The role of annexin A1 in the modulation of the NLRP3 inflammasome

Abstract

Annexins are well-known Ca²⁺ phospholipid-binding proteins, which have a wide variety of cellular functions. The role of annexin A1 (AnxA1) in the innate immune system has focused mainly on the anti-inflammatory and proresolving properties through its binding to the formyl-peptide receptor 2 (FPR2)/ALX receptor. However, studies suggesting an intracellular role of AnxA1 are emerging. In this study, we aimed to understand the role of AnxA1 for interleukin (IL)-1β release in response to activators of the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome. Using AnxA1 knockout mice, we observed that AnxA1 is required for IL-1β release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL-1β, NLRP3 and caspase-1, a step called NLRP3 priming. Moreover, we demonstrate that AnxA1 co-localize and directly bind to NLRP3, suggesting the role of AnxA1 in inflammasome activation is independent of its anti-inflammatory role via FPR2. Therefore, AnxA1 regulates NLRP3 inflammasome priming and activation in a FPR2-independent manner.

Keywords: IL-1β; NLRP3; annexin A1; inflammasome.

Figure 1

Annexin A1 (AnxA1) is necessary for the production of interleukin (IL)‐1β in vivo. Wild‐type (WT) and AnxA1^−/− mice were injected with 100 μg/knee of uric acid crystals (MSU) in the tibiofemoral joint and, 12 hr after injection, peri‐articular tissue was collected to measure IL‐1β levels (a). WT and AnxA1^−/− mice were instilled with 10 mg/40 μl of silica and, 24 hr after instillation, lungs were collected to measure IL‐1β levels (b). Bars show the means ± SEM of 5 mice/group and are from one experiment representative of two independent experiments. Significance was calculated using anova followed by Holm‐Sidak’s multiple comparison test.

Figure 2

Effects of annexin A1 (AnxA1) on interleukin (IL)‐1β release seem to be specific for activators of the NLRP3 inflammasome. Bone marrow‐derived macrophages (BMDMs) from wild‐type (WT) and AnxA1^−/− mice were stimulated with lipopolysaccharide (LPS; 1 μg/ml) for 1 hr followed by the indicated stimulus: 6 hr of uric acid crystals (MSU – 300 μg/ml) (a), 30 min of adenosine 5′‐triphosphate disodium salt (ATP – 5 mm) (A), 6 hr of Silica (SIL – 250 μg/ml) (b); 4 hr of Legionella pneumophila bacteria (Leg ‐ strain JR32 – MOI of 10) (c) and 2 hr of Poly(dA:dT) (p(dA:dT) – 5 μg/ml) (d). Supernatants were collected and IL‐1β levels were measured. Bars show the means ± SEM of 4 replicates/group and are from one experiment representative of three independent experiments. Significance was calculated using anova followed by Holm‐Sidak’s multiple comparison test.

Figure 3

Effect of formyl‐peptide receptor 2 (FPR2) and Ac2‐26 on interleukin (IL)‐1β release. Wild‐type (WT) and FPR2/3^−/− mice were injected with 100 μg/knee of uric acid crystals (MSU) in the tibiofemoral joint and, 12 hr after injection, peri‐articular tissue was collected to measure IL‐1β levels (a). Bone marrow‐derived macrophages (BMDMs) from WT and FPR2/3^−/− mice were stimulated with lipopolysaccharide (LPS; 1 μg/ml) for 1 hr followed by the indicated stimulus: 6 hr of uric acid crystals (MSU – 300 μg/ml), 30 min of adenosine 5′‐triphosphate disodium salt (ATP – 5 mm). Supernatants were collected and IL‐1β levels were measured (b). BMDMs from WT mice were treated with the AnxA1 peptidomimetic Ac2‐26 (32 μm) and were stimulated with LPS (1 μg/ml) for 1 hr followed by stimulation with uric acid crystals (MSU – 300 μg/ml for 6 hr) or adenosine 5′‐triphosphate disodium salt (ATP − 5 mm for 30 min). Supernatants were collected to measure IL‐1β level (c). Bars show the means ± SEM of 5 mice/group (in vivo), and 4 replicates/group (in vitro) and are from one experiment representative of three independent experiments. Significance was calculated using anova followed by Holm‐Sidak’s multiple comparison test.

Figure 4

Annexin A1 (AnxA1) is involved in NLRP3 priming. Bone marrow‐derived macrophages (BMDMs) from wild‐type (WT) and AnxA1^−/− mice were stimulated with lipopolysaccharide (LPS; 1 μg/ml) for 1 hr, and cells were collected to RNA extraction and quantitative polymerase chain reaction (qPCR) analysis for Il‐1β (a), Nlrp3 (b), Asc (c) and Caspase 1 (d) normalized by GAPDH. BMDMs from WT and AnxA1^−/− mice were stimulated with LPS (1 μg/ml) for 1 hr and then stimulated with uric acid crystals (MSU – 300 μg/ml – 6 hr) and adenosine 5′‐triphosphate disodium salt (ATP – 5 mm – 30 min). Cells were collected for protein extraction and Western blot analysis for pro‐IL‐1β, NLRP3, ASC and pro‐caspase 1 (e). Supernatant were collected to analyse cleaved‐caspase 1 expression by Western blot (e). For loading control, membranes were re‐probed with anti‐β‐actin. Densitometry was performed, normalized by β‐actin and values (arbitrary units) are represented below respective lines. Bars show the means ± SEM of 4 replicates/group and are from one experiment representative of three independent experiments. Significance was calculated using anova followed by Holm‐Sidak’s multiple comparison test.

Figure 5

Annexin A1 (AnxA1) co‐localizes with NLRP3. Bone marrow‐derived macrophages (BMDMs) from wild‐type (WT) and AnxA1^−/− mice were stimulated with lipopolysaccharide (LPS; 1 μg/ml) for 1 hr and then stimulated with uric acid crystals (MSU – 300 μg/ml ‐ 4 hr). Cells were collected and cytocentrifuged in a coverslip. After fixation and permeabilization, cells were stained with rabbit anti‐AnxA1 (Santa Cruz) and mouse anti‐NLRP3 (Adipogen) overnight. Then, secondary anti‐rabbit IgG Alexa Fluor 647, and anti‐mouse IgG FITC were incubated for 1 hr, and DAPI (1 : 1000 – BD) was used to stain nucleus. Co‐localization of AnxA1 (purple) and NLRP3 (green) were visualized using confocal microscopy. Scale bar: 100 μm. Figure is representative of at least 500 cells/glass slides analysed.

Figure 6

Annexin A1 (AnxA1) interacts directly with NLRP3. Bone marrow‐derived macrophages (BMDMs) from wild‐type (WT) mice were stimulated with lipopolysaccharide (LPS; 1 μg/ml) for 1 hr and then stimulated with uric acid crystals (MSU – 300 μg/ml ‐ 4 hr). Cells were collected to protein extraction. 1 mg of protein was incubated with anti‐AnxA1 (Santa Cruz) and then incubated with protein G Sepharose 4 fast flow beads (GE Healthcare), and Western blot analyses were performed. Representative immunoblot shows AnxA1 co‐immunoprecipitates NLRP3, and also shows AnxA1 and NLRP3 expression in the cell lysate (input). Immunoprecipitation control (p‐MYPT) is represented as CT‐. The immunoblots are representative of two independent experiments.