Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells

Abstract

Gouty arthritis is an inflammatory disease that is caused by an accumulation of monosodium urate (MSU) crystals in the joints. MSU is capable of activating the nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome, leading to interleukin-1β (IL-1β) secretion. Reactive oxygen species (ROS) are major mediators of the NLRP3/IL-1β interaction. Although nuclear factor E2-related factor 2 (Nrf2) is recognized as a transcription factor that is involved in the response to oxidative stress, the effect of MSU on Nrf2 and on Nrf2-mediated antioxidant enzymes remains unclear. The treatment of THP-1 monocytes using phorbol 12-myristate 13-acetate (PMA) was shown to initiate inflammatory responses. Here, we showed that THP-1 cells, following treatment with MSU crystals, significantly increased IL-1β release, NLRP3 inflammasome activation and ROS production. MSU also promoted the nuclear translocation of Nrf2 and activated lysosomal destabilization. Moreover, the levels of heme oxygenase-1 (HO-1) in gene and protein expressions were upregulated by MSU. MSU-induced IL-1β secretion and NLRP3 inflammasome activation were inhibited by the knockdown of Nrf2 and via the HO-1 inhibitor zinc (II) protoporphyrin IX (ZnPP). In addition, HO-1 inhibition increased the level of superoxide anion production and the consumption of glutathione. These findings suggest that Nrf2 and HO-1 mediate redox homeostasis and interact with pro-inflammatory factors in MSU-challenged THP-1 cells, thereby providing new insight into how MSU-induced gouty inflammation is mediated by specific mechanisms that are involved in the Nrf2/Ho-1 antioxidant signaling pathway.Cellular & Molecular Immunology advance online publication, 11 August 2014; doi:10.1038/cmi.2014.65.

Figure 1

MSU triggered IL-1β maturation and intracellular ROS production. THP-1 cells were incubated with or without 100 nM PMA for 3 h, and then the cells were treated with 75 µg/ml MSU for 6 h. (a) The levels of IL-1β secretion were detected via ELISA; (b) the protein expression levels in cell lysates were estimated via immunoblotting. Following treatment with PMA for 3 h, THP-1 cells were cultured in 25, 50 or 75 µg/ml MSU for 6 h. (c) The levels of IL-1β secretion were analyzed by ELISA. (d) The levels of caspase-1 and caspase-1 cleavage products (p20) in the cell culture supernatant (SN) and the whole cell lysate (WCL) were assayed via immunoblotting. (e) The anti-TXNIP antibody was used for the immunoprecipitation (IP) of TXNIP, NLRP3 and ASC, and the binding results were assessed via immunoblotting (IB). The relative protein expression levels of NLRP3 and ASC were compared using the fold difference of each protein compared with the indicated TXNIP signal from the same sample. (f) ROS production was detected via staining with 10 µM DCFH-DA. (g) MtROS production was assessed via staining with 2.5 µM mitosox for 20 min and analyzed by flow cytometry. The fluorescent intensity of MtROS is shown in h. The values represent the mean±s.d.; *P<0.05 and **P<0.01 indicate significant differences from the PMA treatment. MFI, mean fluorescence intensity.

Figure 2

MSU up-regulated the nuclear translocation of Nrf2 and caused lysosomal destabilization. THP-1 cells were incubated with or without 100 nM PMA for 3 h and were then treated with 75 µg/ml MSU for 6 h. (a) The levels of Nrf2 and Keap-1 in the nuclear and cytosolic fractions were analyzed by immunoblotting. (b) The anti-p62 antibody was used for the immunoprecipitation (IP) of p62, Nrf2, and Keap-1, and the binding results were assessed via immunoblotting (IB). The relative protein expression levels of Nrf2 and Keap-1 were compared using the fold difference of each protein compared to the indicated internal control from the same sample. The levels of lysosomal stability in the MSU treatment for 1 h (c) and in a dose-dependent manner (d) were assayed via staining with 10 µM acridine orange (AO) using flow cytometry. The fluorescent intensity of AO was shown in a time-dependent manner (e) and in a dose-dependent manner (f). The values are presented as the mean±s.d.; *P indicates a significant difference (P<0.05) from the PMA treatment. MFI, mean fluorescence intensity.

Figure 3

The expressions of genes that are regulated by Nrf2 in a time-dependent course of MSU treatment were analyzed by RT-PCR amplification. THP-1 cells were incubated with 100 nM PMA for 3 h and then treated with 75 µg/ml MSU for 1, 3 or 6 h. (a) PCR products were analyzed using a gel assay. The transcription levels of (b) SOD and (c) HO-1 were represented by the target gene/18S rRNA ratio. Blank bars refer to the PMA treatment. Black bars refer to the 75 µg/ml MSU treatment. The values are presented as the mean±s.d.; *P indicates a significant difference (P<0.05) from the concurrent PMA treatment.

Figure 4

Nrf2-mediated gene expression under dose-dependent MSU treatment was analyzed by RT-PCR. THP-1 cells were incubated with 100 nM PMA for 3 h and then treated with 20, 50 or 75 µg/ml MSU for 6 h. (a) PCR products were separated on a 1.5% agarose gel. The transcription levels of (b) SOD and (c) HO-1 were represented by the target gene/18S rRNA ratio. The values are presented as the mean±s.d.; *P indicates a significant difference (P<0.05) from the PMA treatment.

Figure 5

The dose-dependent effect of MSU on (a) SOD activity, (b) catalase activity, (c) GPx activity, (d) GSH/GSSG ratio, (e) GRd activity and (f) HO-1 protein expressions of THP-1 cells. THP-1 cells were incubated with 100 nM PMA for 3 h and then treated with 20, 50 or 75 µg/ml MSU for 6 h. The relative protein expression levels of HO-1 were compared using the ratio of each protein to the indicated β-actin levels from the same sample. The values are presented as the mean±s.d.; *P indicates a significant difference (P<0.05) from the PMA treatment.

Figure 6

Nrf2 is essential for NLRP3 inflammasome activation. (a) THP-1 cells were transfected with non-specific siRNA (si-control) or si-Nrf2 for 24 h. Following transfection, Nrf2 gene expression was analyzed via RT-PCR and agarose gel electrophoresis. After transfection with si-Nrf2, THP-1 cells were incubated with 100 nM PMA for 3 h, and the cells were then treated with 75 µg/ml MSU. (b) IL-1β secretion levels were analyzed by ELISA. (c) Immunoprecipitation (IP) of TXNIP with NLRP3 was performed prior to immunoblotting (IB). The relative protein expression levels of NLRP3 were compared using the ratio of each protein to the indicated TXNIP signal from the same sample. The values are presented as the mean±s.d.; *P indicates a significant difference (P<0.05).

Figure 7

The HO-1 inhibitor ZnPP reduced the MSU-induced inflammatory response. THP-1 cells were incubated with 100 nM PMA for 3 h and then treated with 2.5 µM ZnPP and 75 µg/ml MSU for 6 h. (a) IL-1β production was analyzed via ELISA. (b) The immunoprecipitation (IP) of TXNIP with NLRP3 was performed prior to immunoblotting (IB). (c) Superoxide anion production was determined using flow cytometry with 8 µM DHE; (d) the fluorescence intensity was quantified. (e) Cellular GSH levels were determined using a flow cytometer with 4 µM CMFDA; (f) the fluorescence intensity was quantified. The values are represented as the mean±s.d.; *P indicates a significant difference (P<0.05), and **P indicates a significant difference (P<0.01). The relative protein expression levels of NLRP3 were compared using the ratio of each protein to the indicated TXNIP signal from the same sample. DHE, dihydroethidium; MFI, mean fluorescence intensity; n.s., non-significant.