, Drees BM, Qureshi AA, Van Way CW, Morrison DC, Silswal N. Levels of proteasome subunit expression provide information about host's immune system status. Intern Med Rev 3 11:1–22, 2017.
5. Shen J, Reis J, Morrison DC, Papasian C, Raghavakaimal S, Kolbert C, Qureshi AA, Vogel SN, Qureshi N. Key inflammatory signaling pathways are regulated by the proteasome. Shock 25 5:472–484, 2006.
6. Qureshi N, Morrison DC, Reis J. Proteasome protease mediated regulation of cytokine induction and inflammation. Biochim Biophys Acta 1823 11:2087–2093, 2012.
7. Rajaiah R, Perkins DJ, Ireland DD, Vogel SN. CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance. Proc Natl Acad Sci U S A 112 27:8391–8396, 2015.
8. Fitzgerald KA, Rowe DC, Barnes BJ, Caffrey DR, Visintin A, Latz E, Monks B, Pitha PM, Golenbock DT. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 198 7:1043–1055, 2003.
9. Chen Z, Shao Z, Mei S, Yan Z, Ding X, Billiar T, Li Q. Sepsis up-regulates CD14 expression in a MyD88 dependent and trif independent pathway. Shock 49 1:82–89, 2018.
10. Silswal N, Reis J, Qureshi AA, Papasian C, Qureshi N. Of mice and men: proteasome's role in LPS-induced inflammation and tolerance. Shock 47 4:445–454, 2017.
11. Qureshi N, Perera PY, Shen J, Zhang G, Lenschat A, Splitter G, Morrison DC, Vogel SN. The proteasome as a lipopolysaccharide-binding protein in macrophages: differential effects of proteasome inhibition on lipopolysaccharide-induced signaling events. J Immunol 171 3:1515–1525, 2003.
12. Borissenko L, Groll M. 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev 107 3:687–717, 2007.
13. Groettrup M, Khan S, Schwarz K, Schmidtke G. Interferon-gamma inducible exchanges of 20S proteasome active site subunits: why? Biochimie 83 (3–4):367–372, 2001.
14. Qureshi N, Takayama K, Mascagni P, Honovich J, Wong R, Cotter RJ. Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry. J Biol Chem 263 24:11971–11976, 1998.
15. Reis J, Tan X, Yang R, Rockwell CE, Papasian CJ, Vogel SN, Morrison DC, Qureshi AA, Qureshi N. A combination of proteasome inhibitors and antibiotics prevents lethality in a septic shock model. Innate Immun 14 5:319–329, 2008.
16. Qureshi AA, Guan XQ, Reis JC, Papasian CJ, Jabre S, Morrison DC, Qureshi N. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor. Lipids Health Dis 11:76, 2012.
17. Csiszar A. Anti-inflammatory effects of resveratrol: possible role in prevention of age-related cardiovascular disease. Ann N Y Acad Sci 1215:117–122, 2011.
18. de la Lastra CA, Villegas I. Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. Biochem Soc Trans 35 (pt 5):1156–1160, 2007.
19. Marchal J, Pifferi F, Aujard F. Resveratrol in mammals: effects on aging biomarkers, age-related diseases, and life span. Ann N Y Acad Sci 1290:67–73, 2013.
20. Kutil Z, Temml V, Maghradze D, Pribylova M, Dvorakova M, Schuster D, Vanek T, Landa P. Impact of wines and wine constituents on cyclooxygenase-1, cyclooxygenase-2, and 5-lipoxygenase catalytic activity. Mediators Inflamm 2014:178931, 2014.
21. Liu Y, Ma W, Zhang P, He S, Huang D. Effect of resveratrol on blood pressure: a meta-analysis of randomized controlled trials. Clin Nutr 34 1:27–34, 2015.
22. Kolgazi M, Sener G, Cetinel S, Gedik N, Alican I. Resveratrol reduces renal and lung injury caused by sepsis in rats. J Surg Res 134:315–321, 2006.
23. Zhang HX, Duan GL, Wang CN, Zhang YQ, Zhu XY, Liu YJ. Protective effect of resveratrol against endotoxemia-induced lung injury involves the reduction of oxidative/nitrative stress. Pulm Pharmacol Ther 27 2:150–155, 2014.
24. Yang Y, Li S, Yang Q, Shi Y, Zheng M, Liu Y, Chen F, Song G, Xu H, Wan T, et al. Resveratrol reduces the proinflammatory effects and lipopolysaccharide- induced expression of HMGB1 and TLR4 in RAW264.7 cells. Cell Physiol Biochem 33 5:1283–1292, 2014.
25. Huber EM, Groll M. Inhibitors for the immuno- and constitutive proteasome: current and future trends in drug development. Angew Chem Int Ed Engl 51 35:8708–8720, 2012.
26. Kuhn DJ, Chen Q, Voorhees PM, Strader JS, Shenk KD, Sun CM, Demo SD, Bennett MK, van Leeuwen FW, Chanan-Khan AA, et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood 110 9:3281–3290, 2007.
27. Singh AV, Bandi M, Aujay MA, Kirk CJ, Hark DE, Raje N, Chauhan D, Anderson KC. PR-924, a selective inhibitor of the immunoproteasome subunit LMP-7, blocks multiple myeloma cell growth both in vitro and in vivo. Br J Haematol 152 2:155–163, 2011.
28. Muchamuel T, Basler M, Aujay MA, Suzuki E, Kalim KW, Lauer C, Sylvain C, Ring ER, Shields J, Jiang J, et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat Med 15 7:781–787, 2009.
29. Hubbard PB, Sinclair AD. Small molecule SIRT1 activators for the treatment of aging and age-related diseases. Trends Pharmacol Sci 35 3:146–154, 2014.
30. Kraaij MD, Vereyken EJ, Leenen PJ, van den Bosch TP, Rezaee F, Betjes MG, Baan CC, Rowshani AT. Human monocytes produce interferon-gamma upon stimulation with LPS. Cytokine 67 1:7–12, 2014.
31. Bassermann F, Eichner R, Pagano M. The ubiquitin proteasome system—implications for cell cycle control and the targeted treatment of cancer. Biochim Biophys Acta 1843 1:150–162, 2014.
32. Qureshi AA, Tan X, Reis JC, Badr MZ, Papasian CJ, Morrison DC, Qureshi N. Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models. Lipids Health Dis 10:177, 2011.
33. Qureshi AA, Khan DA, Mahjabeen W, Papasian CJ, Qureshi N. Nutritional supplement-5 with a combination of proteasome inhibitors (resveratrol, quercetin, delta-tocotrienol) modulate age-associated biomarkers and cardiovascular lipid parameters in human subjects. J Clin Exp Cardiol 4 3:238, 2013.
34. Sergides C, Chirila M, Silvestro L, Pitta D, Pittas A. Bioavailability and safety study of resveratrol 500 mg tablets in healthy male and female volunteers. Exp Ther Med 11 1:164–170, 2016.
Keywords:

Cytokines; IL-8; inflammation; macrophages; proteasome subunits; resistin; VCAM1

© 2018 by the Shock Society