Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells

Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells

Atherosclerosis 153 (2000) 303 – 313 www.elsevier.com/locate/atherosclerosis Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase...

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Atherosclerosis 153 (2000) 303 – 313 www.elsevier.com/locate/atherosclerosis

Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells Marı´a Teresa Montero a, Osvaldo Herna´ndez a, Yajaira Sua´rez a, Joaquı´n Matilla a, Antonio J. Ferruelo a, Javier Martı´nez-Botas a,1, Diego Go´mez-Coronado a, Miguel A. Lasuncio´n a,b,* a

Ser6icio de Bioquı´mica-In6estigacio´n, Hospital Ramo´n y Cajal, Ctra. Colmenar, km 9, 28034, Madrid, Spain b Departamento de Bioquı´mica y Biologı´a Molecular, Uni6ersidad de Alcala´, Alcala´ de Henares, Spain Received 24 September 1999; received in revised form 24 January 2000; accepted 31 January 2000

Abstract T cells are prominent components of both early and late atherosclerotic lesions and the role of Th1/Th2 cells subsets in the evolution and rupture of the plaque is currently under investigation. Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exert actions beyond that of simply lowering cholesterol levels, and some effects on immune function have been reported. We studied in vitro the effects of fluvastatin on Th1/Th2 cytokine release in relation to caspase-1 activation, in human peripheral-blood mononuclear cells (PBMC) stimulated or not with Mycobacterium tuberculosis. Fluvastatin treatment resulted in the activation of caspase-1 and in a small secretion of interleukin (IL)-1b, IL-18, and IFNg (Th1). In the presence of bacteria, the release of these cytokines was highly increased by the statin in a synergistic way. By contrast, production of IL-12, IL-10 and IL-4 were unaffected by the statin. Not only did mevalonate abolish the effects of the statin but it also prevented the caspase-1 activation induced by the bacteria, suggesting the involvement of isoprenoids in the response to M. tuberculosis. It is proposed that inhibition of HMG-CoA reductase may be immunoprotective by enhancing the Th1 response, which has therapeutical potential not only in atherosclerosis but also in infectious diseases. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Infection; Interleukins; Caspase-1; Cholesterol

1. Introduction Arteriosclerosis is a chronic inflammatory-fibrotic response to the accumulation of cholesterol in the artery wall [1,2], which has been regarded as a cell-mediated hypersensitivity reaction [3,4] that evoke the immune response akin to Mycobacterium tuberculosis [5,6]. Several reports demonstrated that IFN-g but not IL-4 is preferentially detected in atherosclerotic lesions, suggesting that T lymphocytes present in the plaque corre* Corresponding author. Tel.: +34-1-3368077; fax: +34-13369016. E-mail address: [email protected] (M.A. Lasuncio´n). 1 Present address: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.

spond predominantly to the Th1 phenotype [7,8]. In spite of that, a cross-regularory role of IL-12 and IL-10 also occurs in human atherosclerotic lesions, which may influence the differential development of T lymphocytes to the Th1 and Th2 phenotypes [7,9,10]. Interestingly, Zhou et al. reported that, in mouse, severe hypercholesterolemia is associated with a switch from Th1 to Th2, resulting in the appearance of Th2 cytokines both in lymphoid organs and atherosclerotic lesions [11], which may have important consequences for the immune process that takes place in the plaque [7,12]. On the other hand, immunization of rabbits with M. tuberculosis heat shock protein 65 has been reported to result in the development of atherosclerotic lesions [13], which are aggravated when immunized animals are put on a hypercholesterolemic diet [14]. All this suggests a possi-

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ble link between cholesterol metabolism and the cellmediated response pattern. Additional evidence for this comes from the pharmacological effects of statins, which are competitive inhibitors of 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol biosynthesis. These drugs are being widely used to lower cholesterol levels aimed to reduce atherosclerosis, yet the benefit of such treatment is greater than expected in terms of the ensuing reduction in LDL-cholesterol levels, which has led to the hypothesis that statins exert actions beyond that of simply lowering cholesterol levels [15,16]. In keeping with this, some effects of statins on the inflammatory response have been reported [17 – 23]. Human Th1 cells induce IL-1b production upon cell – cell contact with monocytes [24], a cytokine that has been found in abundance in atherosclerotic lesions [3] and, on the other hand, has been referred as a mediator of the inflammatory process seen in tuberculosis [25]. IL-1b is synthesized as a 31-kDa precursor devoid of a conventional signal sequence and, unlike most other peptide hormones, is not released via the classic secretion pathway. This cytokine is processed to its proinflammatory 17-kDa form by caspase-1, formerly IL-1b converting enzyme (ICE) [26], the founder member of a growing family of cysteine proteases (caspases) with a substrate cleavage specificity for Asp at P1 [27]. In the monocyte/ macrophage, caspase-1 has been also shown to activate the so-called interferon g (IFNg)-inducing factor (IGIF or IL-18) [28,29], which is a 18 kDa polypeptide that is essential for the effective induction and activation of Th1 (IFNg) cells in conjunction with IL-12 [30,31]. In turn, IFNg is a major activator of monocytes that increases their antigen-presenting capacity and primes for the production of proinflammatory cytokines [32]. Therefore, caspase-1 appears to play a central role in the coordinated function of lymphocytes and monocytes in the Th1 immune response, by triggering the secretion of both IL-1b and IL-18. Present study sought to explore the possibility that statins influence the Th1/Th2 cell-mediated response by evaluating the action of this drug on the production of cytokines by human peripheral blood mononuclear cells (PBMC) stimulated with M. tuberculosis.

2. Methods

2.1. PBMC isolation and culture conditions PBMC were isolated from buffy coats from normal donors over a Ficoll – Hypaque gradient, according to the method of Boyum [33], and cultured on 12 well

plates at 2 × 106 cells/ml in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, L-glutamine, penicillin, streptomycin and gentamycin. Incubations were performed at 37°C in a humidified atmosphere containing 5% CO2 in air. The cultures were or not supplemented with fluvastatin dissolved in DMSO (final concentration in the media 0.04%), or mevalonate as indicated. After a 12 h incubation in the mentioned conditions, the cultures were supplemented with heat inactivated M. tuberculosis H37 RA (10 mg/ml) or placebo, without any other change in the medium, and the incubation was prolonged for an additional 24 h. At the end of the incubation, the media were collected, cells in suspension were removed by centrifugation, and the supernatants were frozen until cytokine determinations.

2.2. Immunoblot analysis For these analyses both cells adhered and not adhered were combined. First, cells in suspension were sedimented by centrifugation and the supernatants were reserved. Then, adherent cells were scraped and combined with the non-adherent cells. The whole cells were washed with PBS, resuspended in lysis buffer (62.5 mM Tris ClH pH 6.8, 2% SDS, supplemented with a suitable antiprotease cocktail) and sonicated at 0°C. The cell supernatants were treated with affigel blue (Bio Rad, He´rcules, California) to remove the albumin, and then concentrated three times by using Ultrafree concentrators, cut-off 10 000 (Millipore, Bedford, MA). Proteins from both cell lysates and supernatants were subjected to electrophoresis according to Laemmli [34], on a discontinuous 6–11% SDSPAGE, using a mini-gel system (Hoeffer Scientific Instruments, CA) and then transferred to Immobilon membranes (pore size 0.45 mm, Millipore), using a Semi-phor semi-dry transfer unit (Hoeffer Scientific Instruments). Buffers used for transfer were: Anode 1: 300 mM Tris, 10% methanol, pH 10.4; Anode 2: 25 mM Tris, 10% methanol, pH 10.4; Cathode: 25 mM Tris, 40 mM 6-aminocaproic acid, 20% methanol, pH 9.4. A constant current of 0.22 mA/cm2 was applied for 18 h. The blots were blocked overnight at 4°C in 5% non-fat dry milk in 20 mM Tris, 500 mM NaCl, pH 7.5. Then the blots were probed for 6 h with anti-human IL-1b serum (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1/500 in 2% non-fat dry milk in TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5). After four, 10 min washes in TTBS, the blots were incubated with anti-goat alkaline phosphatase-conjugated serum (Santa Cruz Biotechnology), diluted 1/1000 in 2% non-fat dry milk in TTBS for 45 min and finally washed and developed according to the supplier. Incubations were performed at room temperature.

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2.3. Cytokine determinations At the end of incubations, cell supernatants were collected by centrifugation as mentioned above and cytokines measured by ELISA techniques. Immunoassay kits for IL-1b and IFNg were obtained from R&D Systems (Minneapolis, MN), IL-4 was from ICN Pharmaceuticals (Costa Mesa, CA), IL-12 and IL-10 were purchased from Diaclone Research (Besanc¸on, France) and IL-18 was from Medical & Biological Laboratories (Nagoya, Japan). Assays were performed according to the manufacturer’s instructions.

2.4. Caspase-1 acti6ity At the end of the incubation, both the adhered and not adhered cells were collected, washed with PBS and lysed in a lysis buffer (0.5% Nonidet P-40, 0.5 mM

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EDTA, 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 50 mg/ml antipain) [35]. The lysates were centrifuged at 10 000× g for 10 min. at 4°C, and the supernatants were collected. A volume of 200 ml aliquots of the extracts were incubated with the fluorogenic peptide DABCYL-YVADAPV-EDANS (Bachem AG, Bubendorf, Switzerland), a specific substrate for caspase-1, at a final concentration of 10 mM in 2.5 ml of reaction buffer (100 mM HEPES, pH 7.5, 10% (w/v) sucrose, 0.1% (w/v) 3-[(3-cholamido-propyl) dimethylammonio]1-propane sulfonate (CHAPS), 10 mM dithio-threitol (DTT), 0.1 mg/ml ovalbumin) for 1 hour at 37°C, and the released EDANS was measured on a fluorescence spectrophotometer (Hitachi, Model 244-A) using an excitation wavelength of 340 nm and emission of 490 nm [36]. Activity was calculated as the change in fluorescence, and expressed as arbitrary units of fluorescence (a.u.f) per mg of protein. Specificity of the reaction was assessed by adding equimolar amounts of the specific inhibitor Ac-YVAD-CHO (Bachem AG, Bubendorf, Switzerland).

2.5. Analysis of apoptosis Apoptosis was assessed by using the Apoptosis Detection System kit (Promega Corporation, Madison, WI) following the manufacturer instructions, and it was analyzed by flow cytometry.

2.6. Statistical analysis Values are means9 SEM. The statistical significance of changes was assessed at the 5% level using one-way repeated measures analysis of variance (parametric) or the Friedman repeated measures ANOVA method on ranks (non-parametric), where appropriate, and pairwise multiple comparisons were run using the Student– Newman–Keuls method, by using the Sigma Stat Statistical Analysis System package (Jandel Scientific).

3. Results

3.1. HMG-CoA reductase inhibition promotes IL-1b and IFNg release by PBMC Fig. 1. Dose-response effect of fluvastatin on IL-1b (A) and IFNg (B) release by human peripheral blood mononuclear cells (PBMC) treated or not with M. tuberculosis. PBMC were incubated in the presence of increasing concentrations of fluvastatin for 12 h and then for an additional 24 h, after supplementing the medium with heat-inactivated M. tuberculosis H37 RA (10 mg/ml) ( ) or placebo – – control-("). At the end of the 36-h incubation, both IL-1b and IFNg were determined in the supernatants by specific ELISA. Data correspond to the means 9S.E.M. of four independent experiments. The effect of fluvastatin on IL-1b and IFNg release, both in control and M. tuberculosis-stimulated conditions, was statistically significant as determined by ANOVA.

PBMC were used instead of isolated leukocyte populations to preserve the regulatory interactions between lymphocytes and monocytes/macrophages. Treatment of PBMC with fluvastatin was observed to slightly induce the release of IL-1b to the medium in a dose-dependent manner (Fig. 1A, control conditions). M. tuberculosis induced the release of IL-1b, yet in cultures pre-treated with the statin, this stimulation was synergistic, reaching values up to ten times higher than those

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Fig. 2. Effects of fluvastatin and M. tuberculosis on IL-1b (A) and IFNg (B) release and their reversion by mevalonate. PBMC were incubated in the presence of fluvastatin and mevalonate, as indicated, for 12 h and then for an additional 24 h, after supplementing the medium with heat-inactivated M. tuberculosis H37 RA or placebo. At the end of the 36-h incubation, both IL-1b and IFNg were determined in the supernatants by specific ELISA. Data correspond to the means 9 SEM of eight independent experiments. Statistical comparisons by Friedman repeated measures ANOVA on ranks between treatments are indicated by letters above the bars, different letters denoting statistically significant differences (P B0.05).

observed with the bacterium alone (Fig. 1A). We then measured the release of IFNg, as an indication of the biological interaction between monocytes/macrophages and lymphocytes. On resting conditions, IFNg was barely detectable in the culture medium, and fluvastatin stimulated a slightly but statistically significant, release (Fig. 1B). IFNg levels increased in cultures containing M. tuberculosis, with fluvastatin pretreatment enhancing this a further twofold. As compared to the effect of fluvastatin on IL-1b, which was linear within the range of concentrations used, in the case of IFNg the maximum effect was reached with 2.5 mM, thus indicating

the saturation of the process (Fig. 1B). Similar results were obtained for both cytokines with other statins such as lovastatin (data not shown). Bearing in mind the mechanism of action of statins and the fact that the incubation media contained lipoprotein-cholesterol, the results suggested that the observed immune stimulating effect be due to the depletion of mevalonic acid, rather than that of cholesterol. To directly ascertain whether the effect of fluvastatin was specific to the inhibition of HMG-CoA reductase, the medium was supplemented with 1 mM mevalonate. As shown in Fig. 2, mevalonic acid totally

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prevented fluvastatin-induced stimulation of IL-1b and IFNg secretion, both in the presence and in the absence of M. tuberculosis. Surprisingly, mevalonate supplementation also reduced the release of IL-1b promoted by the bacteria alone, thus suggesting that this immunological response to M. tuberculosis is dependent on mevalonic acid availability. In order to elucidate the mechanism underlying this phenomenon, the effect of inhibitors of prenyl:protein transferases on IL-1b release was determined. Both manumycin A and R( + )limonene, separately, at a concentration of 0.1 mM, enhanced the release of IL-1b in PBMC stimulated with M. tuberculosis, to 186 930% (P B 0.05, n = 3) and to 191 930% (PB0.05, n = 3), respectively, suggesting that inhibition of protein prenylation is involved in the stimulation of IL-1b release.

3.2. HMG-CoA reductase inhibition acti6ates caspase-1 enzyme To determine whether statin-induced IL-1b release involved normal processing of the cytokine IL-1b, forms appearing in cell lysates and culture supernatants were examined by gel electrophoresis and immunoblotting (Fig. 3). As a general observation, intracellular IL-1b corresponded to the 34 kDa pro-form, while that in the culture supernatants corresponded to the 17 kDa active form. Under control conditions, without immunological stimulation, pro-forms were scarcely detected within the cells, with a slight increase in those treated with mevalonate (Fig. 3a), whereas negligible amounts of mature IL-1b were detected in the supernatants by this method regardless of the study conditions (Fig. 3c). In contrast, M. tuberculosis induced a signifi-

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cant increase in intracellular pro-IL-1b (Fig. 3b), and the mature 17-kDa form was also detected in the supernatants (Fig. 3d). In cultures exposed to the bacteria, fluvastatin pretreatment yielded a marked decrease in the content of intracellular pro-IL-1b, along with a concurrent increase in the mature form in cell supernatants. This effect of fluvastatin proved to be specific, since it was totally prevented by mevalonic acid supplementation. The result reflects that inhibition of mevalonate biosynthesis affects the processing and release of IL-1b. Moreover, in cultures stimulated with M. tuberculosis alone, provision of exogenous mevalonate decreased the amount of mature IL-1b in the supernatant and simultaneously increased the cellular content of the pro-form, thus firmly suggesting that mevalonic acid or some isoprene derivative modulates the post-translational processing of IL-1b. We therefore proceeded to determine caspase-1 activity in cell lysates, after incubating PBMC under the above conditions. In Fig. 4A we show that both fluvastatin and M. tuberculosis, separately, increased caspase-1 activity significantly, and their effects were additive although not significantly different from those of fluvastatin alone (Fig. 4A). Mevalonic acid circumvented the effect of the statin and, most interestingly, also reduced M. tuberculosis-induced activation, an effect that was detected even at lower concentrations of mevalonic acid (Fig. 4B). These results are in line with those on IL1-b release and maturation described above, and indicate that mevalonic acid deprivation results in caspase-1 activation. Based on the involvement of caspase enzymes on programmed cell death [37], we analyzed the induction of apoptosis in our conditions. Neither fluvastatin treatment nor the exposure to M. tuberculosis stimulated DNA fragmentation, as assessed by TUNEL assay, or increased the proportion of cells with DNA content lower than 2n (subG1), as measured by labeling with propidium iodide and flow cytometry analysis (data not shown). These results indicate that none of the treatments induced apoptosis in PBMC at the time studied. Therefore, the changes in cytokine production mentioned above could not be ascribed to apoptosis induction.

3.3. Effect of HMG-CoA reductase inhibition on the production of IL-18, IL-12 and IL-4

Fig. 3. Fluvastatin stimulates the processing of pro-IL-1b. PBMC were incubated for 12 h in the presence of 5 mM fluvastatin and/or 1 mM mevalonate, as indicated, then were or were not stimulated with M. tuberculosis H37 RA (10 mg/ml) and incubated for an additional 24-h period. Detection of pro-IL-1b (34 kDa) and mature IL-1b (17 kDa) forms in cell extracts (a, b) and supernatants (c, d) by SDSPAGE and immunoblot analysis using anti-human IL-1b antibody. Lane 1, control; lane 2, fluvastatin (5 mM); lane 3, mevalonate (1 mM); lane 4, fluvastatin (5 mM) plus mevalonate (1 mM).

Caspase-1 also participates in the maturation and release of IL-18 [8,9], a potent co-stimulatory factor for T lymphocyte production of IFNg [38,39]. As shown in Fig. 5, both fluvastatin and M. tuberculosis, separately, stimulated the release of IL-18 and, when used in combination, their effect was greater than additive. On the other side, mevalonic acid supplementation abrogated the effect of fluvastatin, both in stimulated and in unstimulated cells, indicating it was specific (Fig. 5).

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Fig. 4. Effects of fluvastatin and M. tuberculosis on caspase-1 activity. PBMC were incubated for 12 h in the presence of 5 mM fluvastatin and/or 1 mM mevalonate, as indicated, then were or were not stimulated with M. tuberculosis H37 RA (10 mg/ml) and incubated for an additional 24-h period. (A) Caspase-1 activity in cell lysates as determined by measuring the hydrolysis of fluorogenic peptide DABCYL-YVADAPV-EDANS. The results are expressed as the percentage of the control incubation. Specificity of the reaction was assessed by adding equimolar amounts of the specific inhibitor Ac-YVAD-CHO, which resulted in an inhibition \78%. Data correspond to the means9SEM of four independent experiments. Statistical comparisons by Friedman repeated measures ANOVA on ranks between treatments are indicated by letters above the bars, different letters denoting statistically significant differences (PB 0.05). (B) Cells incubated in the presence of increasing concentrations of mevalonate in the medium were exposed to M. tuberculosis H37 RA (10 mg/ml) ( — ) or placebo – – control-(- - - -) and processed for caspase-1 activity determination.

These effects on IL-18 paralleled those observed for IL-1b, which are both produced by monocytes/ macrophages under the action of caspase-1. In synergy with IL-18, IFNg production by lymphocytes may be also stimulated by IL-12 (another cytokine released by monocytes/macrophages), albeit acting via a different signal transduction pathway [30,31]. We therefore measured IL-12 in the supernatants of the above-mentioned cultures (Fig. 6A). As expected from previous studies elsewhere [40], M. tuberculosis stimulated the release of IL-12. Fluvastatin failed to affect this parameter and mevalonic acid supplementation also proved ineffective. As a whole, these results suggest that, while the effect of M. tuberculosis in stimulating the release of IFNg could be mediated by both IL-12 and IL-18, that of fluvastatin appears to be

exclusively associated with the activation of caspase-1. To finally evaluate whether cholesterol synthesis inhibition could also affect the Th2 response, IL-10 and IL-4 were measured in the supernatants of cultured PBMC. Similarly to IL-12, IL-10 production was induced by the bacteria, but not affected by fluvastatin, mevalonate or both (Fig. 6B). In contrast, M. tuberculosis and/or fluvastatin did not stimulate IL-4 release, and mevalonic acid was also ineffective (data not shown).

4. Discussion In the present work we studied the effect of pharmacological inhibition of HMG-CoA reductase on cytokine release by PBMC, both in basal conditions and

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under the stimulation with M. tuberculosis, with the aim to evaluate whether treatment with statins could affect the Th1 immune response. In principle, Th1 response is characterized by release of IFNg and IL-2 by T lymphocytes, which is usually accompanied by IL-1b secretion from monocyte/ macrophages [24], all defining a proinflammatory response. Release of mature IL-1b requires two signals: the primary stimulus to promote transcription and translation of the pro-form, the second to activate caspase-1 [41,42]. This second stimulus provides an additional checkpoint whereby cells regulate the biosynthesis of this interleukin. It is worth mentioning that several pathogens act upon caspase-1, either activating or inhibiting it [43 – 45], which highlights the importance of this enzyme in the inflammatory response to infection. In our conditions we observed that the response to M. tuberculosis comprised both the induction of pro-IL-1b synthesis and caspase-1 activation. However, enzyme activation induced by the bacteria was of a relatively small magnitude and, as in the case of lipopolysaccharide (LPS) [46], a significant proportion of pro-IL-1b is accumulated within the cells. Pretreatment with fluvastatin further increased caspase-1 activity when cells were exposed to the bacterium and the amount of IL-1b released was greatly enhanced, the cell becoming depleted of pro-forms. The synergistic nature of this effect could thus be attributed to action at different levels, namely, the pathogen preferentially inducing pro-IL-1b expression and the statin activating caspase-1. Indeed, fluvastatin treatment would not seem to induce pro-IL-1b expression, as indicated by the scant detection of the pro-form in lysates from non-

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stimulated cells (Fig. 2A), which resulted in limited secretion of mature IL-1b despite the fact that caspase1 was activated. In keeping with this interpretation, when fluvastatin-pretreated cells were exposed to M. tuberculosis, release of IL-1b was very high, likely because pro-forms were induced and proteolytic processing was maximally stimulated. Accordingly, these results suggest that inhibition of mevalonate synthesis constitutes a second signal that triggers IL-1b processing and release. This response appeared not to be associated with apoptosis induction, since in our conditions programmed cell death was not stimulated by either fluvastatin or the bacteria. Apparently, this last result is in contrast with previous ones by us [18] and others [47,48], who reported the induction of apoptosis in established cell lines by effect of statins. It should be noted, however, that this effect of HMG-CoA reductase inhibitors is detectable in proliferating but not in quiescent cells [48], thus, our results in unstimulated PBMC are in line with the latter observations. Similarly to caspase-3 [48], caspase-1 activation by effect of fluvastatin herein shown appears to be related, at least in part, to the inhibition of protein prenylation secondary to mevalonate deficiency, since direct inhibitors of protein prenylation stimulated IL-1b secretion, as shown in the present work. Interestingly, bisphosphonates, which are antiresorptive drugs that co-laterally affect protein prenylation [49], have been shown to activate caspase-3 [50] and to enhance the IL-1b release induced by LPS in macrophages [51,52]. One of the most intriguing observations was the interaction of mevalonic acid with the response to M. tuberculosis. Supplementation with mevalonate slightly,

Fig. 5. Effects of fluvastatin and M. tuberculosis on IL-18 release. PBMC were incubated in the presence of fluvastatin and mevalonate, as indicated, for 12 h and then for an additional 24 h, after supplementing the medium with heat-inactivated M. tuberculosis H37 RA or placebo. At the end of the 36-h incubation, IL-18 was determined in the supernatants by specific ELISA. Data correspond to the means 9SEM of eight independent experiments. Statistical comparisons by Friedman repeated measures ANOVA on ranks between treatments are indicated by letters above the bars, different letters denoting statistically significant differences (P B 0.05).

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Fig. 6. Effects of fluvastatin and M. tuberculosis on IL-12 (A) and IL-10 (B) release. PBMC were incubated in the presence of fluvastatin and mevalonate, as indicated, for 12 h and then for an additional 24 h, after supplementing the medium with heat-inactivated M. tuberculosis H37 RA or placebo. At the end of the 36-h incubation, both IL-12 p70 and IL-10 were determined in the supernatants by specific ELISA. Data correspond to the means 9 SEM of eight (IL-12) or four (IL-10) independent experiments. Statistical comparisons by Friedman repeated measures ANOVA on ranks between treatments are indicated by letters above the bars, different letters denoting statistically significant differences (P B0.05).

yet consistently, prevented caspase-1 activation and reduced the secretion of IL-1b induced by the pathogen. This further indicates that caspase-1 activation is responsive to the availability of mevalonic acid. It is not known yet whether M. tuberculosis inhibits the mevalonate pathway to induce caspase-1 activation in the host, a possibility which will have to be expressly evaluated by further studies. In this regard, a reduction in HMG-CoA reductase activity has been reported in murine macrophages primed in vivo with Corynebacterium parvum [53]. IL-18 is a cytokine that acts as a costimulatory factor for the production of IFNg [28 – 31,38,39], one of the

most important cytokine that seems to be involved in the development of the atherosclerotic lesion [8,11,54]. As for IL-1b, cleavage by caspase-1 of pro-IL-18 is required before the mature form is secreted. We observed that treatment with fluvastatin gave rise to an increase of IL-18 secretion in non-stimulated cells and, more intensely, in M. tuberculosis-stimulated cells, likely due to caspase-1 activation. It is to note that, IL-18 release in response to fluvastatin was as high as to M. tuberculosis when added separately to the cultures. This relatively high response to fluvastatin may be attributed to constitutive gene expression of IL-18, as recently reported in unstimulated freshly isolated

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human PBMC [55]. Despite that IL-18 levels were similar, the release of IFNg was higher in response to M. tuberculosis than to the statin, likely because the bacteria also stimulated the release of IL-12 (Fig. 6), and both interleukins cooperate together for the stimulation of IFNg release, as recently reported by others [30]. Given that in our cell system, release of IL-12/IL10 was not affected by the statin, the increased production of IFNg induced by the drug in the presence of bacteria must be mainly ascribed to the increment of IL-18 and/or IL-1b rather than to changes in IL-12 or IL-10. This is the first evidence that statins could enhance Th1 cytokine production. IFNg has been implicated in the development of atherosclerotic, since mice lacking IFNg receptors are more resistant to atherosclerosis [54]. This together with the frequent detection of this cytokine in human atherosclerotic plaques has led to the notion that first steps in lesion formation are governed by a Th1-cell response [7,8,10,54]. However, once the lesion has been established, IFNg may be beneficial since this cytokine reduces smooth muscle cell and macrophage proliferation [56,57] and collagen synthesis [58], prevents metalloproteinase activation in the plaque [59], and downregulates scavenger receptor expression [60]. Besides, since IFNg plays a crucial role as activator of diverse macrophage effector functions, it could protect the lesion against intracellular infectious agents that would complicate the lesions, as is the case of Chlamydia pneumonie. Thus, the influence of local secretion of IFNg on atherosclerotic plaque disruption is not evident. In advanced lesions, the presence of type Th2 (IL-10 and IL-4) in conjunction with type Th1 cytokines have been recently reported [7,9,10]. Since Th1 and Th2 cells reciprocally inhibit each other, it has been hypothesized that IL-10 locally produced may protect the atherosclerotic lesion from excessive inflammatory response, by downregulating Th1-cell activation [9,10]. However, Th2 cytokines induce a humoral response, implying a change in IgG isotype, IgE production, activation of complement components that mediate cytolysis [61] and activation of different effector cells, such as basophils, eosinophils and mast cells, which release powerful vasoactive substances and potent proteolytic enzymes [62,63]. Interestingly, mast cells were recently shown to accumulate in coronary plaques at the site of rupture [64] and an increase of degranulated mast cells in the adventicia backing ruptured plaques has been reported [65]. Moreover, there is evidence from other systems, that inflammatory lesions mediated by mixed Th1+ Th2 (or Th0) T cell activity are susceptible to necrosis, and disease progression is associated to a worse prognostic [66,67]. We, therefore, hypothesize that a type Th1-driven inflammatory lesions, while promoting lesion growth, ensues the stabilization of the plaque,

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whereas the combination of cytokines type Th1-Th2 correlates with an increase in tissue damage, resulting in necrosis and plaque rupture. In the light of present results, we suggest that statins could be immunoprotective by preserving and/or reestablishing a Th1 cytokine profile in the atheroma, which helps to stabilize the plaque. Systemic administration of cytokines for the restoration of a protective cytokine profile is currently regarded as a therapeutic strategy to resolve critical diseases [68,69]. Results presented herein, besides illustrating a link between cholesterol metabolism and the inflammatory response, provide a tool for modulating the cytokine secretion pattern Th1, which has a potential to be used for therapeutic purposes. Since this modulation is synergistic with an immune stimulus, cytokine regulation could be expected to be principally affected at the lesion site, hence side effects due to systemic action would be avoided. Accordingly, pharmacological inhibition of mevalonate synthesis could be an alternative adjuvant to boost the immune Th1-type response to M. tuberculosis, but until more direct clinical assays are performed, the benefit of such a pharmacological intervention must remain speculative.

Acknowledgements This study was supported by grants from the Fondo de Investigacio´n Sanitaria (FIS 97/0389 and 99/0286) and Comisio´n Interministerial de Ciencia y Tecnologı´a (SAF 96/0011), Spain. We thank Miguel Martı´n for his excellent technical assistance and Novartis for providing us with fluvastatin. We are deeply indebted to all Donor Unit personnel, at the Service of Hematology, Hospital La Paz, Madrid, Spain, for supplying the buffy coats, and to Dr. Enrique Go´mez Mampaso, at the Service of Microbiology, Hospital Ramo´n y Cajal, Madrid, for providing the heat-inactivated M. tuberculosis H37 RA.

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