The Flavonoid, Quercetin, Inhibits HIV-1 Infection in Normal Peripheral Blood Mononuclear Cells

Problem statement: Flavonoids and related polyphenols, have been known to possess cardioprotective, anti-tumor, anti-inflammatory, an ti-allergic and anti-viral activities. Previous stu dies have shown that flavonoid, quercetin significantly downregulates expression of pro-inflammatory cytokines in cultured cells via modulation of NF κB and p38MAPK signaling pathways. In the current study, we hypothesize that quercetin exerts anti-HI V activity by differential modulation of pro-and anti-inflammatory cytokine expression in normal PBM Cs. Approach: Cultures of PBMC received either different concentrations of quercetin (1-50 μM) or media alone. The anti-HIV effects of quercetin was evaluated in an in vitro infection model by quantitaing the HIV-1 LTR gene s uppression by quantitative Real Time PCR, HIV-1 p24 antigen pr oduction by ELISA and viral infectivity by MAGI cell assay. Results: Our results showed that quercetin significantly do wnregulates p24 antigen production, LTR gene expression and viral infectivi ty in a dose dependent manner (5-50 μM) as compared to HIV infected untreated control PBMCs. F urther, we reported that quercetin significantly downregulated the expression of the pro-inflammator y cytokine, TNFα with concomitant upregulation of anti-inflammatory cytokine IL-13 as determined b y measurement of gene expression and protein production. A higher level of IL-13 is known to inh ibit TNF-α production and also HIV-1 infection. Thus, differential modulation of pro-and anti-infla mmatory cytokines could be one of the possible mechanisms for the anti-HIV effects of quercetin. Conclusion: Better understanding of the mechanisms underlying the anti-HIV effects of querc etin may help to develop a new neutraceutical agent useful in the treatment of HIV-1 infected sub jects in conjunction with conventional therapeutic regimens.


INTRODUCTION
Discovery of effective and less toxic antiviral agents is a high priority in the search for more effective therapies against HIV. Flavonoids comprise a large class of low molecular weight polyphenolic compounds ubiquitously distributed in food plants. Flavonoids are enriched in certain vegetables, fruits, seeds and beverages (e.g., tea and wine) and are regarded as a class of semi essential nutrients for humans. Dietary intake of flavonoids is highly variable, with estimation ranging from 23 [1] to more than 500 mg day −1 [2] . Flavonoids display a diverse array of biological effects including antioxidant, anti-inflammatory and anti-tumor activities with no apparent toxic side effects on cells [3][4][5][6] .
These beneficial effects of flavonoids have been attributed to their antioxidant and anti-inflammatory properties.
Quercetin, one of the polyphenolic flavonoids, is known to have effects on a variety of inflammatory processes and immune functions [7][8][9][10][11][12][13][14] . Tumor Necrosis Factor (TNF)-α is a major pro-inflammatory cytokine that regulates the growth, proliferation, differentiation and viability of activated leukocytes. Previous studies have shown that quercetin suppresses TNF-α induced IL-8 and MCP-1 expression in cultured human synovial cells [15] . Likewise, several studies have demonstrated the inhibitory effects of quercetin on the expression of inflammatory cytokines by cultured cells [16,17] . Recent study reported in HMC-1 human mast cell line has indicated that quercetin effects on inflammatory cytokines are mediated through the modulation of NF-κB and p38 MAPK signaling pathways [18] . Previously, we have also demonstrated the inhibitory effects of quercetin on TNF-α production in normal Peripheral Blood Mononuclear Cells (PBMCs) and this effect is mediated by downregulation of NFκB and IκB [19] . Moreover, our laboratory has also shown that quercetin inhibits HIV-1 entry co-receptor CCR5 in normal PBMC in a dose dependant manner [20] .
Although, earlier reports showed the antiviral activity of quercetin, the pharmacological action of quercetin on inflammatory cytokines and the underlying mechanisms are not sufficiently investigated. Therefore, we hypothesize that quercetin exerts anti-HIV activity by differential modulation of pro-and antiinflammatory cytokine expression in normal PBMCs. In the current study, we report the inhibition of HIV-1 replication and virus infectivity by quercetin in normal PBMC as evaluated by HIV-1 p24 antigen production, HIV-1 LTR gene suppression and MAGI cell assay and the mechanism may involve suppression of proinflammatory cytokine (TNF-α), with a reciprocal upregulation of anti-inflammatory cytokine, IL-13.

HIV-1 IIIB infection:
HIV-1 IIIB, CXCR4-tropic, X4 virus strain (Cat #398) was procured through NIH AIDS Research and Reference Reagent Program. PBMCs (3×10 6 cells mL −1 ) were infected with HIV-1 IIIB virus at a concentration of 10 3.0 TCID 50 mL −1 cells for 3h as described by the supplier. Cells were washed and cultured in the presence of quercetin (Sigma-Aldrich, St. Louis, MO) at concentrations ranging between 0-50 µM for 7 days at 37°C in 5% CO 2 incubator. These concentrations selected for the in vitro studies are similar to levels found in plasma of human subjects that have ingested 150 or 300 mg of quercetin [21] . The culture supernatants were harvested and stored at-70°C until further analysis for HIV-1 p24 antigen protein production by commercially available ELISA kit as per manufacturer's instructions (Zeptometrix, Buffalo, NY) and cell pellets were used for RNA extraction and analyzed by qRT-PCR for LTR gene expression.
Quantitative Real Time PCR (qRT-PCR): RNA from cell pellets was extracted using RNAeasy mini kit (Qiagen, GmbH, Germany) followed by cDNA synthesis using high capacity reverse transcriptase cDNA kit (Applied Bio systems, Foster City, CA, USA) to perform qRT-PCR. Relative abundance of each mRNA species was assessed using SYBR green master mix from Stratagene using Mx3000P instrument which detects and plots the increase in florescence versus PCR cycle number to produce a continuous measure of PCR amplification. To provide precise quantification of initial target in each PCR reaction, the amplification plot is examined at a point during the early log phase of product accumulation. This is accomplished by assigning a fluorescence threshold above background and determining the time point at which each sample's amplification plot reaches threshold (defined as the threshold cycle number or C T ). Differences in threshold cycle number are used to identify the relative amount of PCR target contained within each tube [22] . Relative mRNA species expression was quantitated and expressed as transcript accumulation index (TAI = 2 −∆∆CT ), calculated using the comparative C T method [23] .

MAGI Assay to measure HIV-1 infectivity:
To observe the effects of quercetin on HIV-1 infectivity in PBMC, we used the MAGI cell assay. This assay was intended to quantitatively measure virus infectivity [24] . The MAGI (multinuclear activation of a galactosidase indicator) cells are HeLa derived cells stably transfected with CD4 and a reporter construct consisting of the β-galactosidase gene (which is modified to localize to the nucleus) driven by a truncated HIV-1 LTR. Expression of the β-galactosidase gene is Tat-dependent such that an incoming virus must produce active Tat protein to drive expression of the reporter. MAGI cells (4×10 4 cells well −1 ) were plated in a 24 well plate and were treated in duplicate with 100 µL cell suspension of PBMC's that were infected with HIV-1 IIIB virus and subsequently treated with quercetin (5-50 µM). A total of 200 µL of DMEM supplemented with 10% FCS, 100 U mL −1 penicillin, 100 µg mL −1 streptomycin, 0.25 µg mL −1 fungizone and 300 µg mL −1 glutamine containing DEAE-Dextran at a concentration of 15mg mL −1 was added to the MAGI cells and infected cells were incubated for 3 days at 37°C, 5% CO 2. Cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) and blue cells were counted as infected cells.
Stimulated cytokine gene expression and protein secretion: PBMCs (3×10 6 cells mL −1 ) were stimulated with Phorbol Myristate Acetate (PMA) (5 ng mL −1 ) and Ca 2+ ionophore (50 ng mL −1 ) for 24-96 h and simultaneously treated with quercetin (1-50 µM). Proinflammatory cytokine, TNF-α and anti-inflammatory cytokine, IL-13 gene expression was studied by qRT-PCR after 24 h of stimulation, whereas protein secretion was determined in cell culture supernatants by ELISA after 96 h of stimulation. TNF-α and IL-13 protein secretion was quantitated using a commercially available ELISA kit obtained from Bio Source International (Camarillo, CA) as per manufacturer's instructions.

Statistics:
Experiments were performed at least three times and the values obtained were averaged. Data are represented as Mean ± SE. Comparisons between two groups were conducted using Student's paired t-test. Differences were considered significant at p≤0.05, with two-tailed test. Data analysis was performed with the Statistical Program, Graphpad prism software (La Jolla, CA).

Quercetin inhibits HIV-1 LTR gene expression in PBMCs:
In the present study, the anti-HIV activity of flavonoid, quercetin was evaluated using an in vitro infection model in PBMCs. Data showed in Fig. 1 shows the effect of quercetin on HIV-1 LTR gene suppression in PBMC infected with HIV-1. PBMC (3×10 6 cells mL −1 ) were cultured with and without quercetin at different concentrations for 7 days after the HIV-1 IIIB infection. Cells were harvested, RNA was extracted and reverse transcribed to obtain cDNA which was used further to quantitate LTR gene expression by qRT-PCR. Results obtained were transformed to obtain Transcript Accumulation Index (TAI) and plotted as % LTR expression with quercetin treatment. Our data shows that quercetin significantly downregulated LTR gene expression in a dose dependent manner at concentrations of 5 µM (17%, p = 0.069), 10 µM (40%, p = 0.014), 25 µM (72%, p = 0.0005) and 50 µM (88%, p = 0.000006) compared to HIV infected untreated control cultures (100%) (Fig. 1a). Cells were harvested, RNA was extracted and reverse transcribed followed by quantitative real time PCR for LTR gene. Relative expression of mRNA species was calculated using the comparative CT method. The data represents the means ± SE of 3 independent experiments. Statistical significance was calculated by students "t" test

Differential modulation of pro-and antiinflammatory cytokine by quercetin in PBMC:
Previously, we have reported that quercetin inhibits expression of pro-inflammatory cytokine TNF-α in normal PBMC via modulation of the NFκB system [19] . In the current study, we investigated whether quercetin exerts differential effect on expression of pro-and antiinflammatory cytokines in normal PBMC. PBMCs were stimulated with PMA and calcium ionophore and simultaneously treated with quercetin (1-50 µM) for 24-96 h. After 24 h incubation, cells were harvested to determine pro-inflammatory cytokine, TNF-α and antiinflammatory cytokine, IL-13 gene expression by qRT-PCR. Result indicates that quercetin downregulated TNF-α gene expression by PBMCs in a dose dependent manner in the concentration range of 10-50 µM (Fig. 3a). . Cells were harvested, RNA was extracted and reverse transcribed followed by qRT-PCR for TNF-α (Fig. 3a) and IL-13 (Fig. 3b) gene expression. Relative expression of mRNA species (TAI) was calculated using the comparative CT method. The data represents the means ± SE of 3 independent experiments. Statistical significance was calculated by students "t" test On the contrary, IL-13 gene expression was significantly upregulated in the quercetin (1-50 µM) treated cultures compared to untreated cultures (Fig. 3b).
To substantiate gene expression results, we measured cytokine protein secretion from culture supernatants stimulated with PMA and treated with quercetin (1-50 µM) for 96h. Data showed in Fig. 4a shows that quercetin at 5 µM (200 pg mL −1 , p = 0.026) and 10 µM (189pg mL −1 , p = 0.016) significantly inhibited secretion of TNF-α cytokine, while 25 µM and 50 µM quercetin treatment resulted in complete inhibition of TNF-α secretion compared to untreated cultures. Interestingly, IL-13 protein secretion was significantly upregulated (p<0.001) in a dose dependent manner in the concentration range of 1-50 µM of quercetin (Fig. 4b). After incubation, culture supernatants were collected and analyzed by ELISA for TNF-α (Fig. 4a) and IL-13 ( Fig. 4b) protein levels. The data represents the means ± SE of 3 independent experiments. Statistical significance was calculated by students "t" test

DISCUSSION
Flavonoids represent a group of phytochemicals that are known to offer significant health benefits to humans. Flavonoids have been recognized to exert antibacterial and antiviral activity, anti-inflammatory, antiangiogenic and antiallergic effects, analgesic, hepatoprotective, cytostatic, apoptotic, estrogenic and antiestrogenic properties [3][4][5][6][7][8][9][10][11][12][13][14] . Quercetin is the most ubiquitous flavonoid in nature that exhibits a wide range of biological activities arising mainly from its antioxidant property and ability to modulate several enzymes or cell receptors. The current study shows that quercetin exerts significant anti-HIV activity by inhibiting HIV replication and reducing virus infectivity in PBMCs as measured by HIV-LTR gene expression, p24 antigen production and MAGI cell assay. Further, we also report that quercetin downregulates the expression of pro-inflammatory cytokine, TNF-α with a concomitant upregulation of anti-inflammatory cytokine, IL13. The differential modulations of pro-and anti-inflammatory cytokines play a major role in the infection and pathogenesis of HIV disease.
Previous studies from our laboratory demonstrate that quercetin differentially regulates gamma interferon (IFN-γ) and IL-4 expression in PBMCs [25] . Further, we have also shown that flavonoids from grape seed extracts downregulates the expression of HIV-1 entry co-receptors, CCR2b, CCR3 and CCR5 in normal PBMCs [26] , which suggests that flavonoids can interfere with the binding of the virus to the cell receptor and, thus can prevent HIV entry into the normal lymphocyte. In addition, the findings from this study support data demonstrating the ability of quercetin to modulate a variety of inflammatory cytokines and their role in HIV infection. HIV-1 infection and virus replication are regulated by a complex network of cytokines secreted by a variety of cells. TNF-α is one of the major proinflammatory cytokines that significantly upregulate HIV-1 production in Monocytes-Derived Macrophages (MDM) through the activation of NF-κB signaling [27] . On the other hand, IL-13 exerts anti-inflammatory activity by inhibiting production of pro-inflammatory cytokines such as TNF-α and inhibits HIV-1 production [28] . Previous studies have reported that IL-13 acts on macrophages before and after HIV-1 infection and blocks the completion of reverse transcription, inhibits virus production and reduces the infectivity of the progeny virions [29] . Further, IL-13 is also known to suppress TNF-α induced activation of NF-κB, Activation Protein-1 and apoptosis, which describes its immunosuppressive and anti-inflammatory effects [30] . Our results showing significant downregulation of TNF-α by quercetin could probably be attributed to increased levels of IL-13. These findings suggest that in addition to downregulation of HIV entry co-receptors by quercetin, differential modulation of pro-and antiinflammatory cytokines expression could be the potential mechanisms for the anti-HIV activity of quercetin.

CONCLUSION
Quercetin displays a diverse array of biological effects, which is mainly attributed to its antioxidant and anti-inflammatory properties. Elucidating the molecular mechanisms of quercetin-induced anti-HIV effects may help to develop novel flavonoid-based neutraceutical agents useful in the treatment of HIV infection. Furthermore, these studies may provide new information and impetus for developing a potential clinical trial of quercetin as an adjunct nutritional supplement, along with the existing antiviral therapeutic regimen, in the treatment of HIV infection.