Leptin is a cytokine-like hormone secreted by adipocytes, which serves to control energy expenditure and metabolism. In addition, leptin may modulate the innate and adaptive immune responses. The innate immune cell sensor nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is mainly expressed in myeloid immune cells, including macrophages. The NLRP3 inflammasome serves a pivotal role in the development and maintenance of autoimmunity and inflammation. The expression levels of caspase-1, apoptosis-associated speck-like protein containing a CARD, interleukin (IL)-18, IL-1β and leptin are significantly reduced in the white adipose tissue of nonsteroidal anti-inflammatory drug-activated gene-1 transgenic mice. However, the association between leptin and the NLRP3 inflammasome has not yet to be determined. The aim of the present study, was to explore the role of leptin on NLRP3 inflammasome. In order to do this, IL-1β and IL-18 expression levels were investigated in RAW 264.7 cells after incubation with leptin of increasing doses by Elisa or reverse transcription-quantitative polymerase chain reaction, and to assess whether IL-1β and IL-18 were affected after caspase-1 activity being inhibited by an inhibitor or by silencing NLRP3 expression. The results of the present study demonstrated that leptin enhanced the mRNA and protein expression levels of IL-18 in RAW 264.7 cells via activation of the NLRP3 inflammasome. This is achieved partly by enhancing the production of reactive oxygen species and K+ efflux. Therefore, leptin may be considered a novel activator and modulator of the NLRP3 inflammasome.
The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome can stimulate the innate or adaptive immune system in response to certain signals, including endogenous metabolites and microorganisms. It is understood that the underlying mechanism employed by the NLRP3 inflammasome involves the recruitment of apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. As a result of inflammasome activation, the proinflammatory cytokines interleukin (IL)-1β and IL-18 are released via pyroptosis and proteolytic cleavage (
Secreted by adipocytes, leptin is a cytokine-like hormone that has been reported to control energy expenditure and metabolism, and modulate the innate and adaptive immune responses. The activation of natural killer cells, chemotaxis of neutrophils, and secretion of tumor necrosis factor (TNF)-α, IL-6 and IL-12 from macrophages (
RAW 264.7 murine macrophage cells (The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose) supplemented with 10% fetal bovine serum, 100 ng/ml streptomycin and 100 U/ml penicillin (Gibco; Thermo Scientific, Inc., Waltham, MA, USA) at 37°C, 5% CO2 and humidity. Various doses of leptin (10, 100 and 500 ng/ml; PeproTech China, Suzhou, China) were added and the cells were cocultured for 24 h in the presence or absence of Ac-YVAD-cmk (18.4 µM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), KCl (100 µM) or diphenyleneiodonium chloride (DPI; 50 µM, Sigma-Aldrich; Merck KGaA). Lipopolysaccharide (LPS, 100 ng/ml, Sigma-Aldrich; Merck KGaA) and/or adenosine 5′triphosphate (ATP, 5 mM, Sigma-Aldrich; Merck KGaA) were cultured for 3 h respectively and used as positive controls. Cells were centrifuged at 400 × g, for 5 min, 4°C, and supernatants and sediments were collected and analyzed by ELISA or reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Caspase-1 activity was detected using a Fluorochrome-Labeled Inhibitor of caspase-1 kit: FAM-FLICA®Caspase assay kit (cat. no. 655) (ImmunoChemistry Technologies, LLC, Bloomington, MN, USA). ROS synthesis was investigated using a ROS detection assay kit, CFDA Cellular ROS Detection assay kit (cat. no. ab113851, Abcam, Shanghai, China). THBP was used as a positive control. All kits were performed according to the manufacturer's protocol. Results were analyzed using a FACSCanto FlowJo 7.6 (BD Biosciences, Franklin Lakes, CA, USA).
IL-1β and IL-18 expression levels in the supernatant of RAW 264.7 cells were measured using ELISA kits (Mouse IL-18 cat. no. BMS618, Mouse IL-1β cat. no. BMS6002) and were purchased from eBioscience; Thermo Fisher Scientific, Inc., according to the manufacturer's protocols.
Total cellular RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was synthesized from 500 ng total RNA in 10 ul volume using a Superscript kit (Invitrogen; Thermo Fisher Scientific, Inc.). The conditions used were 37°C for 15 min and 85°C for 5 sec. RT-qPCR reactions were performed using 1ul cDNA, 10 ul SYBR Green mater mix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), 2 ul of primer mix in a total volume of 20 ul. Thermocycling conditions were set up as follows: 5 min at 95°C, 40 cycles of denaturation (5 sec at 95°C), and combined annealing/extension (34 sec at 64°C). The housekeeping gene GAPDH was used as the internal standard. Analysis of relative gene expression data using the 2−ΔΔCq method (
RAW 264.7 cells were nucleofected with 20 µM NLRP3 specific small interfering (si)RNA (Shanghai Biotend, Shanghai, China) or negative control (NC) siRNA (Shanghai Biotend) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Nucleofected cells were incubated for 24 h at 37°C, 5% CO2 and humidity in the presence or absence of leptin, 6 h post-transfection. Knockdown of the NLRP3 gene was determined via RT-qPCR with the following primers: NLRP3 siRNA forward, 5′-GCAGGUUCUACUCUAUCAAdTdT-3′ and reverse, 5′-UUGAUAGAGUAGAACCUGCdTdT-3′. The NC siRNA sequences were as follows: NC siRNA forward, 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAAdTdT-3′.
A paired t-test was employed for two group analyses and Kruskal-Wallis one-way analysis of variance was used for analyses of >3 groups using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Results are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference and the experiments were repeated three times.
To study the effects of leptin on macrophages, leptin was applied at increasing doses to RAW 264.7 cells for 24 h, using LPS and/or ATP as positive controls. Supernatants or cell sediments were collected and analyzed by ELISA or RT-qPCR. The present study reported a leptin-induced increase in IL-1β mRNA expression levels only, whereas a dose-dependent increase was observed in IL-18 mRNA and protein expression levels (
Caspase-1 contributes to the NLRP3 inflammasome complex and regulates the synthesis and secretion of IL-18 by proteolytically digesting pro-IL-18 (
The NLRP3 inflammasome comprises NLRP3, and the adapter and effector proteins, ASC and caspase-1, respectively. The effects of leptin on capsase-1 activation prompted an investigation into the association between leptin and the NLRP3 inflammasome. The results demonstrated that leptin upregulated the mRNA expression levels of NLRP3 (
ROS have been reported to be key mediators in the activation of the NLRP3 inflammasome (
Previous studies have suggested that K+ efflux appears to be an important mediator in the activation of the NLRP3 inflammasome (
A previous study revealed that leptin induced the upregulation of phagocytic function-associated markers, and stimulated the secretion of proinflammatory cytokines, including TNF-α, IL-6 and IL-12 (
In the present study, leptin was reported to increase IL-1β gene expression; however, protein secretion remained unaffected within RAW 264.7 cells. Additionally, IL-1β was unaffected by caspase-1 inhibition. Martin
Reactive oxygen species and K+ efflux appear to be important mediators for the activation of the NLRP3 inflammasome. The present study revealed that leptin promotes IL-18 secretion by increasing ROS synthesis and K+ efflux, which may activate the NLRP3 inflammasome in RAW 264.7 cells. Numerous studies have indicated that ROS and K+ efflux may serve as strong activators for the NLRP3 inflammasome (
In conclusion, the findings of the present study revealed that leptin promotes IL-18 secretion by enhancing ROS synthesis and K+ efflux, which activates the NLRP3 inflammasome in RAW 264.7 cells. Leptin may therefore serve as a novel activator of the NLRP3 inflammasome through blocking leptin may be considered as a target for future antimicrobial and anti-inflammatory treatment.
The present study was supported by grants from the National Science Foundation for Young Scholars of China (grant no. 81401345) and the Young Physician Training Plan of North Huashan Hospital, Fudan University (grant no. 0000077).
Leptin promotes IL-18, but not IL-1β secretion, in RAW 264.7 cells. (A) IL-1β and (B) IL-18 ELISA of culture supernatants from RAW 264.7 cells incubated with LPS, ATP, L+A or leptin at increasing doses (10, 100 and 500 ng/ml) for 24 h. Results are presented as the mean ± standard error of the mean from four independent experiments. Relative mRNA expression levels of (C) IL-1β and (D) IL-18 in RAW 264.7 cells treated as aforementioned. Results are from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 vs. the control. ATP, adenosine 5′triphosphate; IL, interleukin; LPS, lipopolysaccharide; L+A, LPS and ATP.
Caspase-1 activation facilitates IL-18 secretion in response to leptin. (A) Caspase-1 activity was measured by Fluorochrome-Labeled Inhibitor of Caspases staining of RAW 264.7 cells incubated with L+A or leptin at increasing doses (10, 100 and 500 ng/ml) for 24 h. Cumulative data are from three experiments. (B) IL-1β and (C) IL-18 expression in the culture supernatants from RAW 264.7 cells following treatment with or without the caspase-1 inhibitor Ac-YVAD-cmk (20 µM) for 24 h, as determined by ELISA. Results are expressed as pg/ml and are presented as the mean ± standard error of the mean from four independent experiments. *P<0.05, **P<0.01 vs. the control group. ATP, adenosine 5′triphosphate; IL, interleukin; LPS, lipopolysaccharide; L+A, LPS and ATP.
NLRP3 upregulation promotes IL-18 expression in response to leptin. (A) Relative mRNA expression levels of NLRP3 in RAW 264.7 cells incubated with LPS, ATP, L+A or leptin at increasing doses (10,100 and 500 ng/ml) for 24 h. (B) Relative mRNA expression levels of NLRP3 in RAW 264.7 cells following nucleofection with NLRP3 specific siRNA or negative control siRNA. (C) IL-1β or (D) IL-18 expression in the culture supernatants from RAW 264.7 cells treated as aforementioned, as determined by ELISA. *P<0.05, **P<0.01, ***P<0. 001 vs. the control. ATP, adenosine 5′triphosphate; IL, interleukin; LPS, lipopolysaccharide; L+A, LPS and ATP; NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3; siRNA, small interfering RNA.
ROS synthesis and K+ efflux is involved in producing IL-18 in response to leptin. (A) Flow cytometric analysis of ROS in RAW 264.7 cells incubated with leptin at increasing doses (10, 100 and 500 ng/ml) for 24 h. THBP was used as a positive control. Representative data from three independent experiments are presented. (B) Cumulative data of ROS in RAW 264.7 cells from three experiments. (C) IL-18 ELISA of culture supernatants from RAW 264.7 cells incubated with leptin (500 ng/ml) for 24 h in the presence or absence of the ROS inhibitor DPI (50 µM). (D) IL-1β or (E) IL-18 expression in the culture supernatants from RAW 264.7 cells incubated with leptin at increasing doses (10, 100 and 500 ng/ml) for 24 h in the presence or absence of KCl (100 mM), as determined by ELISA. *P<0.05, **P<0.01, ***P<0.001 vs. the control. ATP, adenosine 5′triphosphate; DPI, diphenyleneiodonium chloride; IL, interleukin; LPS, lipopolysaccharide; L+A, LPS and ATP; ROS, reactive oxygen species.