S100A8 and S100A9 induce cytokine expression and regulate the NLRP3 inflammasome via ROS-dependent activation of NF-κB(1.)

Abstract

S100A8 and S100A9 are cytoplasmic proteins expressed by phagocytes. High concentrations of these proteins have been correlated with various inflammatory conditions, including autoimmune diseases such as rheumatoid arthritis and Crohn's disease, as well as autoinflammatory diseases. In the present study, we examined the effects of S100A8 and S100A9 on the secretion of cytokines and chemokines from PBMCs. S100A8 and S100A9 induced the secretion of cytokines such as IL-6, IL-8, and IL-1β. This secretion was associated with the activation and translocation of the transcription factor NF-κB. Inhibition studies using antisense RNA and the pharmacological agent BAY-117082 confirmed the involvement of NF-κB in IL-6, IL-8, and IL-1β secretion. S100A8- and S100A9-mediated activation of NF-κB, the NLR family, pyrin domain-containing 3 (NLRP3) protein, and pro-IL-1β expression was dependent on the generation of reactive oxygen species. This effect was synergistically enhanced by ATP, a known inflammasome activator. These results suggest that S100A8 and S100A9 enhance the inflammatory response by inducing cytokine secretion of PBMCs.

Figure 1. S100A8 and S100A9 induce the secretion of several cytokines in human PBMCs.

Cytokine production was evaluated using proteome profiler arrays (A and B) or ELISA (C), as described in Materials and Methods. Cells were stimulated for 24 hours, and supernatants were harvested and used for experiments. (B) Densitometry analyses of antibody arrays in PBMCs. Data represent supernatants pooled from 10 different donors (A and B). Black bars, upregulated cytokines; white bars, unchanged cytokines. Data are the mean ± SEM of at least five experiments performed on cells from different donors (C). ***p≤0.005, Dunnett’s multiple comparison test.

Figure 2. S100A8 and S100A9 increase translocation of NF-κB in human PBMCs.

NF-κB translocation and binding were assessed by EMSA (A) and by a NF-κB p50 TransFactor ELISA kit (B). Nuclear proteins were isolated from cells stimulated for 4 h. Lanes 1, 3, 5, and 7 (c, competitive assay). Lanes 2, 4, 6, and 8 (non-competitive assay). Phosphorylation of the IKK complex and degradation of IκB-α were assessed by immunoblotting (C). Results shown are of one experiment that is representative of at least four other experiments (A and C). Data are the mean ± SEM of three experiments performed on cells from different donors (B). *p≤0.05, Dunnett’s multiple comparison test.

Figure 3. Inhibition of p50 and p65 using an antisense (AS) strategy in human PBMCs.

Cells were treated for 4 h with p50 AS (10 or 40 µM), p65 AS (10 or 40 µM), the scrambled sequence AS (Scr 40 µM), or the equivalent volume of diluent (No). Cells were then fixed on coverslips for fluorescence microscopy (A) or lysed for immunoblotting of p50 (B) and p65 (C). Results are from one experiment out of 4 total experiments are shown. *p≤0.05, Dunnett’s multiple comparison test.

Figure 4. S100A8 and S100A9 induce IL-6 and IL-8 secretion via activation of NF-κB in human PBMCs.

Secretion of IL-6 (A and B) and IL-8 (C and D) was evaluated using ELISA. Cells were pre-treated with 1–10 µM of BAY-117082 (or the equivalent amount of diluent) for 30 min or with antisense (Scr, p50, p65) for 4 h, followed by incubation with the indicated agonist for a total of 24 h. Supernatants were harvested and used for ELISA. Data are the mean ± SEM of at least four experiments performed on cells from different donors. *p≤0.05, **p≤0.01, ***p≤0.005, Dunnett’s multiple comparison test.

Figure 5. NF-κB translocation and IL-8 production in S100A8- or S100A9-treated cells are dependent on ROS production.

Cells were stimulated for different time periods and ROS production was assessed by oxidation of H₂DCFDA (A and B). In some experiments, cells were pre-treated with DPI for 30 min (B-F). ROS production was measured after 180 min (B). IL-6 and IL-8 secretion was assessed using ELISA (C and D). Translocation of NF-κB was assessed using the TransFactor p50 kit (E). NF-κB activity was followed with THP1-XBlue cells (F). Data are the mean ± SEM of three experiments performed on cells from four different donors (A-E). Data are the mean ± SEM of three independent experiments (F). *p≤0.05, **p≤0.01, ***p≤0.005, Dunnett’s multiple comparison test.

Figure 6. S100A8 and S100A9 regulate the NLRP3 inflammasome.

Cells were incubated for 4 h with S100A8 (10 µg/ml), S100A9 (10 µg/ml), LPS (1 µg/ml), or the equivalent volume of buffer (Ctrl). For some experiments, ATP (1 mM) was added for 30 min (D–G). Cells were harvested and lysed with Laemmli’s buffer for immunoblotting of pro-IL-1β (A), NLRP3 (B), ASC (C) or caspase-1 (E). Cells were lysed with caspase-1 lysis buffer for the caspase assay (F). ASC immunoprecipitates were loaded on SDS-PAGE for NLRP-3 and ASC western blot analysis (D). Supernatants were harvested before performing IL-1β ELISA (G). Results are from one experiment that is representative of at least three others (A–E). Data are the mean ± SEM of three experiments performed on cells from different donors (F–G). ?, non-specific band (as indicated in the manufacturer’s datasheet). *p≤0.05, **p≤0.01, ***p≤0.005, Dunnett’s multiple comparison test.

Figure 7. Role of ASC and NLRP-3 in the production of IL-1β by S100A8 and S100A9-treated human PBMCs.

Cells were transfected with ASC siRNA, NLRP-3 siRNA, Ctrl siRNA or the equivalent volume of buffer (No) for 24 hours. Cells were then harvested and lyzed for western blot analysis (A and B) or stimulated for 4 hours with the indicated agonists and another 30 min with ATP 1mM. Supernatants were then harvested before performing ELISA (C). Results are from one experiment representative of three others (A and B). Data are the mean ± SEM of four experiments performed on cells from different donors (C). *p≤0.05, **p≤0.01, ***p≤0.005, Dunnett’s multiple comparison test.

Figure 8. ROS- and NF-κB-mediated NLRP3 inflammasome priming by S100A8 and S100A9 in human PBMCs.

Cells were pre-treated with various inhibitors (DPI, NAC, BAY-117082 or the equivalent volume of DMSO), then stimulated for 4 h with the indicated agonists. ATP (1 mM) was added for 30 min for caspase-1 immunoblots and for IL-1β ELISA (B and D). Cells were harvested and lysed in Laemmli’s buffer for immunoblotting of NLRP-3, pro-IL-1β, ASC and caspase-1.(A-C). Supernatants were harvested and IL-1β secretion was evaluated using ELISA (C). *p≤0.05, ***p≤0.005, Dunnett’s multiple comparison test.