GREEN TEA INHIBITORS OF HIV REACTIVATION BY ORAL PATHOGENS.doc

此文檔收集于網(wǎng)絡(luò)，如有侵權(quán)，請(qǐng)聯(lián)系網(wǎng)站刪除Green Tea Inhibitors of HIV Reactivation by Oral PathogensY. Zhou1 and C.B. Huang2*(1.Department of Pharmaceutical Engineering, College of Life Sciences, Guizhou University, Guiyang, 550025, P. R. China; 2. College of Dentistry, University of Kentucky, Lexington, Kentucky 40503, U.S.A.)ABSTRACTBackground: The introduction of HAART regimens (Highly Active Antiretroviral Therapy) has significantly modified the course of HIV disease, with longer survival rates and improvement of life quality in HIV-infected individuals. However, complete eradication of HIV infection cannot be achieved with currently available antiretroviral regimens. This persistence of HIV-1 within various host immune cells constitutes a major obstacle in the control of HIV-1 infection. Various exogenous stimuli have been shown to exacerbate HIV-1 infections, including Gram-negative bacteria and LPS, or cytokines/chemokines induced by these stimuli. Evidence of viral recrudescence, ie. plasma HIV RNA levels (viral load), generally reflects a loss of responsiveness to HAART. Continued progress in development of additional therapeutics for managing chronic HIV infections remains important. Objective: This study is designed to (1) screen and test the possible inhibition of HIV-1 reactivation by green tea and other natural products using our cell model and investigate the possible inhibition mechanism through the toll-like receptors (TLR); (2) Base on the aim (1), to elucidate the green teas function as adjunctive treatments for a potential risk of HIV-1 recrudescence from latently infected macrophages induced by mucosal infections, including periodontal infections. Methods: We used a model macrophage cell line, BF24, with the HIV LTR promoter driving CAT production to screen for inhibitors which can block the activation of this promoter. The BF24 cells were challenged with various oral microorganisms in the presence of potential natural product inhibitors. The inhibitors can block CAT production in the absence of cytotoxicity to the macrophages.The concentration of chloramphenicol acetyltransferase (CAT) to be detect to get the information of the interaction of oral microorganisms and other mucosal pathogens with macrophages leading to activation of the HIV-1 promoter in the presence/absence of the extract. Results: We found that (1) green tea extract is able to inactivate the HIV promoter in BF24 cells; We showed that green tea extract is capable of inhibiting HIV promoter reactivation stimulated by oral bacteria. (2) Green tea components could function as potential natural product inhibitors to block the capacity of microbial challenge to activate the HIV-1 promoter. We identified green tea fractions that block the ability of the infections to stimulate macrophages for reactivation of HIV-1. Conclusion: The green tea extract exhibited inhibition of the HIV-1 LTR which could be used as indicator for anti-HIV reactivation by oral pathogens. Green tea exhibited the strongest activity and the active fraction was obtained. The inhibition mechanism of HIV-1 promoter reactivation by green tea extract is possibly through TLRs. The green tea leave extract has potential usage to treat HIV reactivation in the future.INTRODUCTIONThe introduction of HAART regimens (Highly Active Antiretroviral Therapy) has significantly modified the course of HIV disease, with longer survival rates and improvement of life quality in HIV-infected individuals. However, complete eradication of HIV infection cannot be achieved with currently available antiretroviral regimens. This primarily results from the establishment of a pool of latently infected CD4+ T cells, macrophages, and other host cells (eg. dendritic cells, mast cells) during the very earliest stages of acute HIV infection(1) that persists with an extremely long half-life(2-6). This persistence of HIV-1 within various host immune cells, including macrophages constitutes a major obstacle in the control of HIV-1 infection. Periodontal disease represents a chronic, Gram-negative bacterial infection of the oral cavity. In addition, a variety of microbial species present as commensal opportunistic or exogenous pathogens at mucosal sites throughout the body, ie. respiratory, genitourinary, gastrointestinal. Various exogenous stimuli have been shown to exacerbate HIV-1 infections, including Gram-negative bacteria and LPS, or cytokines/chemokines induced by these stimuli. Evidence of viral recrudescence, plasma HIV RNA levels (viral load), generally reflects a loss of responsiveness to HAART. We have previously shown that the presence of this oral chronic infection may serve as a catalyst for the subsequent loss of responsiveness to HAART by exacerbating HIV-1 production from latently infected cells, both T-cells and macrophages(7-8). It has been noted that monocyte/macrophages, just as many other cell types, express toll-like receptors (TLRs). Human and murine TLR proteins have been identified, with 10-11 of the TLR having been sequenced or cloned(30-36). The TLR can loosely be classified into three groups based upon expression pattern: ubiquitous (TLR1), restricted (TLR2, TLR4, TLR5, TLR6, TLR7, TLR9) and specific (TLR3, TLR8, TLR10). The TLR family are type I transmembrane proteins that act as receptors for pathogen-specific molecular patterns (PAMPs) generally limited to microorganisms and thereby activate innate immune cells. These receptors are coupled to a signaling pathway that is conserved in mammals, insects, and plants, resulting in the activation of genes that mediate innate immune defenses. TLR signaling represents a key feature of innate immune responses to pathogen invasion.In HIV-infected patients, concurrent bacterial infections are known to induce HIV replication, leading to transient bursts of HIV viremia. New strategies to block the ability of various exogenous infectious agents of mucosal surfaces, including those in the complex microbial ecology of the oral cavity, to stimulate HIV latently infected macrophages, would appear to provide a unique benefit in the arsenal of agents for controlling the consequences of HIV infection. This paper provides an innovative strategy to screen natural product agents from green tea that focus on blocking the ability of exogenous/endogenous infectious agents to reactivate HIV in patients controlled/managed by HAART. MATERIALS AND METHODSInstruments: High-performance liquid chromatography (HPLC): Waters 1525 binary HPLC pump (Waters, USA), Waters 2487 dual absorbance detector (Waters, USA) and a XTerra RP18 column (250 mm 4.6 mm i.d., particle size 5 m; Waters, USA). Tissue Culture: The BF24 is a subclone of the monocytic leukemia cell line THP-1. BF24 cells are transfected with the HIV LTR driving the CAT reporter gene. A cell density of 1 X 105/well was seeded into each well of a 96-well plate and cells were challenged with microbial preparations in the presence/absence of various natural product extracts. After 16 hr incubation, the cells will be centrifuged, washed, and lysed in the 96-well plates. The resulting supernatants were analyzed for the concentration of CAT.Bacteria: Porphyromonas gingivalis ATCC 33277; Fusobacterium nucleatum ATCC 25586; Treponema denticola ATCC 35404; Streptococcus mutans ATCC 25175 . The bacteria were cultured overnight, and the cells were harvested and sonicated. Sonication is carried out on ice for 6 one-minute intervals followed by protein concentration determination using Coomassie Plus-The Better Bradford Assay Kit (Pierce).BF24 Stimulation: BF24 monocytes were stimulated in duplicate with various concentrations of sonicated oral bacteria or 1g E.coli O127:B8 LPS. Cells were harvested using 3ml of PBS. The cells were lysed using 500l lysis buffer, frozen in liquid nitrogen and stored at -80C until CAT activity was measured. ELISA Reading: BF24 cell lysate samples were added in duplicate to a 96 well microtiter plate pre-coated with anti-CAT antibody. Expression of CAT driven by HIV LTR was measured using an MRX plate reader (Dynatech Laboratories).The Toxicity test of the tea samples:The dry tea samples were dissolved in DMSO to get the 2mg/mL solvents. Then the dilution was made to get the 1mg/mL 1g/mL samples. BF-24 cells were seated in 96-well plate with cell density of 105 cell/well. Two l of diluted samples were added to each well. After 16 hrs of incubation at 37 degree, the cell culture media were harvested and centrifuged. The remaining BF24 cells were stained with Trypan Blue (Sigma Cat T8154) and cell survival were estimated by percentage of live cells under the microscope.HPLC Purification: 1 ml of fraction will be (150mg/ml dissolved in methanol) filtered with Ultrafree-MC 0.45um Filter Unit and injected into the Waters 1525 HPLC system. An RP18 Symmetry 4.6150mm HPLC Column will be used for the purification with a step gradient using mixture of methanol/water 1% to 100% gradient. The anti-HIV bioactivity of each HPLC fraction will be tested as described above. Real-time PCR: Real-time PCR was conducted as following: bacteria treated DCs were harvested, washed twice in PBS and lysed in 350 l RLT buffer with fresh addition of 2-mercaptoethanol in each well. Total RNA was isolated using RNeasy Minikits (Qiagen), according to the manufacturers protocol. RNA quality and quantity was estimated spectrophotometrically at 260/280 nm (Beckman DU-640). Real time PCR (qPCR) was performed following standard procedures. Briefly, a master mix was prepared containing 11.6 l of H2O, 2l of Primer Mix (Table 1) final concentration 4mM, 2.4l of MgCl2 (final concentration 0.5M), and 2l Light Cycler FastStart DNA master SYBR GreenI. After gentle mixing, 18l was pipetted into a precooled LightCycler Capillary, 2L of c-DNA (or DNA) was added. qPCR was performed with pre-incubation for 20 sec. at 95oC, amplification for 45 cycles at target temperatures specific for each primer, melting curve analysis and than cooled using a LightCycler 2.0 (Roche). Each reaction was duplicated and the number was the average value.RESULTSGreen tea extract significantly inhibited HIV-1 promoter reactivation: We demonstrated that a variety of oral bacteria are able to activate the HIV promoter in BF24 cells by differing doses and time course requirements exist for maximal responses, reflecting effectiveness of HIV promoter activation. Fig. 1 demonstrates the effect of sonicates from F. nucleatum, P. gingivalis, and T. denticola, as well as E. coli LPS, on HIV promoter activation, ie. CAT production. The results demonstrated that the doses of individual bacteria that induced optimal CAT production varied, and that the maximum CAT production demonstrated some species-specific features (ie. T. denticola elicited the highest level), suggesting that this HIV model can possibly be used for anti-HIV inhibitor screening. We have initiated the screening of over 300 natural product extracts in our library for anti-HIV activity. In our preliminary screening we identified four extracts that inhibited CAT production; however, 2 extracts demonstrated sufficient cytotoxicity that they were eliminated from further study. The remaining nontoxic extract of Green tea (Camellia sinensis), showed significant inhibitory activities and continued to be analyzed in our study. Fig. 2 is a representation of the CAT-ELISA for detecting inhibition activity of the natural product extracts. The blue color indicates CAT production in BF24, while the clear wells indicate the lack of CAT production following a similar challenge in the presence of extracts. Purification of Camellia sinensis extract by HPLC and the inhibition of HIV-1 promoter reactivation: We purified the Camellia sinensis raw extract by HPLC. Fig. 3A shows the HPLC profile of the Camellia sinensis (400 l) raw extract of 100mg/ml concentration. The extract was separated into 80 fractions of 1 ml. The 80 fractions were dried and re-dissolved in 50 l (50% EtOH/DH2O; v/v) solution. Ten l of each fraction was used for determining the ability to inhibit CAT production, ie. HIV promoter activation. The specificity of Green tea (C. sinensis) fractions was examined for inhibition of the ability of oral bacteria and E. coli LPS to stimulate the HIV promoter. Green tea (C. sinensis) extract showed significant inhibition activity, primarily contained in fractions 27, 32, and fraction 38 of an HPLC separation of the raw extract. Fig. 3B showed that significant inhibition activities were found primarily at fractions 27, 28, 32, 38. Fig. 4 demonstrates the specificity of the inhibition by the fractions following cell stimulation for various oral bacteria and the LPS. The results supported some variations across the various fractions, as well as differences in the breadth of effectiveness of individual fractions.Green tea extracts inhibited HIV-1 promter possibly through influencing the TLR expression: To study the potential mechanism of green tea extract, real-time PCR was conducted. QPCR data indicated that C. sinensis extracts could differentially influence the expression of TLRs in macrophage BF24 induced by oral bacteria (Table 2). From the table 2, we know that the P. gingivalis, T. denticola, as well as E. coli LPS increased the expression of the TLRs in macrophage BF24, while C. sinensis extracts could modify the expression of TLRs in macrophage BF24 induced by oral bacteria. The C. sinensis extracts up-regulated the expression of TLR5 and MyD88, it down-regulated the expression of TLR2 and TLR4. DISCUSSIONThis study used a model macrophage cell line, BF24, with the HIV LTR promoter driving CAT production to screen for inhibitors which can block the activation of this promoter. The BF24 cells were challenged with various oral microorganisms in the presence of potential natural product inhibitors. The inhibitors were identified that could block CAT production in the absence of cytotoxicity to the macrophages. We found that one of the extracts, green tea extract, was able to inactivate the HIV promoter in BF24 cells because we showed that green tea extract was capable of inhibiting HIV promoter reactivation stimulated by oral bacteria. This is the first study designed to look for HIV-1 reactivation inhibitor. Our study approved that this is a plausible for future study and a useful method to screen anti-HIV drugs.Natural products, such as plant extracts, represent a useful resource for drug discovery. Many modern drugs were first discovered and isolated from natural products. We have generated a plant extract library of over 300 plant species, including plants from the U.S. and China. From this plant extract library, we find that green tea extract could contain inhibitors with the potential to block the ability of oral microorganisms to stimulate HIV-latently infected macrophages, thus preventing HIV-1 virion production and associated health consequences of the resulting immunosuppression. we have identified that fractions from green tea exhibit the capacity to block HIV promoter activation in macrophages. The green tea extract exhibited inhibition of the HIV-1 LTR which could be used as indicator for anti-HIV reactivation by oral pathogens. Green tea exhibited the strongest activity and the active fraction was obtained. The inhibition mechanism of HIV-1 promoter reactivation by green tea extract is possibly through TLRs. The green tea leave extract has potential usage to treat HIV reactivation in the future.The findings of this study documented a unique strategy to determine the ability of green tea extracts to block the ability of oral bacteria to trigger HIV reactivation in latently infected macrophages. Coupling these strategy could result in an innovative adjunctive strategy to ameliorate the impact of chronic oral infections on HIV recrudescence in these patients, or other related HIV reactivation. REFERENCES1Chen R, Westfall A, Cloud G, et al. 2001. Long-term survival after initiation of antiretroviral therapy. 8th Conference on Retroviruses and Opportunistic Infections. Chicago, IL. (Abstract 341). 2Sterling TR, Bartlett JG, Moore RD. 2001. CD4+ lymphocyte level is better than HIV-1 plasma vial load in determining when to initiate HAART. 8th Conference on Retroviruses and Opportunistic Infections. Chicago, IL. (Abstract 519). 3Egger M. 2001. ART Cohort Collaboration. 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