3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin)-induced 28-kDa interleukin-1β interferes with mature IL-1β signaling

Abstract

Multiple clinical trials have shown that the 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors known as statins have anti-inflammatory effects. However, the underlying molecular mechanism remains unclear. The proinflammatory cytokine interleukin-1β (IL-1β) is synthesized as a non-active precursor. The 31-kDa pro-IL-1β is processed into the 17-kDa active form by caspase-1-activating inflammasomes. Here, we report a novel signaling pathway induced by statins, which leads to processing of pro-IL-1β into an intermediate 28-kDa form. This statin-induced IL-1β processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1β cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1β signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1β. These results may provide new clues to explain the anti-inflammatory effects of statins.

Keywords: Atherosclerosis; Caspase; Immunology; Inflammation; Innate Immunity; Interleukin.

FIGURE 1.

The mevalonate pathway and the action of statins. Statins inhibit the conversion of HMG-CoA to mevalonate by competitively inhibiting the rate-limiting enzyme HMG-CoA reductase. PP, pyrophosphate.

FIGURE 2.

Statin treatment induces IL-1β processing into 28-kDa form. BMDMs were primed with 200 ng/ml pure LPS for 2 h followed by stimulation with 10 μm simvastatin for an additional 6 h. Concentrations of IL-1β (A) and TNFα (B) in the culture supernatants were determined by ELISA (N.D., not detected). C, BMDMs were stimulated as described in A, IL-1β in supernatants as well as total cell lysate was determined by Western blot. GAPDH was used as a loading control. D, BMDMs were primed with 200 ng/ml LPS for 2 h followed by stimulation with simvastatin at 10 μm for different time periods as indicated in the figure. Culture supernatant were harvested, concentrated by methanol/chloroform precipitation, and probed with anti-IL-1β antibody. E, BMDMs were primed with 200 ng/ml LPS for 2 h and then stimulated with simvastatin or cerivastatin at concentrations indicated in the figure, and culture supernatants were concentrated with methanol/chloroform precipitation and subjected to Western blot with anti-IL-1β antibody. F, human PBMCs were primed with LPS for 2 h followed by stimulation with 10μM simvastatin for 6 h. Culture supernatants were concentrated and subject to Western blot with anti-human IL-1β antibody.

FIGURE 3.

Statin-induced IL-1β processing into 28-kDa form is independent of caspase-1-activating conventional inflammasomes. A, mouse BMDMs were primed or not with 200 ng/ml LPS followed by stimulation with simvastatin for 6 h or nigericin for 1 h. Concentrated culture supernatant and cell lysate were subjected to Western blot with antibodies against mouse IL-1β and mouse caspase-1. GAPDH was used as a loading control. B, caspase-1/caspase-11 double knock-out macrophages were primed with LPS and stimulated with simvastatin for an additional 6 h and then concentrated culture supernatant, and cell lysates were probed with anti-IL-1β, anti-caspase-1, and anti-caspase-11. C, BMDMs were stimulated as described in B, and IL-1β and Rantes (regulated on activation normal T cell expressed and secreted) in the culture supernatants were measured using ELISA.

FIGURE 4.

Statin-induced IL-1β processing depends on caspase activity. A, BMDMs were preincubated with 50 μm pan-caspase inhibitor Z-VAD for 30 min. Cells were then primed with 200 ng/ml LPS followed by stimulation with simvastatin for 6 h or nigericin for 1 h. Processing of IL-1β and caspase-1 were determined by Western blot. B, wild type and ASC^−/− BMDMs were primed with 200 ng/ml LPS followed by stimulation with 10 μm simvastatin for 6 h. IL-1β in the culture supernatants was determined by Western blot.

FIGURE 5.

Statin-induced IL-1β processing is independent of caspase-6 or -8. Wild type and caspase-6 (A) or caspase-8 (Casp-8)/Rip3 DKO and Rip3 KO BMDMs (B) were primed with 200 ng/ml LPS followed by stimulation with simvastatin for 6 h. IL-1β secretion and proteins in the cell lysate was determined by Western blot.

FIGURE 6.

Simvastatin-induced IL-1β processing is independent of its HMG-CoA reductase inhibitory effects. BMDMs were preincubated with mevalonate lactone at different final concentrations for 2 h followed by priming with 200 ng/ml LPS and 10 μm simvastatin stimulation for 6 h. Processing of IL-1β was determined by Western blot.

FIGURE 7.

Simvastatin-induced IL-1β is independent of Rac1 or Rac2. Quantitative RT-PCR of Rac1 and Rac2 gene expression in cDNAs prepared from wild type, Rac1^−/−, Rac2^−/−, or Rac1 and Rac2 DKO BMDMs (A). IL-1β (B) and TNFα (C) production upon LPS or LPS and simvastatin stimulation in wild type, Rac1^−/−, Rac2^−/−, or Rac1 and -2 DKO BMDMs. Western blot analysis of IL-1β processing from wild type or Rac1 and -2 DKO BMDMs after stimulation with LPS, simvastatinm, or LPS together with simvastatin. GAPDH is used as a loading control.

FIGURE 8.

The 28-kDa form of IL-1β interferes with mature IL-1β signaling. A, a schematic graph of pro-IL-1β, the putative 28-kDa IL-1β, and the mature, 17-kDa IL-1β. Asp-26 and Asp-116 are the two cleavage sites on pro-IL-1β by caspase-1. B, PVDF membrane showing the band corresponding to the 28-kDa IL-1β purified by affinity chromatography and partial amino acid sequence of the N terminus of pro-IL-1β with the cleavage site at Asp-26. C, Western blot of culture supernatant from 293T cells stably transfected with plasmids expressing pro-IL-1β, the 28-kDa intermediate IL-1β, and the mature IL-1β. D, TFF2-deficient BMDMs were stimulated with control culture supernatant or 293T cell supernatants containing pro-IL-1β, 28-kDa IL-1β, or mature, 17-kDa IL-1β for 24 h, production of IL-6 in the supernatants was determined by ELISA. E, TFF2-deficient BMDMs were stimulated with mature IL-1β in the presence or absence of different concentrations of recombinant 28-kDa IL-1β for 24 h. IL-6 production was determined by ELISA. N.S., not significant.