Statin synergizes with LPS to induce IL-1β release by THP-1 cells through activation of caspase-1

Abstract

Mevalonate kinase deficiency (MKD) is a hereditary syndrome characterized by recurring episodes of fever and inflammation. Peripheral blood mononuclear cells from MKD patients secrete high levels of interleukin (IL)-1β when stimulated with lipopolysaccharide (LPS), which is thought to be a primary cause of the inflammation. However, the link between a deficient mevalonate kinase and excessive IL-1β release remains unclear. To investigate this we made use of a model in which monocytic cells (THP-1) were treated with simvastatin. Statins are compounds that inhibit 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and thereby artificially impair the isoprenoid biosynthesis pathway, mimicking mevalonate kinase deficiency.

Our study revealed that LPS-stimulated THP-1 cells treated with simvastatin had an increased caspase-1 mediated processing of proIL-1β. This increased processing was caused by enhanced autoprocessing of caspase-1, rather than enhanced transcription or translation of caspase-1 or proIL-1β. Simvastatin-induced activation of caspase-1 was caused by an impairment of non-sterol isoprenoid biosynthesis, as the isoprenyl intermediate GGPP could block activation of caspase-1 and mIL-1β release. In addition, inhibition of both farnesyl pyrophosphate synthase and geranylgeranyltransferase I also induce mIL-1β release.

Taken together, these results demonstrate that simvastatin augments LPS-induced IL-1β release post-translationally, by inducing caspase-1 activity. These findings suggest that MKD patients may have overactive caspase-1, causing enhanced IL-1β processing and subsequent inflammation in response to bacterial components.

Introduction

The hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM#260920), is an autosomal recessive disorder, characterized by recurrent fever attacks and an elevated level of serum IgD (>100 IU/ml) (Drenth et al., 1994). The febrile attacks are accompanied by painful cervical lymphadenopathy and often by abdominal pain, vomiting and diarrhea. A variety of other symptoms including headache, skin rashes, mucosal ulcers, myalgia and arthralgia may also occur (Frenkel et al., 2001, Frenkel et al., 2000, Drenth et al., 1994). During the fever episodes an acute phase response is observed, with leukocytosis and elevated acute-phase reactants. Serum levels of proinflammatory cytokines, such as interleukin-6 (IL-6) and interferon-γ (IFN-γ), rise during fever attacks (Drenth et al., 1995a, Drenth et al., 1995b). Also, between attacks, isolated peripheral blood mononuclear cells (PBMC) from HIDS patients secrete large amounts of IL-1β (Drenth et al., 1996), which further increases during fever (Drenth et al., 1995b).

In 1999, the genetic defect of HIDS was identified: patients were shown to have mutations in the gene MVK, which codes for the enzyme mevalonate kinase (Houten et al., 1999, Drenth et al., 1999). Since this discovery, HIDS and its more severe allelic phenotype, mevalonic aciduria, are jointly referred to as mevalonate kinase deficiency (MKD). Mevalonate kinase is an important enzyme in the isoprenoid biosynthesis pathway (Houten et al., 2003). This pathway produces cholesterol and a number of non-sterol isoprenoids. The latter play a vital role in the prenylation of a variety of proteins, mostly of the Ras GTPase superfamily. Recently, it has become apparent that impairment of the isoprenoid pathway has widespread effects on immune function, both anti-inflammatory and pro-inflammatory (Kwak et al., 2000, Ikeda et al., 2000, Martinez-Gonzalez et al., 2001, Sadeghi et al., 2000, Greenwood et al., 2006, Montero et al., 2000). Several studies have shown that the secretion of IL-1β by activated PBMC was greatly augmented by the inhibition of isoprenoid biosynthesis using hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors, also known as statins. This increased cytokine release appeared to be specifically due to a lack of isoprenoids, since the addition of mevalonic acid, the product of HMG-CoA reductase, reduced cytokine production to control levels (Montero et al., 2004, Montero et al., 2000, Frenkel et al., 2002). In PBMC from patients suffering from mevalonate kinase deficiency it is the lack of isoprenoid products, specifically of geranylgeranylated proteins, that raises IL-1β production (Mandey et al., 2006). This IL-1β production may be largely responsible for the inflammation and fever observed in MKD patients. However, it is not known how impaired isoprenoid biosynthesis leads to increased IL-1β release.

Unlike most cytokines, IL-1β is synthesized as an inactive precursor (proIL-1β), lacking a conventional leader sequence. Instead of passing through the endoplasmic reticulum and the Golgi complex, proIL-1β is translated in the cytosol. There, the inactive proform requires processing by caspase-1, which cleaves proIL-1β directly after the aspartic acid residue at position 116 (Thornberry et al., 1992, Cerretti et al., 1992). In addition to IL-1β, caspase-1 can also cleave interleukin-18 (Ghayur et al., 1997, Gu et al., 1997) and recently, interleukin-33 was also identified as a caspase-1 substrate (Schmitz et al., 2005). Caspase-1 itself is synthesized as an inactive zymogen of ∼45 kDa that, via induced proximity to another caspase-1 zymogen, can undergo autocleavage, creating 10 kDa and 20 kDa subunits. Two p10 and two p20 subunits form the fully functional heterodimeric enzyme. In analogy to the apoptotic caspase-9 (Acehan et al., 2002), caspase-1 auto-activates itself in a complex of proteins termed the inflammasome (Martinon et al., 2002). Caspase-1 contains an N-terminal caspase recruitment domain (CARD), which forms a homotypic interaction (CARD-CARD interaction) with apoptosis-associated speck-like protein containing a CARD (ASC). This adaptor protein then binds to other members of the inflammasome (Srinivasula et al., 2002) via similar homotypic interactions, enabling oligomerization and autocleavage. Active caspase-1 can then process proIL-1β into mature IL-1β (mIL-1β), which is subsequently secreted. The exact export mechanism of mIL-1β remains unclear.

To investigate the regulation of increased mIL-1β production in mevalonate kinase deficiency, we studied the monocytic cell line THP-1 in which the isoprenoid biosynthesis pathway was artificially impaired using simvastatin. We examined the effect of this impairment on transcription and translation of (pro)caspase-1 and (pro)IL-1β and on caspase-1 enzyme activity. Our study revealed that simvastatin treatment induced an increase in caspase-1 mediated processing of proIL-1β by LPS-stimulated THP-1 cells. This increased processing was caused by enhanced autoprocessing of caspase-1, rather than enhanced transcription or translation of either caspase-1 or proIL-1β. The simvastatin-induced activation of caspase-1 was caused by an impairment of non-sterol isoprenoid biosynthesis, as the isoprenyl intermediate GGPP could completely block activation of caspase-1 and mIL-1β release and the inhibition of geranylgeranyltransferase I enhanced IL-1β release, similar to simvastatin.