Fluoxetine Inhibits NLRP3 Inflammasome Activation: Implication in Depression

Abstract

Background: Emerging evidence indicates that NLRP3 inflammasome-induced inflammation plays a crucial role in the pathogenesis of depression. Thus, inhibition of NLRP3 inflammasome activation may offer a therapeutic benefit in the treatment of depression. Fluoxetine, a widely used antidepressant, has been shown to have potential antiinflammatory activity, but the underlying mechanisms remain obscure.

Methods: We used a chronic mild stress model and cultured primary macrophage/microglia to investigate the effects of fluoxetine on NLRP3 inflammasome and its underlying mechanisms.

Results: We demonstrated that fluoxetine significantly suppressed NLRP3 inflammasome activation, subsequent caspase-1 cleavage, and interleukin-1β secretion in both peripheral macrophages and central microglia. We further found that fluoxetine reduced reactive oxygen species production, attenuated the phosphorylation of double-stranded RNA-dependent protein kinase, and inhibited the association of protein kinase with NLRP3. These data indicate that fluoxetine inhibits the activation of NLRP3 inflammasome via downregulating reactive oxygen species-protein kinase-NLRP3 signaling pathway. Correspondingly, in vivo data showed that fluoxetine also suppressed NLRP3 inflammasome activation in hippocampus and macrophages of chronic mild stress mice and alleviated chronic mild stress-induced depression-like behavior.

Conclusions: Our findings reveal that fluoxetine confers an antidepressant effect partly through inhibition of peripheral and central NLRP3 inflammasome activation and suggest the potential clinical use of fluoxetine in NLRP3 inflammasome-driven inflammatory diseases such as depression.

Keywords: NLRP3 inflammasome; depression; double-stranded RNA-dependent protein kinase; fluoxetine; reactive oxygen species.

Figure 1.

Fluoxetine inhibited NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDMs) and RAW264.7 macrophages. BMDMs and RAW264.7 macrophages were incubated with indicated concentrations of fluoxetine (Flx, 0.1 μM, 1 μM, 10 μM) for 1 hour, then stimulated with LPS (100ng/mL) followed by 30 minutes of ATP (5mM) treatment. (A) The cytotoxicity of fluoxetine (Flx, 0.1 μM, 1 μM, 10 μM, 50 μM, 100 μM) was measured by 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) assay (^*** P<.001 vs Flx 0 μM). There were no significant differences among Flx 0 μM, Flx 0.1 μM, Flx 1 μM, and Flx 10 μM groups. (B) Immunoblot analysis of interleukin (IL)-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of BMDMs and immunoblot analysis of the NLRP3, proIL-1β, and procaspase-1 in lysates of those cells (input). (C) ELISA of IL-1β in supernatants of BMDMs. (D-E) Quantitative analysis of NLRP3 (D) and proIL-1β (E) in BMDMs. (F) ELISA of IL-1β secretion in supernatants of RAW264.7 macrophages. (G-I) Representative immunoblots (G) and quantitative analysis of NLRP3 (H) and caspase-1 (I) in RAW264.7 macrophages. Data are represented as means ± SEM from 2 replicates in 3 independent experiments. ^* P<.05, ^** P<.01, ^*** P<.001 vs LPS+ATP; ^# P<.05, ^## P<.01, ^### P<.001 vs LPS+ATP+Flx 0.1 μM. There were no significant differences between -LPS-ATP and -LPS-ATP + Flx 10 μM.

Figure 2.

Fluoxetine suppressed NLRP3 inflammasome activation in microglia. Microglia were incubated with indicated concentrations of fluoxetine (Flx, 0.1 μM, 1 μM, 10 μM) for 1 hour, then stimulated with LPS (100ng/mL) followed by 30 minutes of ATP (5mM) treatment. (A) Immunoblot analysis of interleukin (IL)-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of microglia and immunoblot analysis of the NLRP3, proIL-1β, and procaspase-1 in lysates of those cells (input). (B) ELISA of IL-1β in supernatants of microglia. (C-D) Quantitative analysis of NLRP3 (C) and proIL-1β (D) in microglia. Data are represented as means ± SEM from 2 replicates in 3 independent experiments. ^* P<.05, ^** P<.01, ^*** P<.001 vs LPS+ATP; ^# P<.05, ^## P<.01 vs LPS+ATP+Flx 0.1 μM. There were no significant differences between -LPS-ATP and -LPS-ATP + Flx 10 μM.

Figure 3.

Fluoxetine reduced ATP-induced ROS production, RNA-dependent protein kinase (PKR) phosphorylation and the association of PKR with NLRP3 in macrophages. Bone marrow-derived macrophages (BMDMs) were pretreated with fluoxetine (Flx, 10 μM) for 1 hour, then stimulation with LPS+ATP. (A) Cells were stained with H₂DCF-DA labeling and images captured with a fluorescence microscope. (B) The quantitative analysis of ROS levels by a Titertek Fluoroskan II microtiter fluorescence plate reader. (C) LPS-primed BMDMs were stimulated with ATP at indicated time points and phosphorylated and total PKR were determined by Western-blot analysis (^** P<.01, ^*** P<.001 vs LPS+ATP 30min). (D-E) BMDMs were pretreated with fluoxetine (Flx, 10 μM) for 1h, followed by stimulation with LPS+ATP for 30 minutes. (D) Representative immunoblots and quantitative analysis of phosphorylated and total PKR. (E) Immunoprecipitation and immunoblot analysis of the PKR-NLRP3 interaction. Data are represented as means ± SEM from 2 replicates in 3 independent experiments. ^** P<.01, ^*** P<.001 vs LPS+ATP. There were no significant differences among -LPS-ATP, -LPS-ATP+Flx, and +LPS+ATP+Flx.

Figure 4.

Fluoxetine inhibited chronic mild stress (CMS)-induced NLRP3 inflammasome activation via suppression of the association RNA-dependent protein kinase (PKR) with NLRP3 in hippocampus of mice. (A) Sucrose preference (n=18/groups; ^* P<.05, ^# P<.05 vs CMS). (B) Tail suspension test (n=18/groups). (C) Forced swim test (n=18/groups). Representative immunoblots (D) and quantitative analysis of NLRP3 (E), caspase-1 (F) and IL-1β (G) in hippocampus of CMS mice (n=4/groups). (H) Representative immunoblots and quantitative analysis of phosphorylated and total PKR (n=4/groups). (I) Immunoprecipitation and immunoblot analysis of the PKR-NLRP3 interaction (n=4/groups). Data are represented as means ± SEM. ^* P<.05, ^** P<.01 vs CMS.

Figure 5.

Fluoxetine inhibited NLRP3 inflammasome activation in bone marrow-derived macrophages (BMDMs) of chronic mild stress (CMS) mice. (A) Serum interleukin (IL)-1β levels by ELISA (n=18/groups). Representative immunoblots (B) and quantitative analysis of NLRP3 (C), caspase-1 (D), and IL-1β (E) in BMDMs of CMS mice (n=4/groups). Data are represented as means ± SEM. ^** P<.01, ^*** P<.001 vs CMS.

Figure 6.

Schematic layout of showing fluoxetine inhibits NLRP3 inflammasome activation via ROS- RNA-dependent protein kinase-NLRP3 signaling pathway in depression. Fluoxetine decreases the level of ROS, which attenuates PKR phosphorylation and the interaction of PKR with NLRP3, and subsequently inhibits the NLRP3 inflammasome activation.