Nickel induces inflammatory activation via NF-κB, MAPKs, IRF3 and NLRP3 inflammasome signaling pathways in macrophages

NiCl₂ induces cytotoxicity in BMDMs

Our previous work has shown that NiCl₂ can inhibit the immune response in chicken. To evaluate the cytotoxicity of NiCl₂, BMDMs were exposed to various doses of NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24 h. It was found that NiCl₂ suppressed BMDMs viability in a dose-dependent manner (Figure 1A and 1B). Notably, the viabilities of BMDMs were significantly (p < 0.01) decreased in 0.5 and 1.0 mM NiCl₂ exposure groups compared to control group.

Figure 1. Cytotoxicity of NiCl₂ in BMDMs. (A) BMDMs are treated with NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24h, and changes of cell numbers were observed by microscopy. Scale bar 50 μm. (B) Cell viability is analyzed by MTT assay. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

NiCl₂ activates NF-κB, MAPKs and IRF3 pathways in BMDMs

To investigate the inflammatory potential of NiCl₂, NF-κB pathway was tested. The activation of NF-κB transcription factor may trigger an inflammatory response [15]. NF-κB proteins are bound and inhibited by IκBα proteins. NiCl₂ treatment increased the phosphorylation levels of IκBα protein expression, and decreased the total IκBα protein expression levels (Figure 2A and 2B). Phosphorylation of IκBα caused its ubiquitination and proteasomal degradation, freeing NF-κB complexes. In Figure 2C and 2E, the results showed that NF-κB was translocated to the nucleus, and thus inducing target gene expression.

Figure 2. NiCl₂ activates NF-κB pathway in BMDMs. (A and B) BMDMs are treated with NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24h, and immunoblotted for the whole cell lysis IκBα and p-IκBα protein expression. (C and D) cells are treated with NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24h, and immunoblotted for the cytoplasm and nuclear cell lysis NF-κB p65 protein expression. (E) The translocation of NF-κB p65 protein determined by NF-κB p65 staining of NiCl₂-primed BMDMs. Scale bar 50 μm. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

ERK1/2, Jun amino-terminal kinases (JNK) and p38 play crucial roles in cell growth and differentiation as well as inflammatory response and cellular stress [28]. In Figure 3A and 3B, our results showed that the protein expression levels of p-ERK, p-JNK and p-p38 were remarkably (p < 0.01) increased in 0.5 and 1.0 mM NiCl₂ exposure groups.

Figure 3. NiCl₂ activates MAPKs and IRF3 pathway in BMDMs. (A and B) BMDMs are treated with NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24h, and immunoblotted for the whole cell lysis p-p38, p38, p-JNK, JNK, p-ERK and ERK protein expression. (C and D) BMDMs are treated with NiCl₂ (0, 0.1, 0.5 and 1.0 mM) for 24h, and immunoblotted for the p-IRF3 and IRF3 protein expression. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

Interferon regulatory factor 3 (IRF3) can suppress cell proliferation and regulate the expression levels of genes related to innate immunity [29]. As shown in Figure 3C and 3D, NiCl₂ exposure markedly (p < 0.01) increased the protein expression levels of p-IRF3.

NiCl₂ induces pro-inflammatory cytokine production in BMDMs

The above results showed that NiCl₂ activated NF-κB, MAPKs and IRF3 pathways, by upregulating the expression levels of pro-inflammatory cytokines. In Figure 4A, the mRNA expression levels of IL-1β, -6, -8, -18, TNF-α and IFN-β were remarkably (p < 0.01) increased in 0.5 and 1.0 mM NiCl₂ exposure groups compared to those in control group. The ELISA data revealed that NiCl₂ treatment markedly (p < 0.01) increased the production of IL-1β, -6, -8, -18, TNF-α and IFN-β (Figure 4B).

Figure 4. NiCl₂ induces pro-inflammatory cytokine production in BMDMs. (A) The mRNA expression levels of inflammatory cytokines after NiCl₂ (0, 0.1, 0.5 and 1.0 mM) treatment for 24h. (B) Inflammatory cytokine expression is quantified in BMDMs cultural medium by ELISA after NiCl₂ (0, 0.1, 0.5 and 1.0 mM) treatment for 24h. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

NiCl₂ activates NLRP3 inflammasome pathway in BMDMs

NiCl₂ activated caspase-1-mediated inflammatory cytokine production in BMDMs. Inflammasomes are multi-protein signaling complexes that promote inflammatory caspases activation and IL-1β maturation. We tested whether NLRP3-ASC-caspase-1 inflammasome is activated after NiCl₂ treatment. It was found that NiCl₂ induced the cleavage of IL-1β and caspase-1 (Figure 5E).

Figure 5. NiCl₂ activates NLRP3 inflammasome pathway in BMDMs. (A and B) Relative mtROS amounts determined by MitoSOX-red staining of NiCl₂-primed BMDMs. Scale bar 50 μm. (C) Relative cytosolic mtDNA expression in NiCl₂-primed BMDMs. (D) NiCl₂-induced changes in mitochondrial membrane potential (Ψm) in BMDMs measured by TMRM fluorescence. (E) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of NiCl₂-treated BMDMs, and cleaved-caspase-1and cleaved- IL-1β in the supernatant. (F) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3 and ASC in lysates of Mito-TEMPO (500 μM)-pre-treated 1h before 24h of NiCl₂ stimulation. (G) Relative cytosolic mtDNA expression in NiCl₂-treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. (H) Changes of mitochondrial membrane potential (Ψm) in NiCl₂-treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) pre-treatment. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

The mechanism underlying NiCl₂-induced caspase-1 activation was explored in this study. The results showed that NiCl₂ induced mitochondria damage such as increase of mitochondrial ROS (mtROS) production (Figure 5A and 5B), elevation of mitochondrial DNA (mtDNA) release (Figure 5C) and decrease of mitochondrial membrane potential (Figure 5D). We further assessed whether mitochondria-specific ROS production is associated with NiCl₂-induced activation of caspase-1. Mito-TEMPO, a mitochondria-specific ROS scavenger, suppressed the formation of cleaved-IL-1β and cleaved-caspase-1 in BMDMs in response to NiCl₂ (Figure 5F). Additionally, Mito-TEMPO attenuated the elevation of mtDNA release and loss of mitochondrial membrane potential (Figure 5G and 5H). These data demonstrate that NiCl₂-induced caspase-1 activation largely relies on the mitochondrial generation of ROS.

NiCl₂ induces apoptosis in BMDMs

In Figure 6A and 6B, we observed that the protein expression levels of cleaved-caspase-3, -8, -9 and cleaved-poly (ADP-ribose) polymerase (PARP) were significantly (p < 0.01) increased in 0.5 and 1.0 mM NiCl₂ exposure groups (Figure 6A and 6B).

Figure 6. NiCl₂ induces apoptosis in BMDMs. (A and B) Immunoblot analysis of cleaved-caspase-8, cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP in lysates of the NiCl₂-treated BMDMs. (C) Immunoblot analysis of pro-caspase-1, cleaved-caspase-1, pro-IL-1β, cleaved- IL-1β, NLRP3, ASC and cleaved-caspase-3 in the NiCl₂-treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) or Z-YVAD-FMK (100 μM, 1h) pre-treatment, and cleaved-caspase-1and cleaved- IL-1β in the supernatant. (D) Flow cytometry analysis apoptosis in the NiCl₂-treated (24h) BMDMs in the presence/absence of Mito-TEMPO (500 μM, 1h) or Z-YVAD-FMK (100 μM, 1h) pre-treatment. Data are presented with the means ± standard deviation (n=5). *p < 0.05 and **p < 0.01, compared with the control group.

MtROS generation diminishes mitochondrial membrane potential, and subsequently induces mitochondria-mediated apoptosis. Our above-mentioned results showed that NiCl₂ treatment resulted in excessive mitochondrial ROS production and decreased mitochondrial membrane potential. Next, we examined whether mitochondrial ROS and caspase-1 activation are involved in NiCl₂-induced apoptosis.

Cotreatment with NiCl₂ and Mito-TEMPO suppressed the cleavage of caspase-1, -3 and IL-1β (Figure 6C). Cotreatment with NiCl₂ and Z-YVAD-FMK, a potent inhibitor of caspase-1, suppressed the cleavage of IL-1β and caspase-1 (Figure 6C). However, the caspase-1 inhibitor only partly inhibited the cleaved-caspase-3 protein expression (Figure 6C). The percentage of apoptosis was significantly (p < 0.01) higher in NiCl₂ treatment groups and NiCl₂+Z-YVAD-FMK treatment group than that in control group (Figure 6D).

Taken altogether, these findings indicate that NiCl₂-induced apoptosis is dependent on mitochondrial ROS production, and caspase-1 activation may also partly contribute to the apoptotic process.

Materials and reagents

NiCl₂ (451193) was purchased from Sigma Aldrich Corporation. The mitochondria-targeted antioxidant Mito-TEMPO (CAS 1569257-94-8) was bought from Santa Cruz Biotechnology. Tetramethylrhodamine, methyl ester (TMRM) was obtained from AnaSpec Inc. (#CA94555). Z-YVAD-FMK (ALX-260-154-R100) was purchased from Enzo Life Sciences. MitoSOX (M36008) was obtained from Invitrogen.

The antibodies such as mouse anti-IκBα (#4814, 1:1000 for WB), rabbit anti-phospho (p)-IκBα (#2859, 1:1000 for WB), rabbit anti-NF-κB p65 (#8242, 1:1000 for WB, 1:200 for IF), mouse anti-β-Actin (#3700, 1:1000 for WB), rabbit anti-p38 (#8690, 1:1000 for WB), rabbit anti-p-p38 (#4511, 1:1000 for WB), rabbit anti-JNK (#9252, 1:1000 for WB), rabbit anti-p-JNK (#4668, 1:1000 for WB), rabbit anti-ERK (#4695, 1:1000 for WB), rabbit anti-p-ERK (#9101, 1:1000 for WB), rabbit anti-IRF3 (#11904, 1:1000 for WB), rabbit anti-p-IRF3 (#83611, 1:1000 for WB), rabbit anti-NLRP3 (#15101, 1:1000 for WB), rabbit anti-IL-1β (#12426S, 1:1000 for WB), rabbit anti-cleaved-caspase-3 (#9664, 1:1000 for WB), rabbit anti-cleaved-caspase-8 (#8592, 1:1000 for WB), rabbit anti-cleaved-caspase-9 (#9509, 1:1000 for WB), rabbit anti-PARP (#9548, 1:1000 for WB) were supplied by Cell Signaling Technology. Mouse anti-Histone H3 (sc-517576, 1:1000 for WB) and mouse anti-Tubulin (sc-73242, 1:1000 for WB) were obtained from Santa Cruz Biotechnology, while mouse anti-caspase-1 p20 (#AG-20B-0042-C100, 1:1000 for WB) and mouse anti-ASC (#AG-25B-0006-C100, 1:1000 for WB) were purchased from Adipogen.

IL-1β (MLB00C), IL-6 (M6000B), IL-18 (7625), TNF-α (MTA00B) and IFN-β (42400-1) ELISA kits were purchased from R&D system, while IL-8 (MBS261967) ELISA kit was obtained from MyBioSource.

Cell isolation and culture

Preparation of primary BMDM was carried out as proposed by Nakahira et al. [35]. Briefly, BMDMs were collected from the femur and tibia of each mouse, and seeded on a sterile petri dish. The BMDMs were cultured for 7 days in DMEM supplemented with 10% heat-inactivated fetal bovine serum, penicillin, streptomycin and 25% conditioned medium from L929 mouse fibroblasts. Upon reaching confluency, the BMDMs were exposed to different concentrations (0, 0.1, 0.5 and 1.0 mM) of NiCl₂ for 24 h. In the cotreatment with NiCl₂ and Mito-TEMPO experiment, the Mito-TEMPO (500 μM) or Z-YVAD-FMK (100 μM) were treated 1h before 24h of NiCl₂ treatment. In the cotreatment with NiCl₂ and Z-YVAD-FMK experiment, the Z-YVAD-FMK (100 μM) were treated 1h before 24h of NiCl₂ treatment.

Cytotoxicity assessment

The viability of BMDMs was assessed using MTT assay. Briefly, BMDMs (1 × 10⁶ cells/ml) were suspended in complete culture media, and 150 μL of the cell suspension was cultured 48-well plates for 24 h. After treatment with NiCl₂, 0.5 mg/ml MTT solution was added onto the BMDMs, followed by a 4 h incubation period. The obtained formazan crystals were dissolved in dimethyl sulfoxide, and the absorbance was recorded at 540 nm using a microplate reader (PerkinElmer). Data were the mean ± SD of 5 independent experiments conducted in triplicate.

Western blotting

After NiCl₂ treatment, cell lysis was performed with ice-cold RIPA buffer. After centrifuging at 15000 g for 15 min at 4 °C, the protein lysates were separated by 12% SDS-PAGE and then transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% non-fat milk in TBST, and then incubated with primary antibodies, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescent (ECL) detection (GE Healthcare, Piscataway, NJ, USA). The protein bands were visualized using Bio-Rad ChemiDoc XRS+ System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The statistical differences in protein expression levels were computed by an ImageJ2x software.

The supernatant protein is extracted by the method of trichloroacetic acid (TCA) precipitation of proteins according to the reference [38], followed by the detection of protein content using BCA protein assay. The expression levels of cleaved-caspase-1 and IL-1β are detected by western blotting.

Determination of NF-κB p65 protein expression

After treatment with NiCl₂ for 24 h, BMDMs were collected and total protein was isolated from cytoplasm and nucleus by using Cytoplasmic and Nuclear Protein Extraction Kit (SINP001 Viagene Biotech). The protein expression was determined by Western blotting.

BMDMs were seeded and allowed to be adhered on a coverslip for 24 h. After exposure to NiCl₂ for 24 h, the BMDMs were fixed and permeabilized with 4% paraformaldehyde and 0.05% Triton X-100, respectively. After blocking with PBS containing 5% BSA, the BMDMs were stained with rabbit anti-rabbit NF-κB p65, followed by Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch). The nuclei of BMDMs were stained with propidium iodide (PI; Thermo Fisher). All images were acquired with a confocal microscope. For image quantification, 100 cells randomly chosen from 10 high-power fields and pooled from five independent experiments, were evaluated for the distribution pattern of the indicated molecules. Then, we quantified the percentage of cells with NF-kB nuclear translocation.

Determination of mitochondrial ROS

MitoSOX was used to measure mtROS production in BMDMs. After NiCl₂ treatment, the BMDMs were rinsed in PBS and then stained with 5 μM of MitoSOX-red at 37 °C, 5% CO₂ for 15 min. After MitoSOX staining, the BMDMs were washed with PBS, followed by fixing and staining with DAPI. All samples were visualized and recorded using Zeiss Axio Imager A2 fluorescence microscope. For image quantification, 100 cells randomly chosen from 10 high-power fields and pooled from five independent experiments, were evaluated for the distribution pattern of the indicated molecules. The red fluorescence of each cell is detected by image J.

Measurements of mitochondrial membrane potential (Ψm)

Mitochondrial membrane potential (Ψm) was measured using TMRM as proposed by Zhong et al. [39]. Briefly, after NiCl₂ exposure, the BMDMs were rinsed twice in PBS, incubated with 200 nM of TMRM for 30 min, and then rinsed twice in 50 nM of TMRM. The BMDMs were resuspended in PBS, and the fluorescence intensities recorded using FilterMax F5 multimode plate reader (Molecular Devices).

Cellular fractionation and determination of cytosolic mtDNA

MtDNA in cytosol was performed as described previously [35]. Total DNA was extracted from the cytosolic fraction (200 μL) using DNeasy Blood and Tissue kit (Cat. 69504 Qiagen). Quantitative PCR was adopted for the measurement of mtDNA by using the established mitochondrial and nuclear gene primers and SYBR® Premix Ex Taq^TMII (RR820A, Takara, China). The copy numbers of mtDNA were normalized to those of nuclear DNA, and the data were expressed as the ratio of mtDNA (cytochrome c oxidase I) to nuclear DNA (18S rRNA). The accession number, sequences, product size and annealing temperature of each primer are presented in Table 1.

RNA isolation and quantitative Real-Time PCR (qRT-PCR)

Total RNA was isolated from the treated BMDM and was reverse transcribed. QRT-PCR reactions were carried out as proposed by Guo et al. [30]. QRT-PCR data were analyzed by the 2^−ΔΔCT method [40]. The accession number, sequences, product size and annealing temperature of each primer are listed in Table 2.

Enzyme-linked immunosorbent assay

After NiCl₂ treatment, ELISA kit was used to examine the concentration of cytokines in cell culture supernatants as per the manufacturer's protocols.

Apoptosis analysis by flow cytometry

After NiCl₂ treatment, the BMDMs were rinsed twice with ice-cold phosphate buffer saline (PBS, pH 7.2-7.4), and then suspended in PBS to the desired concentration of 1×10⁶ cells/ml. The staining protocol was consistent with the description of Guo et al. [37]. Briefly, 100 μL of the cell suspension was placed into a 5 mL tube. The cell suspension was stained with 5 μL of Annexin V-FITC (Cat: 51-65874X, BD, USA) followed by 5 μL of PI (Cat: 51-66211E, BD, USA) at 25°C in darkness. After 15-min incubation, 400 μl of 1× binding buffer were added, and the stained cells were evaluated by a flow cytometry (BD FACS Calibur) within 40 min of preparation. Flow cytometry data analysis was performed using the ModFit LT v3.0 application software.

Statistical analysis

All data were expressed as mean ± standard deviation. The statistical differences between the three experimental groups and control group were compared using one-way analysis of variance (SPSS 17.0 statistical software). P-value below 0.05 was considered statistically significant.

Ethics statement

The animal protocols and all procedures of the experiment were performed in compliance with the laws and guidelines of Animal Care and Use Committee, Sichuan Agricultural University (Approval No: 2012- 024).