Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer’s disease in rodent models

Introduction

Since their characterization as COX inhibitors1, non-steroidal anti-inflammatory drugs (NSAIDs) have been used to treat a wide variety of diseases with relatively limited side effects2. NSAID inhibition of COX prevents the conversion of arachidonic acid to eicosanoids resulting in a reduction in the synthesis of proinflammatory prostaglandins3. Evidence indicates a polyvalent effect of NSAIDs with some research suggesting COX-independent activity4. One such important action may be to directly limit the production of proinflammatory cytokines.

Many inflammatory diseases are driven by the proinflammatory cytokine interleukin-1β (IL-1β)5. IL-1β is produced in myeloid cells as an inactive precursor (pro-IL-1β) that requires cleavage by the protease caspase-1 for its activation and secretion6. Caspase-1 is also produced as an inactive precursor, which is activated following recruitment to a large multi-protein complex called the inflammasome6. Inflammasomes are defined by the presence of a pattern recognition receptor. The best characterized inflammasome-forming pattern recognition receptor, and the most commonly associated with disease is NLRP3 (NLR family, pyrin domain containing 3). In response to pathogen-associated molecular patterns or damage-associated molecular patterns NLRP3 nucleates the oligomerization of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) molecules7,8 into large ‘specks’ that serve as platforms for caspase-1 activation and subsequent release of IL-1β.

The NLRP3 inflammasome is an important contributor to inflammatory diseases, including Alzheimer’s disease9, atherosclerosis10, metabolic diseases such as type 2 diabetes11 and others12. Hence, there is substantial interest in the discovery of potentially therapeutic inflammasome inhibitors. One such compound, MCC950, has been identified as a potent NLRP3-selective inhibitor13, but is not yet available for clinical use. Fenamate NSAIDs have been shown to inhibit IL-1β secretion from macrophages14, although the significance of COX inhibition remains unclear15. Here we show that the fenamate class of NSAIDs inhibit the NLRP3 inflammasome via reversible blockade of volume-regulated anion channels (VRAC) in the plasma membrane, and inhibit cognitive impairments in models of Alzheimer’s disease in rodents, thus offering a safe and rapidly translatable option to treat NLRP3-related inflammatory diseases.

Results

Fenamate NSAIDs selectively inhibit the NLRP3 inflammasome

Immortalized mouse bone marrow-derived macrophages (iBMDMs) were primed with lipopolysaccharide (LPS; 1 μg ml−1, 2 h), after which the media was replaced with serum-free media. At this point the cells were incubated with a range of NSAIDs (Supplementary Fig. 1) for 15 min before 1 h stimulation with 5 mM ATP to activate the P2X7 receptor and induce NLRP3 inflammasome activation16. ELISA analysis of cell supernatants revealed that of the NSAIDs tested the fenamates (N-phenyl-substituted anthranilic acid derivatives such as flufenamic acid, meclofenamic acid, mefenamic acid) were most effective at inhibiting IL-1β release (Fig. 1a). The selective COX-2 inhibitor celecoxib did not inhibit IL-1β release, nor did ibuprofen, even at concentrations supra-maximal for COX inhibition. Western blot analysis of the supernatants also showed that caspase-1-dependent processing of IL-1β was also inhibited by the fenamates (Supplementary Fig. 2). Fenamate NSAIDs had no effect on ATP-induced cell death (Supplementary Fig. 3) suggesting that their effects were specific to IL-1β release, and independent of the stability of ATP. As early studies indicated multiple sites of action for the fenamate NSAIDs17, these data reveal the fenamates as inhibitors of IL-1β processing and release and suggest that this effect is independent of COX inhibition.

Figure 1: Fenamate NSAIDs inhibit IL-1β processing and release.

(a) iBMDMs were primed with LPS (1 μg ml−1, 2 h) then pre-treated with NSAID at indicated concentration before stimulating with ATP (5 mM, 1 h). (b–d) Murine primary BMDMs from WT (b) or NLRP3−/− (c,d) mice were primed with LPS (1 μg ml−1, 4 h) and pre-treated with NSAID (100 μM, 15 min) before stimulating with monosodium urate (MSU) crystals (250 μg ml−1, 4 h) (b), transfected ultrapure flagellin from Salmonella typhimurium (1 ng per 1,000 cells, 2 h) (c), or transfected DNA (0.66 ng per 1,000 cells, 4 h) (d). Supernatants were analysed by ELISA. Data are presented as mean % IL-1β release versus vehicle (DMSO) control+s.e.m (n=3 or 4). NS, not significantly different, *P<0.05, **P<0.01 determined by one-sample t-test versus hypothetical value of 100%.