Aggregatibacter actinomycetemcomitans cytolethal distending toxin activates the NLRP3 inflammasome in human macrophages, leading to the release of proinflammatory cytokines

Abstract

The cytolethal distending toxin (Cdt) is produced from a number of bacteria capable of causing infection and inflammatory disease. Our previous studies with Actinobacillus actinomycetemcomitans Cdt demonstrate not only that the active toxin subunit functions as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase but also that macrophages exposed to the toxin were stimulated to produce proinflammatory cytokines. We now demonstrate that the Cdt-induced proinflammatory response involves the activation of the NLRP3 inflammasome. Specific inhibitors and short hairpin RNA (shRNA) were employed to demonstrate requirements for NLRP3 and ASC as well as caspase-1. Furthermore, Cdt-mediated inflammasome activation is dependent upon upstream signals, including reactive oxygen species (ROS) generation and Cdt-induced increases in extracellular ATP levels. Increases in extracellular ATP levels contribute to the activation of the P2X7 purinergic receptor, leading to K+ efflux. The relationship between the abilities of the active toxin subunit CdtB to function as a lipid phosphatase, activate the NLRP3 inflammasome, and induce a proinflammatory cytokine response is discussed. These studies provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aggregatibacter actinomycetemcomitans.

FIG 1

Cdt-induced proinflammatory cytokine release from macrophages is dependent upon caspase-1 activation. (A) THP-1-derived macrophages were treated with 200 ng/ml Cdt for 2 and 4 h. Cell extracts were assessed for caspase-1 activity as described in Materials and Methods. The inset shows Western blot analysis of extracts obtained from THP-1-derived macrophages treated with 0 to 500 ng/ml Cdt; the mature form of caspase-1 (p20 subunit) is shown, along with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. Levels of expression are shown below the blot as a percentage of levels in untreated control cells. OD, optical density. (B) THP-1-derived macrophages were treated with Cdt (0 to 200 ng/ml) for 5 h. Supernatants were then analyzed for the presence of caspase-1 (anti-p20) by an ELISA. Results are the means ± standard errors of the means for three experiments, each performed in triplicate. (C) Effects of caspase-1 inhibition on release of proinflammatory cytokines. Macrophages were exposed to various amounts of the caspase inhibitor Z-WEHD-FMK for 60 min; 50 ng/ml Cdt was then added to the cells for 5 h. Culture supernatants were analyzed for IL-1β (circles), IL-18 (diamonds), and TNF-α (triangles) by an ELISA. Results are plotted as the mean ± the standard error of the mean pg/ml cytokine released versus the Z-WEHD-FMK concentration. Levels of cytokines release from untreated control cells were 9.8 ± 7.8 pg/ml (IL-1β), 38.1 ± 14.0 pg/ml (IL-18), and 120.9 ± 2.6 pg/ml (TNF-α). Asterisks indicate statistical significance (P ≤ 0.05) compared to untreated control cells (A and B) or compared to cells treated with toxin alone (C). (D) Cells were assessed for the expression of pro-IL-1β after 5 h of exposure to Cdt following solubilization, fractionation by SDS-PAGE, and analysis by Western blotting. Actin is shown as a loading control. Western blots are representative of results from three experiments.

FIG 2

Cdt-induced cytokine release from THP-1-derived macrophages involves activation of the NLRP3 inflammasome. THP-1 cells were stably transfected with an shRNA that targets caspase-1, NLRP3, or ASC or with nontargeted control shRNA. (A to C) The effect of protein knockdown in cells exposed to 0 to 200 ng/ml Cdt was determined, and culture supernatants were analyzed for IL-1β (A), IL-18 (B), and caspase-1 (p20) (C) release by an ELISA. Results are the means ± standard errors of the means for three experiments performed in triplicate; asterisks indicate a statistically significant difference from the shRNA control P ≤ 0.05). (D) Cells were also analyzed by Western blotting for the presence of pro-IL-1β. Actin is shown as a loading control. WT, wild type.

FIG 3

Cdt-induced inflammasome activation involves ROS, K⁺ efflux, and extracellular ATP. NAC (10 mM) and DPI (250 nM) were used to determine the requirement for ROS, elevated extracellular levels of K⁺ (70 mM) and glibenclamide (Gli) (50 μg/ml) were used to determine the requirement for K⁺ efflux, and AZ11645373 (AZ) (1 μM) was used to determine the requirement for an ATP-PTXR₇ interaction. (A and B) Effects of these agents on Cdt (50 ng/ml)-induced production of IL-1β and IL-18 (A) and on caspase-1 release (B). Results are the means ± standard errors of the means for three experiments, each performed in triplicate; asterisks indicate statistical significance compared to the Cdt-only control (P ≤ 0.05). (C) Cells were also analyzed for the presence of pro-IL-1β by Western blotting. Actin is shown as a loading control.

FIG 4

The proinflammatory response induced by Cdt is dependent upon extracellular ATP. (A) Macrophages were treated with 0 to 200 ng/ml Cdt for 2 h, and supernatants were then analyzed for ATP. (B and C) Cells were also analyzed for the effect of apyrase (Pyrase) on extracellular ATP levels (B) as well as IL-1β release (open bars) and caspase-1 release (solid bars) (C). Results represent the means ± standard errors of the means for three experiments, each performed in triplicate; asterisks indicate statistical significance compared to control cells (P ≤ 0.05).

FIG 5

GSK3β activation contributes to Cdt-induced expression of pro-IL-1β and increased extracellular ATP levels. (A) THP-1-derived macrophages were pretreated with GSK inhibitors for 1 h, followed by the addition of 200 ng/ml Cdt. After 4 h, cells were fractionated by SDS-PAGE and analyzed by Western blotting for pro-IL-1β. Results are representative of three experiments. (B) Macrophages were pretreated with GSK inhibitors as described above and then treated with 200 ng/ml Cdt for 4 h. Supernatants were analyzed for ATP as described in Materials and Methods. Results represent the means ± standard errors of the means for three experiments; asterisks indicate statistical significance compared to control cells treated with Cdt alone (P ≤ 0.05).

FIG 6

Summary diagram depicting the mechanism by which Cdt induces a proinflammatory cytokine response in macrophages. Two signals are proposed. The first signal (pink arrows) involves the upregulation of inflammatory cytokine gene and protein expression; this signal is dependent upon Cdt's abilities to function as a PIP3 phosphatase, block PI-3K signaling, and activate GSK3β, leading to the expression and synthesis of pro-IL-1β and IL-18. The second signal (blue arrows) involves the activation of the NLRP3 inflammasome, which requires the generation of extracellular ATP, an ATP-P2X₇ interaction, K⁺ efflux, and utilization of endogenous ROS. Inhibitors of GSK3β also block ATP generation, suggesting that Cdt-induced activation of this kinase is also critical to ATP-dependent inflammasome activation.