Knockdown of MVK does not lead to changes in NALP3 expression or activation

Celsi et al. Journal of Inflammation Knockdown of MVK does not lead to changes in NALP3 expression or activation Fulvio Celsi 0 2 Elisa Piscianz 0 2 Maurizio Romano 1 Sergio Crovella 0 1 2 0 Institute for Maternal and Child Health - IRCCS Burlo Garofolo , via dell'Istria 65/1, 34137 Trieste , Italy 1 Department of Life Sciences, University of Trieste , Via A. Valerio 28, 34127 Trieste , Italy 2 Institute for Maternal and Child Health - IRCCS Burlo Garofolo , via dell'Istria 65/1, 34137 Trieste , Italy Background: Mutations in the Mevalonate Kinase gene (MVK) are causes of a rare autoinflammatory disease: Mevalonate Kinase Deficiency and its more acute manifestation, Mevalonic Aciduria. The latter is characterized, among other features, by neuroinflammation, developmental delay and ataxia, due to failed cerebellar development or neuronal death through chronic inflammation. Pathogenesis of neuroinflammation in Mevalonate Kinase Deficiency and Mevalonic Aciduria has not yet been completely clarified, however different research groups have been suggesting the inflammasome complex as the key factor in the disease development. A strategy to mimic this disease is blocking the mevalonate pathway, using HMG-CoA reductase inhibitors (Statins), while knock-out mice for Mevalonate Kinase are non-vital and their hemyzygous (i.e only one copy of gene preserved) littermate display almost no pathological features. Findings: We sought to generate a murine cellular model closely resembling the pathogenic conditions found in vivo, by direct silencing of Mevalonate Kinase gene. Knockdown of Mevalonate Kinase in a murine microglial cellular model (BV-2 cells) results in neither augmented NALP3 expression nor increase of apoptosis. On the contrary, statin treatment of BV-2 cells produces an increase both in Mevalonate Kinase and NALP3 expression. Conclusions: MKD deficiency could be due or affected by protein accumulation leading to NALP3 activation, opening novel questions about strategies to tackle this disease. Immunology; Inflammation; Autoimmunity; Mevalonate kinase deficiency - Introduction Mevalonate Kinase is a key enzyme in the cholesterol biosynthetic pathway and is responsible for converting mevalonic acid to mevalonate-5-phosphate, an early intermediate in sterol and isoprenoid synthesis. Mutations in the MVK gene (12q24) encoding Mevalonate Kinase enzyme (MVK) cause Mevalonate Kinase Deficiency (MKD, OMIM #260920) and its more severe form, Mevalonic Aciduria (MA, OMIM #610377). The latter is characterized by recurrent attacks of fever, developmental delay, ataxia, dysmorphic features, failure to thrive, cataracts, and retinal dystrophy. The prognosis is poor: few patients survive until adolescence and more than 50% of them succumb to an inflammatory crisis during the first years of life [1]. One of the characteristics of this disease is cerebellar atrophy, due to neuronal apoptosis [2]. Although the genetic defects are well known and characterized, the pathogenic mechanism remains elusive, and treatment of the disease remains mainly supportive, with poor efficacy. For an evidence-based therapy, it is crucial to determine the exact pathogenic mechanisms occurring in the disease. Animals or cell-based models are thus necessary; in the past a mouse model of MVK deficiency was generated [3] however it was unable to correctly represent the pathology. Then a cellular model have been developed, where the mevalonate pathway is pharmacologically targeted using HMG-CoA blockers (statins), mimicking some of the inflammatory features observed in cells from patients [4]. Using this strategy, our group and other authors have found that, in different cell lines, NALP3 inflammasome activation is responsible for initiating inflammation [4-6]. This multi-molecular complex, constituted by NALP3, ASC and Caspase-1, cleaves proIL-1 to release its active form, starting inflammation [7]. Accordingly, NALP3 activation could represent a possible starting point for MA pathogenic progression. A number of issues need to be clarified, such as how the inflammasome pathway is incorrectly activated in cells presenting the pathological mutations. In the attempt to better mimic MA pathology we developed a novel murine cell model in which MVK expression is specifically down-regulated, using the silencing RNA (siRNA) technology. We then compared the results obtained by siRNA transfection with those achieved with Lovastatin and LPS treatment, in order to determine the better model suitable for mimicking the pathologic condition. Materials and methods BV-2 cell culture, transfection and treatment During febrile attacks, MVK deficient patients experience an acute phase response showing increased plasma levels of IL-1, IL-6 and TNF-, and elevated urinary leukotriene E4 and neopterin excretion [8,9]. These observations have suggested that activation of macrophages might play a crucial pathogenic role, during as well as between attacks [10]. Evidences suggest that also the nervous system is involved in the most severe forms of MKD and that microglia might be activated consequently triggering neuroinflammation [11]. For these reasons, we used the murine microglial BV-2 cell line for the MVK RNAi experiments. The microglial cell line BV-2 was maintained in RPMI medium (Euroclone) supplemented with 10% FBS, Pen/Strep. All experiments were conducted in passages between 2 and 8-10 to avoid effects due to cellular senescence. siRNA transfection was performed using Lipofectamine 2000 (LifeTechnologies) following manufacturers instructions. siRNA was obtained from Origene, (cat#SR413228), three different siRNA duplexes named 8a,8b,8c were used. Only 8b and 8c were able to produce significant reduction in MVK expression. Lovastatin was dissolved in 99% Ethanol at a concentration of 10mM and diluted to working concentration (50 M) in cellular media. LPS (Sigma) from E. Coli 055:B5 has been dissolved in water in a stock concentration of 1 mg/ml and diluted to working concentration of 1 g/ml. Lovastatin was applied for 24h and at the final 4h LPS was added in combined experiments Lova + LPS. Real-time PCR Total cellular RNA was extracted using TRI-reagent (Sigma) following manufacturers instructions and retrotranscribed using High-Capacity cDNA Reverse transcription kit (Applied Biosystems). To quantify mRNA, Taq-man probes for MVK, NALP3 and -Actin (as calibrator), were used (Applied Biosystems). Raw fluorescent data were converted to fold-increase using the software LinRegPCR [12] corrected for the analysis of hydrolysis probe [13], using the Ct method. In these calculations, -Actin was used as internal standard and scrambled samples used as reference. Western blot Cells were lysed in Lysis Buffer (LB: 300mM NaCl, 1%NP40, 50mM Tris-HCl pH 7.4, 1mM EDTA) for 15 minutes in ice. Proteins were then quantified, using Bio-Rad Bradford protein assay (Bio-Rad), loaded onto a 4-20% gradient gel (Bio-rad) and electrophoretica (...truncated)