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Red wine, resveratrol, Chlamydia pneumoniae and the French connection
Atherosclerosis 171 (2003) 379–380
Letter to the Editor
Red wine, resveratrol, Chlamydia pneumoniae and the French connection Chlamydia pneumoniae is an intracellular gram-negative pathogen, responsible for 5–30% of acute respiratory tract infections worldwide [1,2]. However, while infection begins in the respiratory tract, the bacterium is disseminated systemically in the blood stream within alveolar macrophages, and can lead to the development of chronic infection [2]. Recent investigations have suggested that chronic C. pneumoniae infection may be a causative factor in the development of atherosclerosis and coronary heart disease (CHD) [2,3]. This association has been demonstrated in seroepidemiological investigations, as well as by immunocytochemical staining, polymerase chain reaction (PCR) and electron microscopy of atherosclerotic plaque [3]. Thus, there is a growing body of evidence that C. pneumoniae is associated with the development or progression of atherosclerotic plaque and CHD [2,3]. Interestingly, despite a diet that is relatively high in fat, the French have one of the lowest incidence of CHD in the western world [4]. This phenomenon is commonly referred to as the “French Paradox”, and has been linked to the high consumption of red wine. It has been suggested that resveratrol (3,4 ,5-trihydroxystilbene), one of the polyphenolic compounds in red wine may be responsible for the protective effects on CHD [5]. In a recent report we have shown that a red wine extract and resveratrol inhibit the growth (in vitro) of Helicobacter pylori, a gram-negative bacterium associated with the development of peptic ulcers and gastric cancer [6]. This was the first time that resveratrol had been reported to be active against a human pathogen. In this investigation the activity of a red wine extract and resveratrol was assessed on the proliferation of C. pneumoniae in HEp-2 cells in vitro. Pinot noir (red wine) extracts were prepared by extraction of 1500 ml of Pinot noir with 3 × 500 ml of methanol. The extracts were filtered to separate methanol soluble from methanol insoluble fractions and each fraction was dried under reduced pressure. Pure resveratrol was obtained from Sigma Chemical (St Louis, MO). Stock solutions of resveratrol and concentrated pinot noir extracts (methanol soluble and methanol insoluble) were prepared by dissolving resveratrol or the two concentrated pinot noir fractions in sterile, tissue culture grade DMSO (Sigma Chemical) and were prepared according to NCCLS guidelines [7]. Azithromycin was included as a control (United States Pharmacopeia,
Rockville, MD). The solutions were further diluted 1:10 in Eagle’s MEM with 10% fetal calf serum and cycloheximide (1 g/ml) to give a final concentration of 1000 g/ml. Final DMSO concentrations in resveratrol and concentrated pinot noir samples did not exceed 0.25%. C. pneumoniae isolates, TW 183 (ATCC VR-2822) and AR 39 (ATCC 53592) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Minimal inhibitory concentrations (MICs) and minimal chlamydicidal concentrations (MCCs) were performed as outlined by Hammersclag et al. [8]. HEp-2 cells (ATCC) were grown to near confluence, harvested, and plated in 96-well microtiter plates at a final concentration of 0.5 × 106 cells/ml. After a 24 h incubation, each well was inoculated with 0.1 ml of each chlamydial test strain at a concentration of 103 −104 inclusion-forming units (IFUs)/ml. Microtiter plates were then centrifuged at 1700 × g for 1 h at room temperature. After centrifugation, plates were incubated for 1 h at 35 ◦ C and 5% CO2 . Following incubation, the medium was carefully aspirated from each well and overlaid with 100 l of resveratrol, concentrated pinot noir extract or azithromycin. Concentrations tested ranged from 500 to 3.6 g/ml for the pinot noir extract, 100 to 0.78 g/ml for resveratrol, and 1.0 to 0.0078 g/ml for azithromycin. The concentrations were obtained by serial dilutions in Eagle’s MEM with 10% fetal calf serum and cycloheximide (1 g/ml). All susceptibility assays were run in triplicate. Microtiter plates were placed at 35 ◦ C and 5% CO2 and allowed to incubate for 72 h. Chlamydia were fixed with methanol and stained with a genus-specific monoclonal antibody (Pathfinder® Chlamydia Culture Confirmation System, BioRad, Redmond, WA). The MIC and MCC was preformed as described by Suchland et al. [9]. The MTT cytotoxicity assay was performed according to Mosmann to determine the effect of resveratrol and DMSO on HEp-2 cell viability [10]. The MICs and MCCs for resveratrol and wine extracts are shown in Table 1. Both resveratrol and the concentrated pinot noir extract displayed antichlamydial activity against the 2 test strains. The methanol soluble extract did not demonstrate any noticeable activity up to a concentration of 500 g/ml. The azithromycin control demonstrated MICs and MCCs similar to those reported in the literature. The MTT cytotoxicity assay demonstrated there was no decrease in HEp-2 cell viability at the concentration of resveratrol associated with the MIC and MCC. Previous studies have indicated that the consumption of red wine is associated with a significant reduction in CHD,
0021-9150/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2003.08.026
380
Letter to the Editor / Atherosclerosis 171 (2003) 379–380
Table 1 MICs and MCCs for resveratrol, pinot noir extracts, and control antibiotic (azithromycin) Chlamydial Strain
Extract
MICTP (g/ml)
MIC (g/ml)
MCC (g/ml)
AAR-39 TW-183 AR-39 TW-39 AR-39 TW-183 AR-39 TW-183
Pinot noir (methanol-soluble fraction)
>500 >500 125 125 6.25 6.25 0.0625 0.0625
>500 >500 250 250 12.5 12.5 0.125 0.125
>500 >500 250 250 12.5 12.5 0.125 0.125
Pinot noir (concentrated fraction) Resveratrol Azithromycin (0.0625-0.25)
however, the mechanism by which red wine and resveratrol exert their beneficial effects in CHD has not been completely elucidated. The results from this study demonstrate that both red wine and resveratrol have in vitro activity against C. pnemoniae. Thus, the French Paradox may be due in part to the beneficial effects of red wine consumption on development and progression of CHD through its antimicrobial activity on C. pnemoniae. References [1] Halm EA, Teirstein AS. Management of community-acquired pneumonia. N Eng J Med 2002;347(25):2039–45. [2] Grayston JT. Background and current knowledge of Chlamydia pneumoniae and atherschlerosis. J Infect Dis 2000;181(Suppl 3):S402–10. [3] Noll G. Pathogenesis of atherosclerosis: a possible relation to infection. Atherosclerosis 1998;140:S3–9. [4] Renaud S, de Lorgeril M. The French paradox: dietary factors and cigarette smoking-related health risks. Ann NY Acad Sci 1993;686:299–309. [5] Kopp P. Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the French Paradox? Eur J Endocrinol 1998;138:619–20. [6] Mahady GB, Pendland SL. Resveratrol inhibits the growth of Helicobacter pylori in vitro. Am J Gastroenterol 2001;96(12):3454–5. [7] National Committee for Clinical and Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing; Twelfth Informational Supplement, January 2002.
[8] Hammerschlag MR, Qumei KK, Roblin PM. In vitro activities of azithromycon, clarithromycin, l-ofloxacin, and other antibiotics against Chlamydia pneumoniae. Antimicrob Agents Chemother 1992;7:1573–4. [9] Suchland RJ, Geisler WM, Stamm WE. Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother 2003;47:636–42. [10] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferration and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
Christopher Schriever, Susan L. Pendland Microbiology Research Laboratory Department of Pharmacy Practice, College of Pharmacy University of Illinois at Chicago, Chicago, IL 60612, USA Gail B. Mahady Department of Pharmacy Practice Program for Collaborative Research in the Pharmaceutical Sciences, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy University of Illinois at Chicago Chicago, IL 60612, USA Corresponding author. Tel.: +1-312-996-1669 fax: +1-312-996-1797 E-mail address: [email protected] (G.B. Mahady) 25 August 2003
Letter to the Editor
Red wine, resveratrol, Chlamydia pneumoniae and the French connection Chlamydia pneumoniae is an intracellular gram-negative pathogen, responsible for 5–30% of acute respiratory tract infections worldwide [1,2]. However, while infection begins in the respiratory tract, the bacterium is disseminated systemically in the blood stream within alveolar macrophages, and can lead to the development of chronic infection [2]. Recent investigations have suggested that chronic C. pneumoniae infection may be a causative factor in the development of atherosclerosis and coronary heart disease (CHD) [2,3]. This association has been demonstrated in seroepidemiological investigations, as well as by immunocytochemical staining, polymerase chain reaction (PCR) and electron microscopy of atherosclerotic plaque [3]. Thus, there is a growing body of evidence that C. pneumoniae is associated with the development or progression of atherosclerotic plaque and CHD [2,3]. Interestingly, despite a diet that is relatively high in fat, the French have one of the lowest incidence of CHD in the western world [4]. This phenomenon is commonly referred to as the “French Paradox”, and has been linked to the high consumption of red wine. It has been suggested that resveratrol (3,4 ,5-trihydroxystilbene), one of the polyphenolic compounds in red wine may be responsible for the protective effects on CHD [5]. In a recent report we have shown that a red wine extract and resveratrol inhibit the growth (in vitro) of Helicobacter pylori, a gram-negative bacterium associated with the development of peptic ulcers and gastric cancer [6]. This was the first time that resveratrol had been reported to be active against a human pathogen. In this investigation the activity of a red wine extract and resveratrol was assessed on the proliferation of C. pneumoniae in HEp-2 cells in vitro. Pinot noir (red wine) extracts were prepared by extraction of 1500 ml of Pinot noir with 3 × 500 ml of methanol. The extracts were filtered to separate methanol soluble from methanol insoluble fractions and each fraction was dried under reduced pressure. Pure resveratrol was obtained from Sigma Chemical (St Louis, MO). Stock solutions of resveratrol and concentrated pinot noir extracts (methanol soluble and methanol insoluble) were prepared by dissolving resveratrol or the two concentrated pinot noir fractions in sterile, tissue culture grade DMSO (Sigma Chemical) and were prepared according to NCCLS guidelines [7]. Azithromycin was included as a control (United States Pharmacopeia,
Rockville, MD). The solutions were further diluted 1:10 in Eagle’s MEM with 10% fetal calf serum and cycloheximide (1 g/ml) to give a final concentration of 1000 g/ml. Final DMSO concentrations in resveratrol and concentrated pinot noir samples did not exceed 0.25%. C. pneumoniae isolates, TW 183 (ATCC VR-2822) and AR 39 (ATCC 53592) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Minimal inhibitory concentrations (MICs) and minimal chlamydicidal concentrations (MCCs) were performed as outlined by Hammersclag et al. [8]. HEp-2 cells (ATCC) were grown to near confluence, harvested, and plated in 96-well microtiter plates at a final concentration of 0.5 × 106 cells/ml. After a 24 h incubation, each well was inoculated with 0.1 ml of each chlamydial test strain at a concentration of 103 −104 inclusion-forming units (IFUs)/ml. Microtiter plates were then centrifuged at 1700 × g for 1 h at room temperature. After centrifugation, plates were incubated for 1 h at 35 ◦ C and 5% CO2 . Following incubation, the medium was carefully aspirated from each well and overlaid with 100 l of resveratrol, concentrated pinot noir extract or azithromycin. Concentrations tested ranged from 500 to 3.6 g/ml for the pinot noir extract, 100 to 0.78 g/ml for resveratrol, and 1.0 to 0.0078 g/ml for azithromycin. The concentrations were obtained by serial dilutions in Eagle’s MEM with 10% fetal calf serum and cycloheximide (1 g/ml). All susceptibility assays were run in triplicate. Microtiter plates were placed at 35 ◦ C and 5% CO2 and allowed to incubate for 72 h. Chlamydia were fixed with methanol and stained with a genus-specific monoclonal antibody (Pathfinder® Chlamydia Culture Confirmation System, BioRad, Redmond, WA). The MIC and MCC was preformed as described by Suchland et al. [9]. The MTT cytotoxicity assay was performed according to Mosmann to determine the effect of resveratrol and DMSO on HEp-2 cell viability [10]. The MICs and MCCs for resveratrol and wine extracts are shown in Table 1. Both resveratrol and the concentrated pinot noir extract displayed antichlamydial activity against the 2 test strains. The methanol soluble extract did not demonstrate any noticeable activity up to a concentration of 500 g/ml. The azithromycin control demonstrated MICs and MCCs similar to those reported in the literature. The MTT cytotoxicity assay demonstrated there was no decrease in HEp-2 cell viability at the concentration of resveratrol associated with the MIC and MCC. Previous studies have indicated that the consumption of red wine is associated with a significant reduction in CHD,
0021-9150/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2003.08.026
380
Letter to the Editor / Atherosclerosis 171 (2003) 379–380
Table 1 MICs and MCCs for resveratrol, pinot noir extracts, and control antibiotic (azithromycin) Chlamydial Strain
Extract
MICTP (g/ml)
MIC (g/ml)
MCC (g/ml)
AAR-39 TW-183 AR-39 TW-39 AR-39 TW-183 AR-39 TW-183
Pinot noir (methanol-soluble fraction)
>500 >500 125 125 6.25 6.25 0.0625 0.0625
>500 >500 250 250 12.5 12.5 0.125 0.125
>500 >500 250 250 12.5 12.5 0.125 0.125
Pinot noir (concentrated fraction) Resveratrol Azithromycin (0.0625-0.25)
however, the mechanism by which red wine and resveratrol exert their beneficial effects in CHD has not been completely elucidated. The results from this study demonstrate that both red wine and resveratrol have in vitro activity against C. pnemoniae. Thus, the French Paradox may be due in part to the beneficial effects of red wine consumption on development and progression of CHD through its antimicrobial activity on C. pnemoniae. References [1] Halm EA, Teirstein AS. Management of community-acquired pneumonia. N Eng J Med 2002;347(25):2039–45. [2] Grayston JT. Background and current knowledge of Chlamydia pneumoniae and atherschlerosis. J Infect Dis 2000;181(Suppl 3):S402–10. [3] Noll G. Pathogenesis of atherosclerosis: a possible relation to infection. Atherosclerosis 1998;140:S3–9. [4] Renaud S, de Lorgeril M. The French paradox: dietary factors and cigarette smoking-related health risks. Ann NY Acad Sci 1993;686:299–309. [5] Kopp P. Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the French Paradox? Eur J Endocrinol 1998;138:619–20. [6] Mahady GB, Pendland SL. Resveratrol inhibits the growth of Helicobacter pylori in vitro. Am J Gastroenterol 2001;96(12):3454–5. [7] National Committee for Clinical and Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing; Twelfth Informational Supplement, January 2002.
[8] Hammerschlag MR, Qumei KK, Roblin PM. In vitro activities of azithromycon, clarithromycin, l-ofloxacin, and other antibiotics against Chlamydia pneumoniae. Antimicrob Agents Chemother 1992;7:1573–4. [9] Suchland RJ, Geisler WM, Stamm WE. Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother 2003;47:636–42. [10] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferration and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
Christopher Schriever, Susan L. Pendland Microbiology Research Laboratory Department of Pharmacy Practice, College of Pharmacy University of Illinois at Chicago, Chicago, IL 60612, USA Gail B. Mahady Department of Pharmacy Practice Program for Collaborative Research in the Pharmaceutical Sciences, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy University of Illinois at Chicago Chicago, IL 60612, USA Corresponding author. Tel.: +1-312-996-1669 fax: +1-312-996-1797 E-mail address: [email protected] (G.B. Mahady) 25 August 2003