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The Journal of Immunology, 2004, 173: 4936-4944.
Copyright © 2004 by The American Association of Immunologists

Geranylgeraniol Regulates Negatively Caspase-1 Autoprocessing: Implication in the Th1 Response against Mycobacterium tuberculosis1

María T. Montero2,3,*, Joaquín Matilla2,*, Enrique Gómez-Mampaso{dagger} and Miguel A. Lasunción*,{ddagger}

* Servicio de Bioquímica-Investigación and {dagger} Servicio de Microbiología, Hospital Ramón y Cajal, Madrid, Spain; and {ddagger} Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caspase-1 is a cysteine protease composed by two 20-kDa and two 10-kDa subunits that processes pro-IL-1{beta} and pro-IL-18 to their mature forms. This enzyme is present in cells as a latent zymogen that becomes active through a tightly regulated proteolytic cascade. Activation is initiated by the oligomerization of an adaptor molecule, or by the formation of a multiprotein complex named inflammasome. Negative regulation of caspase-1 activation is exerted by proteins that compete with the adaptor molecule or with the inflammasome formation. We previously reported that fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, increases caspase-1 activity in PBMC. This effect was strengthened by Mycobacterium tuberculosis, rending an exacerbated IL-1{beta}, IL-18, and IFN-{gamma} production. Mevalonate, the product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is a precursor for both nonsterol isoprenoid and sterol formation. In this study, we studied the involvement of mevalonate derivatives in the regulation of caspase-1 activation. Inhibition of sterol formation by SKF-104976 or haloperidol had no effect on IL-1{beta} release. However, the isoprenoid geranylgeraniol prevented both caspase-1 activation and the exacerbated IL production induced by fluvastatin. This isoprenoid significantly reduced the release of IL-18 and IFN-{gamma} by PBMC treated with mycobacteria, even in the absence of fluvastatin. In correlation with the increased caspase-1 activity, fluvastatin stimulated the proforms cleavage, enhancing the formation of active subunit p10. Geranylgeraniol not only prevented this effect, but induced proforms accumulation. Present results suggest that, once the proteolytic cascade is initiated, geranylgeraniol may exert an additional negative regulation on caspase-1 cleavage process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acquired resistance to Mycobacterium tuberculosis is primarily mediated by specific T cells producing IFN-{gamma} (Th1), while the activation of cells producing IL-4 (Th2) results in progressive disease (1, 2, 3, 4, 5, 6). IL-1{beta} and IL-18 are proinflammatory cytokines that act synergistically with IL-12 to induce IFN-{gamma} release through a mechanism still not fully understood (7, 8, 9, 10), and both have been involved in the generation of a protective immunity against M. tuberculosis (11, 12, 13, 14, 15, 16). Unlike most cytokines, IL-1{beta} and IL-18 are synthesized as inactive precursor forms, devoid of a conventional leader sequence. Before their mature forms are secreted, both pro-IL-1{beta} and pro-IL-18 require enzymatic cleavage by caspase-1 (17, 18, 19, 20), which constitutes an additional control whereby cells regulate the formation of these ILs. Like other proteinases, caspase-1 exists in the cell as a latent zymogen, and enzymatic activation occurs through a very tightly regulated proteolytic cascade that rends an heterotetramer composed by two 20-kDa (p20) and two 10-kDa (p10) active fragments (21, 22, 23). The exact mechanism by which caspase-1 activation occurs remains unclear, and different possible routes have been proposed. Concurring with the mechanism suggested for caspase-9, some authors propose that the cleavage process is initiated by oligomerization of an adaptor protein that recruits procaspase-1 via caspase recruitment domain (CARD)-CARD4 domain interaction (23, 24). Several candidate CARD proteins such as RIP2 (receptor-interacting protein 2), Ipaf (IL-1-converting enzyme protease-activating factor), or ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) (25, 26, 27) enhance pro-IL-1{beta} processing and have been proposed as protein adaptors. Recently, Tschopp and colleagues (28) have described the formation of a multiprotein complex termed the inflammasome. These authors provided evidence suggesting that, in this complex, NALP-1 (neuronal apoptosis inhibitory protein/MHC class II transcription activator/incompatibility locus protein from Podospora anserina/telomerase-associated protein-, leucine-rich repeat-, and pyrin domain-containing protein-1), a molecule containing Piryn and CARD domains recruits procaspase-5 and ASC, a protein that binds procaspase-1 through CARD-CARD domain interaction. In this way, procaspases are brought in close contact and both become activated. In addition to positive regulation by adaptor molecules, proteins that negatively regulate caspase-1 processing have been identified. Proteins such as COP (CARD-only protein (also called pseudo-IL-1 converting enzyme)) and ICEBERG contain CARD domains that closely resemble the caspase-1 prodomain, and have been proposed as possible adaptor protein competitors (29, 30). Some proteins have been implicated in the prevention of the inflammasome formation; in this regard, ASCI (also called ASC2) by binding to NALP-1 blocks their association with ASC, and thus prevents caspase-1 recruitment to the inflammasome (31). Pyrin also acts as an anti-inflammatory molecule by binding to ASC (32). In addition to these mechanisms, caspase-1 activity is also regulated by enzymatic inhibitors such as the human serpin PI-9 (33), or by S-nitrosylation mediated by NO (34).

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (statins) are effective lipid-lowering agents widely used to treat hypercholesterolemia. In large clinical trials, it has been shown that the use of statins not only reduces coronary events, but also total mortality rates in patients with coronary heart disease (35, 36, 37, 38, 39, 40). Because the benefit of such a treatment is greater than expected in terms of the reduction of low density lipoprotein cholesterol levels produced, it has been proposed that statins exert actions beyond that of simply lowering cholesterol. In keeping with this, some effects of statins on immune function have been reported (41, 42, 43, 44, 45). In PBMC, we demonstrated that fluvastatin induces caspase-1 activation and a small secretion of IL-1{beta}, IL-18, and IFN-{gamma}. In combination with M. tuberculosis, caspase-1 was synergistically activated, increasing the processing of IL-1{beta} and IL-18 proforms, whereas the release of IL-12, IL-10, and IL-4 (Th2) was unaffected (46). Mevalonate, the product of HMG-CoA reductase, not only abolished the effects of the statin, but also reduced the caspase-1 activation induced by the bacteria alone, suggesting the involvement of the mevalonate pathway (see Fig. 1) in the control of the immune response against M. tuberculosis (46). In the same line of evidence, the aminobisphosphonates, which are antiresorptive drugs that inhibit the mevalonate pathway (47), have been shown to augment LPS-induced cytokine secretion by RAW 264 macrophages (48, 49) and to increase caspase-3 activity (50).



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  		FIGURE 1. The cholesterol biosynthesis pathway. The figure shows the intermediates relevant for this study and the enzymes inhibited by statins, SKF-104976 or haloperidol.

 
Mevalonate is the precursor of sterols (mainly cholesterol) and other nonsterol derivatives, such as ubiquinone, dolichol, farnesol, and geranylgeraniol, with paramount importance in cell physiology. Among mevalonate derivatives, farnesyl pyrophosphate and geranylgeranyl pyrophosphate are used for posttranslational modification of proteins with important cellular functions and have the ability to reverse many of the pleiotropic effects of statins (51, 52, 53). When mevalonate synthesis is inhibited by statins, evidence exists that the availability of these isoprenoid groups is reduced, and consequently, the proteins that usually are prenylated accumulate in an unprenylated form (54, 55)

In this study, we use the experimental model previously described (46) to study the role of isoprenoids on caspase-1 activation, and the subsequent production of IL-1{beta}, IL-18, and IFN-{gamma} in response to M. tuberculosis. As hydrophilic pyrophosphate groups impair the incorporation of the isoprenoids into living cells, farnesol and geranylgeraniol in their alcohol form were used in the present study. These fatty alcohols freely enter the cells and are converted to the pyrophosphorylated forms (56). The results reveal that geranylgeraniol is involved in the regulation of procaspase-1 cleavage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Immunoassay kits for IL-1{beta}, IL-18, and IFN-{gamma} were purchased from Diaclone Research (Besançon, France). Rabbit polyclonal specific anti-human p10 and anti-human p45 were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Farnesol and geranylgeraniol were purchased from Sigma-Aldrich (Poole, U.K.) and dissolved in DMSO. Fluvastatin was kindly provided by Novartis Pharmaceuticals (East Hanover, NJ). SKF-104976, an inhibitor of lanosterol 14{alpha}-demethylase, was a gift from SmithKline Beecham (Dr. R. Dalre, SmithKline Beecham, Madrid, Spain). Haloperidol was purchased from Sigma-Aldrich. Bacto M. tuberculosis H37 RA was obtained from Difco Laboratories (Detroit, MI). Caspase substrate acetyl-WEHD-7-amino-4-methyl coumarin was from Calbiochem (Nottingham, U.K.).

PBMC isolation and culture conditions

PBMC were isolated from buffy coats from normal male donors over a Ficoll-Hypaque gradient, according to the method of Boyum (57), and cultured on 12-well plates at a final density of 2 x 106 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FCS, L-glutamine, penicillin, streptomycin, and gentamicin. Incubations were performed at 37°C in a humidified atmosphere containing 5% CO2 in air. The cultures were supplemented or not with 5 µM fluvastatin dissolved in DMSO (final concentration in the medium 0.04%), and geranylgeraniol or farnesol at various concentrations (100 pM, and 0.1, 1, and 5 µM). After a 12-h incubation under the mentioned conditions, cells were stimulated by adding to the medium heat-inactivated M. tuberculosis H37 RA (25 µg/ml) or vehicle, without any other change in the medium, and the incubation was prolonged for an additional 24-h period. In other instances, to block cholesterol biosynthesis more distally, SKF-104976 (3 µM) or haloperidol (25 µM) was used instead of fluvastatin.

At the end of incubations, adherent cells were scraped and harvested together with cells in suspension. After centrifugation, cell pellets were washed in PBS and frozen for caspase-1 activation study. The supernatants were frozen until cytokine determinations.

Determination of caspase-1 activity

A fluorometric cleavage microassay was designed for microtiter plates Fluoronunc F16 black polysorp (Nalge Nunc International, Rochester, NY), following the method described by Thornberry (58): cell lysates were prepared by three consecutive freeze-thaw cycles in lysis buffer (25 mM HEPES, pH 7.5, 0.5 mM EDTA, 150 mM MgCl2, 0.1% Nonidet P-40, supplemented with 1 mM PMSF, 1 µg/ml aprotinin, and 50 µg/ml antipain) in the proportion 12 x 106 cells/50 µl buffer. Lysates were then centrifuged at 10,000 x g for 10 min at 4°C, the supernatants were collected, and protein concentration was measured. A total of 100 µg of protein in 50 µl of lysis buffer for each sample was added to 175 µl of reaction solution (22.9% glycerol, 0.15% CHAPS, 11.5 mM dithiotreitol, 175.5 mM NaCl) and incubated at 37°C for 2 h in the presence of 100 µM acetyl-WEHD-7-amino-4-methyl coumarin, a specific fluorogenic substrate for caspase-1. The emitted fluorescence was measured on a fluorometric plate reader (Spectrafluor; Tecan, Barcelona, Spain) using an excitation wavelength of 380 nm and an emission wavelength of 465 nm. The results were expressed as percentages of the fluorescence emitted by control lysates.

Cytokine determinations

IL-1{beta}, IL-18, and IFN-{gamma} concentrations in cell supernatants were determined by ELISA using commercially available kits (Diaclone Research) following the manufacturer’s instructions.

Immunoblot analysis

Cells were washed with PBS and resuspended in lysis buffer, as mentioned above. Proteins were subjected to electrophoresis, according to Laemmli (59), on a discontinuous 6–11% SDS-PAGE, using a minigel system (Hoeffer Scientific Instruments, San Francisco, CA), and then transferred to Immobilon membranes (pore size 0.22 µm; Millipore, Bedford, MA), using a Semiphor semidry 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 for 1 h in 5% nonfat dry milk in 20 mM Tris and 500 mM NaCl, pH 7.5. Subsequently, blots were probed with two different polyclonal antisera raised against human caspase-1. Anti-human N-terminal caspase-1 (A-19; Santa Cruz Biotechnology) was used to detect p45 proforms and N-terminal fragments. Transferred membranes were incubated with this Ab diluted 1/1000 in TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5) containing 1% nonfat dry milk for 1 h, and then washed. Then bound Abs were detected by ECL Western blotting detection reagents (Amersham Biosciences, Cardiff, U.K.). A polyclonal antiserum raised against C-terminal sequence of caspase-1 (C-20; Santa Cruz Biotechnology) was used for p45 and p10 detection. Transferred membranes were cut into two pieces at the level of the 25-kDa marker. The piece containing the higher m.w. proteins was incubated for 1 h in 1/500 diluted antiserum (1% nonfat dry milk in TBST), whereas the piece with the lower m.w. proteins was incubated for 2 h in a 1/250 antiserum dilution. ECL Western blotting detection reagents were used to detect specific binding.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of fluvastatin, SKF-104976, and haloperidol on IL-1{beta} production by PBMC exposed to M. tuberculosis

To obtain a deeper insight into the mechanism involved in exacerbation of caspase-1 activity induced by fluvastatin, we first examined the possible participation of sterol derivatives by blocking cholesterol biosynthesis at different levels. For this, PBMC were treated with either fluvastatin (5 µM); SKF-104976, an inhibitor of lanosterol 14{alpha}-desmethylase (60) (3 µM); or haloperidol, an inhibitor of sterol {Delta}8{Delta}7 isomerase (61) (25 µM), and exposed to the bacteria (see Fig. 1). The release of IL-1{beta} into the medium was measured as an indirect, but sensitive method to detect changes in caspase-1 activation. As shown in Fig. 2, inhibition of HMG-CoA reductase with fluvastatin gave rise to a strong increase of IL-1{beta} production, whereas inhibition of sterol formation by the other drugs had no effect in this parameter. These results suggest that the effect of fluvastatin on IL-1{beta} production is mediated by the depletion of mevalonate or some of its nonsterol isoprenoid derivatives.



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  		FIGURE 2. Effect of different cholesterol synthesis inhibitors on IL-1{beta} production induced by M. tuberculosis. IL-1{beta} concentration (pg/ml) measured by ELISA in supernatants of PBMC cultured in the presence of different inhibitors of cholesterol biosynthesis (fluvastatin, 5 µM; SKF-104976, 3 µM; and haloperidol, 25 µM) and challenged with M. tuberculosis. Data correspond to the means ± SEM of four independent experiments. Statistical comparisons by Friedman repeated measures ANOVA on ranks; differences between treatments are indicated by letters above the bars; different letters denote statistically significant differences (p < 0.05).

 
Regulation of IL-1{beta}, IL-18, and IFN-{gamma} release by isoprenoids

The possible implication of farnesol and/or geranylgeraniol in the regulation of caspase-1 activation was first evaluated by determining the effect of these isoprenoids on IL-1{beta} and IL-18 production. PBMC treated with several concentrations of farnesol or geranylgeraniol, alone or in combination with 5 µM fluvastatin, were exposed to M. tuberculosis for 24 h, and IL production was measured in supernatants by ELISA. In our experience, cytokine release by cells exposed to the bacteria showed a high variability among donors, although the relative increase induced by the drug was more homogeneous. Thus, the results were normalized by considering cytokine production by cells exposed to the bacteria as 1 (Fig. 3). Supplementing the medium with farnesol (A) or geranylgeraniol (B) efficiently prevented the effect of fluvastatin on IL-1{beta}, IL-18, and IFN-{gamma} production, in both nonstimulated and mycobacteria-stimulated cells. This effect was dose dependent in the two cases, but geranylgeraniol appeared to be more efficient than farnesol. Interestingly, in cells treated with the bacteria alone, not exposed to the statin, farnesol and geranylgeraniol also reduced the release of IL-18 in a dose-dependent manner, and 5 µM geranylgeraniol was also able to significantly reduce IFN-{gamma} (Th1) secretion (Fig. 3).



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  		FIGURE 3. Dose-response effects of farnesol and geranylgeraniol on IL-1{beta}, IL-18, and IFN-{gamma} production. PBMC were incubated in the presence or absence of 5 µM fluvastatin (Fl) in combination with farnesol (A) or geranylgeraniol (B), at different doses: 0 ({blacksquare}), 0.1 µM ( {permzspch022}), 1 µM ({cjs2113}), and 5 µM ({square}). After 12 h of incubation, cells were stimulated or not with 25 µg/ml M. tuberculosis (Myc. tb.), and the incubation was resumed for additional 24 h. At the end of the incubation, IL-1{beta}, IL-18, and IFN-{gamma} were measured by ELISA in the supernatants. To normalize the results, cytokine production by cells exposed to the bacteria was considered as 1. Data correspond to the means ± SEM of four independent experiments. For each isoprenoid, statistical comparisons against dose 0 were done by Friedman repeated measures ANOVA on ranks: *, p < 0.05.

 
Geranylgeraniol prevents the exacerbated activation of caspase-1 induced by fluvastatin

We previously reported that the strong stimulation of IL-1{beta} and IL-18 production induced by fluvastatin in PBMC exposed to M. tuberculosis was due to augmented caspase-1 activity, an effect that was abrogated by mevalonate (46). The results presented above suggested that geranylgeraniol was a mevalonate derivative involved in the regulation of caspase-1 activity. We therefore proceeded to determine caspase-1 activity in cytosolic fractions from PBMC untreated or treated with fluvastatin, alone or in combination with geranylgeraniol, and exposed to the bacteria. As shown in Fig. 4, the increase of caspase-1 activity induced by fluvastatin in combination with the bacteria was totally prevented by 5 µM geranylgeraniol.



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  		FIGURE 4. Geranylgeraniol prevents the activation of caspase-1-induced fluvastatin in combination with M. tuberculosis. Arbitrary units of caspase-1 activity measured in cytosolic extracts from PBMC treated or not with 5 µM fluvastatin (Fl) or fluvastatin in combination with 5 µM geranylgeraniol (GG), and exposed to 25 mg/ml M. tuberculosis (Myc. tb.). Data correspond to the means ± SEM of four independent experiments. Statistical comparisons by Friedman repeated measured ANOVA on ranks; differences between treatments are indicated by letters above the bars; different letters denote statistically significant differences (p < 0.05).

 
Involvement of geranylgeraniol in procaspase-1 cleavage

Caspase-1 is present in the cytosol of monocytic cells as an inactive 45-kDa precursor form (62), and a tightly controlled cleavage of this proform is required for enzyme activation (21, 22). The stimulatory effect of fluvastatin on caspase-1 activity, as shown above, could be due to either augmented proform production or increased processing of the pre-existing zymogen. According to Yamin et al. (21), procaspase-1 autocatalysis is initiated by the excision of the proform in two fragments: the 12-kDa C-terminal (p12), and the 35-kDa N-terminal (p35), which is more active than the proform. This process is a critical step for caspase-1 activation. Alternatively, caspase-1 could be cleaved to generate a 35-kDa C-terminal fragment, which is inactive (21). With the aim to investigate the role of isoprenoids on this first cleavage stage, we analyzed by immunoblot the effect of fluvastatin and geranylgeraniol on the content of both the proform and the 35-kDa fragments in cytosolic extracts from PBMC treated with fluvastatin, alone or in combination with increasing concentrations of the isoprenoid, and exposed to the bacteria. The immunoblots were performed by using, independently, two polyclonal antisera raised against the N-terminal or the C-terminal sequence of caspase-1. Results shown in Fig. 5 illustrate that fluvastatin treatment promotes a significant reduction in cytosolic content of the proform (p45) and the N-terminal 35-kDa fragment. We observed that geranylgeraniol not only prevented this effect in a dose-dependent way, but it also induced, at the highest isoprenoid concentration used, a significant accumulation of the proform without reduction of 35-kDa fragments, which even increased in some donors.



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  		FIGURE 5. Effects of fluvastatin on caspase-1 cleavage in PBMC exposed to M. tuberculosis and their prevention by geranylgeraniol. Caspase-1 proform and 35-kDa fragments were analyzed by Western blot in cytosolic fractions from PBMC treated or not (control) with 5 µM fluvastatin alone or in combination with 100 pM, 0,1 µM, 1 µM, or 5 µM geranylgeraniol, as indicated, and exposed to M. tuberculosis (25 µg/ml). A, Detection of the proform and C-terminal 35-kDa fragment by using an Ab directed against the C-terminal region of the enzyme. B, The same study in a different donor and using an Ab directed against the N-terminal region. C, Representation of the densitometric scan of bands identified with the anti-N-terminal Ab. Results are expressed as percentage of values measured for each band in the control condition. Data correspond to the means ± SEM of three independent experiments. Statistical comparisons against the control by Friedman repeated measures ANOVA on ranks: *, p < 0.05.

 
First cleavage of p45 is followed by the removal of a 20-aa peptide from p12 to generate the active 10-kDa chain (p10) (21, 22). Using a polyclonal antiserum raised against the C-terminal sequence of caspase-1, we detected p45 and the derived p10 and p12 fragments in cytosolic extracts from every study condition (Fig. 6). It has been reported by some authors that p10 is extremely difficult to detect in cells actively producing mature IL-1{beta}. Possible explanations include the rapid degradation of the active enzyme, rapid secretion to the medium, or minimal production of active enzyme (21, 62, 63, 64, 65). In agreement with this, we found a high disproportion between p45 and p10 levels in cytosolic extracts. To avoid competition for the Ab and thus improve the detection of those scarce polypeptides, we excised the transfer membrane in two parts at the level of the 25-kDa marker, and then these parts were incubated separately with the Ab under different conditions. Membranes containing the lower m.w. proteins were incubated with the primary Ab for a longer period and at a higher concentration than the other. It must be mentioned that we found a high variability among the different polyclonal antiserum batches used (some of them could detect p45, but were unable to detect p10 and p12). By following this protocol and using appropriate Abs, we were able to demonstrate that, first, exposure of PBMC to the bacteria resulted in increased levels of procaspase-1 without clear changes in p10 (Fig. 6). Second, fluvastatin both in nonstimulated as well as in M. tuberculosis-stimulated cells reduced the proform content, whereas it increased the levels of p10. It should be noted that the extent of procaspase-1 processing induced by fluvastatin varied among the different PBMC preparations used (compare blots shown in Figs. 5A, 5B, 6A, and 6B), as it did the release of IL-1{beta} and IL-18 by effect of the drug. In any case, the effect of fluvastatin was abrogated by 5 µM geranylgeraniol added to the culture medium, showing a clear tendency to accumulate p12 (Fig. 6A). Similarly, 1 mM mevalonate prevented the decrease of p45 and the increase of p10 induced by fluvastatin (Fig. 6B).



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  		FIGURE 6. Effect of geranylgeraniol and mevalonate on caspase-1 proform cleavage. A, Analysis by immunoblot of procaspase (p45) and fragments p10 and p12 in cytosolic extracts from PBMC treated or not with 5 µM fluvastatin (Fl), alone or in combination with 5 µM geranylgeraniol (GG), and exposed or not to M. tuberculosis (25 µg/ml), as indicated. B, The same study, but using 1 mM mevalonate (Mv) instead of geranylgeraniol. Results correspond to cells from two different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-1{beta} and IL-18 release is considered to be crucial to resolve the infectious process, although an excess of these cytokines could become harmful to the host. Hence, production of these cytokines needs to be under a tight control. Both ILs are synthesized as propeptides, and their proteolytic processing by caspase-1 represents an additional checkpoint whereby cells regulate their production (66, 67, 68). In turn, caspase-1 is present in cells as a zymogen, which only in certain circumstances acquires activity through a complex enzymatic mechanism (21, 22). In fact, several negative feedback loops prevent caspase-1 activation by controlling proform oligomerization (29, 30, 31, 32), and different enzymatic inhibitors regulate its activity (33, 34). The data provided in this work suggest that, once the proteolytic cascade is initiated, geranylgeraniol may inhibit caspase-1 cleavage, thereby exerting an additional negative regulation of this enzyme.

Induction of proforms proximity is considered to be the initial step in caspase-1 activation. Subsequently, a sequential cascade of conformational changes and cleavages occurs to finally rend the p20 and p10 subunits that form the completely active heterodimer. The first procaspase-1 fragmentation occurs by an intermolecular process with a weak catalytic activity, rending a C-terminally truncated 35-kDa fragment and a 12-kDa fragment that corresponds to the active subunit p10 plus a 20-aa peptide, a linker that is next released (21, 22). As reported by Yamin et al. (21), p12 remains associated with the cleaved 35-kDa fragment and is processed down to p10 just as 35-kDa fragment is processed to the p20 active subunit. This initial 35-kDa/p12 fragment is much more active than proforms and serves both as an intermediate enzyme to cleave new proforms with a higher rate, as well as a substrate for the formation of the final p10/p20 active fragments, amplifying in such a way the cleavage process. In the present work, we show that inhibition of HMG-CoA reductase by fluvastatin leads to a reduction of proforms and N-terminal 35-kDa fragment, whereas this inhibition augments the p10 subunit, which reflects an increased rate of proform processing. When cells treated with the drug are exposed to M. tuberculosis, the enhanced proform cleavage was associated with both an increase of caspase-1 activity (Fig. 4) and intense IL-1{beta}, IL-18, and IFN-{gamma} release (Fig. 3). The effects of fluvastatin were abrogated by mevalonate and by geranylgeraniol (present results and Montero et al. (46)). We have observed that the exacerbated IFN-{gamma} release induced by fluvastatin is prevented by specific Abs directed to IL-18, and by caspase inhibitors (data not shown), which indicates that the mevalonate pathway modulates the IFN-{gamma} release by regulating caspase-1 activity.

Interestingly, 5 µM geranylgeraniol not only abolished the effect of statins, but also induced the accumulation of proforms without interfering with the generation of C- and N-terminal 35-kDa fragments (Fig. 5). This observation reflects that this isoprenoid regulates caspase-1 fragmentation distally to the induction of proforms proximity. In contrast, in the presence of geranylgeraniol, the p12 fragment tended to accumulate, decreasing p10 formation (Fig. 6). The fact that geranylgeraniol reduced p35 fragmentation and prevented p10 formation suggests that this isoprenoid interferes with the association of these fragments, blocking the sequential cascade of cleavages that rends the active heterodimer (see Fig. 7). However, whether this action involves the molecular interaction of geranylgeraniol with the enzyme or the isoprenoid modulates caspase-1 processing through other undefined mechanisms has not been elucidated.



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  		FIGURE 7. Caspase-1 cleavage regulation by geranylgeraniol. Schematic representation of sequential cleavages of caspase-1 proforms based on the induced proximity though CARD-CARD domain interaction (23 ) and according to Yamin et al. (21 ) and Ramage et al. (22 ). A hypothetical negative control point of geranylgeraniol (GG) in the catalytic process that activates caspase-1.

 
There is a set of heritable human diseases characterized by inflammation without evidence of an Ag-specific immune response, which are known as autoinflammatory disorders (69, 70, 71). The hereditary periodic fever syndromes are a subset of them, characterized by inflammatory attacks and febrile peaks that reflect disorders of the innate immune response (72). In some of them, mutations affect genes encoding for important mediators of apoptosis, inflammation, and cytokine processing (71). Cryopyrin mutations are responsible for three of these clinically defined illnesses: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurologic cutaneous articular syndrome, whereas pyrin (or marenostrin) mutation is responsible for familial Mediterranean fever (71). Both pyrin and criopyrin contain an N-terminal pyrin domain that interacts with the pyrin domain of the adaptor protein ASC, and their mutations are associated with augmented caspase-1 activity (32, 71, 73). In hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), the underlying genetic defect appears to affect the MVK gene, leading to deficient mevalonate kinase activity (74). This enzyme phosphorylates mevalonic acid, a key step in isoprenoid biosynthesis (see Fig. 1). Although proteins mutated in HIDS and in familial Mediterranean fever are functionally unconnected, symptoms are very similar in both syndromes (72). By using an approach similar to ours, Frenkel et al. (75) demonstrated that the shortage of isoprenoids, rather than the excess of mevalonic acid, was causative of the increased secretion of IL-1{beta} by PBMC from HIDS-affected patients (75). In keeping with this, the temporary deficiency of isoprenoids has been associated with the induction of inflammation and fever in HIDS (76). Present results demonstrate that depletion of isoprenoids leads to caspase-1 activation and increased production of proinflammatory ILs. Actually, febrile attacks in HIDS are coincident with peaks of mevalonic acid excretion and elevated serum levels of proinflammatory cytokines (74). It is also worthy to mention that therapy with statins, directed to reduce mevalonate production, aggravates rather than alleviates the syndrome in these patients (77). According to this, in patients with HIDS, insufficient synthesis of isoprenoids due to mevalonate kinase deficiency would result in an excessive caspase-1 activity, subsequently giving rise to an inflammatory response similar to that induced by mutations in pyrin and criopyrin.

It has been reported that mycobacterial infection of macrophages results in membrane-permeable phagosomes that facilitate transit of macromolecules between the cytosolic and vacuolar compartments of infected cells (78). Interestingly, several isoprenoid pyrophosphates have been characterized as V{gamma}9/V{delta}2 T cell-stimulating Ags in bioactive fractions from mycobacteria (79, 80, 81, 82), and isoprenoid-specific {gamma}{delta} T cells have been detected in tuberculoid patients (83, 84). All that reflects is that isoprenoids are released to the cytosol of infected macrophages. Based on present results, it could be speculated that isoprenoids released by the bacteria could be used by the macrophage as substrates in the mevalonate pathway, rending extra geranylgeraniol, and hence reducing caspase-1 activation to evade the immune response. In connection with this, we found that both farnesol and geranylgeraniol significantly reduced the release of IL-18 in PBMC stimulated with the bacteria (Fig. 3). Because IL-18 proform is constitutively expressed, these results indicate that the action of isoprenoids in caspase-1 activation is operative in this condition, in the absence of fluvastatin. It is worth mentioning that several pathogens act upon caspase-1, either activating or inhibiting it to evade immunity (85, 86, 87).

Systemic administration of cytokines for the restoration of a protective cytokine profile is currently regarded as a therapeutic strategy to resolve critical diseases (88, 89). The results presented in this work, besides illustrating a link between cholesterol metabolism and the caspase-1 activation process, provide a tool to strengthen the Th1 secretion pattern by means of statins. Because this modulation is synergistic with M. tuberculosis, cytokine regulation could be expected to be affected mainly 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. Obviously, this needs direct experimental confirmation. In this line of evidence, in patients with bacteremia, a significant reduction in mortality has been observed among those taking as compared with those not taking statins (37), which reinforces the tight connection between the cholesterol biosynthetic pathway with the immune response.


   Acknowledgments
 
We thank Garbine Roy for his critical review of the manuscript, and Novartis Pharmaceuticals 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.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported in part by Grant FIS 00/029 from the Fondo de Investigación Sanitaria, Spain. Back

2 M.T.M. and J.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. María T. Montero, Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, Ctra. Colmenar, km 9, 28034-Madrid, Spain. E-mail address: teresa.montero{at}hrc.es Back

4 Abbreviations used in this paper: CARD, caspase recruitment domain; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; HIDS, hyperimmunoglobulinemia D and periodic fever syndrome; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A. Back

Received for publication April 13, 2004. Accepted for publication July 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Simon and J. W. M. van der Meer
Pathogenesis of familial periodic fever syndromes or hereditary autoinflammatory syndromes
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R86 - R98.
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 J. Immunol.Home page
W. R. Coward, A. Marei, A. Yang, M. M. Vasa-Nicotera, and S. C. Chow
Statin-Induced Proinflammatory Response in Mitogen-Activated Peripheral Blood Mononuclear Cells through the Activation of Caspase-1 and IL-18 Secretion in Monocytes
J. Immunol., May 1, 2006; 176(9): 5284 - 5292.
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