KN-62

Plasticizer DBP Activates NLRP3 Inflammasome through the P2X7 Receptor in HepG2 and L02 Cells

ABSTRACT: Ditubyl phthalate (DBP), one of the most widely used plasticizers, can migrate out to contami- nate our bodies and environment. A number of stud- ies have showed that DBP is closely related to liver pathological changes and diseases. Inflammasomes are multiprotein complexes composed of procaspase and pattern recognition receptors such as Nucleotide oligomerization domain (NOD) like receptor fam- ily, pyrin domain containing 3 (NLRP3). Activation of NLRP3 inflammasome is implicated in the patho- geneses of liver damage. The aim of this study was to determine the effects of DBP on NLRP3 inflammasome. We found that DBP triggered the activation of NLRP3 inflammasome in hepatocyte cell lines. By using Ca-074-Me, N-acetylcysteine and KN-62, we observed that the P2X7 receptor partici- pated in the DBP-induced activation of NLRP3 inflam- masome. DBP could also trigger the ATP release. In conclusion, we demonstrated that DBP is one of the activator of NLRP3 inflammasome and may play an important role in liver damage.

INTRODUCTION
Plasticizers are a group of compounds widely used to provide flexibility and resilience to plastic products, such as medical care products, children toys, and food-packaging materials. Phthalate ester accounts for a great part of plasticizers. Di(2- ethylhexyl)phthalate (DEHP) and ditubyl phthalate (DBP) are the most widely used phthalate ester in plastic industry. Plasticizers reach out to environment easily to result in widespread general population exposure [1, 2]. DBP has been identified in plasma and tissues of patients taking medications coated with DBP (1-233 μg/kg/day) [3], undergoing hemodialysis or receiving blood transfusions (34.3–433.2 μM) [4] and 0.1–76 μg/kg/day in occupationally exposed groups [5]. Plasticizers have been found to affect the endocrine system activity. In immune system, DEHP and DBP in- creased the production of inflammatory cytokines, in- cluding tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), IL-18, and IL-6, by inducing the translocation of p65 NF-κB in human macrophages [6, 7]. In the vascu- lar system, plasticizers also increased the concentration of IL-6, IL-8 and intercellular adhesion molecule-1 (ICAM-1) in vascular cells [8, 9]. In addition, DEHP profoundly caused structural and functional properties of the liver. It affected the cell organelles, such as per- oxisomes and mitochondria, and expression of the en- zymes involving in fatty acid transport and β-oxidation [10, 11]. The U.S. Environmental Protection Agency has defined plasticizes as water and air pollutants [12].

Inflammasomes are multiprotein complexes that play an important role in regulating innate immunity and inflammatory response. Among them, NLRP3 inflammasome is composed of Nucleotide oligomerization domain (NOD) like receptor family, pyrin domain containing 3 (NLRP3), the adaptor ASC [apoptosis-associated speck-like protein con- taining caspase activation and recruitment domain (CARD)] and Caspase-1. Many studies showed that a battery of initial signals prime the synthesis of pro-IL-1β and NLRP3 by transcriptional induction, whereas a secondary stimulus leads to inflammasome oligomeization, Caspase-1–dependent cleavage and the subsequent release of the biologically active, ma- ture IL-1β. Such secondary signals may be triggered by a group of chemically and biologically unrelated molecules including pathogen-associated molecular patterns or damage-associated molecular patterns [13–15]. Many recently characterized activators of NLRP3 inflammasome were found to trigger the NLRP3 inflammasomes via three major mechanisms [16]: (1) ATP-gated P2X7 ion channel is stimulated by extracellular ATP, leading to an increased K+ efflux and the activation of NLRP3 inflammasome [17, 18];(2) some particulate or crystalline activators, such as monosodiumurate, alum, and silica, are engulfed via phagocytosis and cause lysosomal damage and release lysosomal contents, such as cathepsin B. Some of these contents can be sensed by the NLPR3 inflam- masome [19–21]; (3) excessive reactive oxygen species (ROS) production is another common pathway leading to the activation of NLRP3 inflammasome [22, 23].

Recent studies have shown that the liver expressed NLRP1, 2, 3, 6, 10, 12 inflammasomes [24]. Aberrant activation of the inflammasomes was considered to be involved in the pathogenesis of both chronic and acute liver injury and diseases [25–27]. In addition, some studies showed that plasticizers caused the accu- mulation of inflammatory cells with hepatocyte degen- eration [28, 29]. However, the effects of DBP on NLRP3 inflammasome activation in the liver and the underly- ing mechanisms remain unknown.Rabbit polyclonal antibodies to NLRP3 and IL-1β were purchased from Abcam (Ann Arbor, MI). Rabbit polyclonal antibody to Caspase-1 was purchased from Cell Signaling Technology (Danvers, MA). Mouse polyclonal antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fatal bovine serum (FBS) was purchased from Hy- Clone (Logan, UT). DMSO was purchased from MP Biomedicals (Santa Ana, CA). DBP, KN-62, Ca-074-Me, N-acetylcysteine (NAC), lipopolysaccharides (LPS), ATP, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis., MO).
HepG2 cell line was purchased from American Type Culture Collection (Manassas, VA). HepG2 is a perpetual cell line derived from the liver tissue of a 15-year-old Caucasian American male with a well-differentiated hepatocellular carcinoma. L02 is a normal human liver cell line and was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, People’s Republic of China). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and at 37◦C in a humidified atmosphere of 5% CO2 in air. Cells were grown to 80% confluence in all experiments. HepG2 and L02 cells in six-well culture plates were stimulated for 36 h with the indicated concentrations of DBP. Cells were treated with DBP at concentrations as previously described [30]. The cells were treated with LPS 500 ng/mL for 12 h as positive control and stimulated with 5 mM ATP for 30 min be- fore cells were lysated. In inhibition experiments, cells were pretreated with different inhibitors (Ca-074-Me 10 μM, NAC 10 mM, or KN-62 10 μM) for 2 h before the exposure to DBP for 36 h.

Cell viability was evaluated by the MTT assay. HepG2 and L02 cells were seeded in 96-well plates and cultured until 80% confluence. Then, cells were treated with the indicated concentrations of DBP for 36 h or treated 10 or 25 μM DBP in different time periods be- fore incubation with 5 mg/mL MTT at 37◦C in 5% CO2 atmosphere for 4 h. Next, the culture medium was re- moved and the formazan formed in the reaction was dissolved in 150 μL DMSO. The optical density of the solution was measured at a wavelength of 490 nm us- ing a multifunctional microplate reader. Cell viability in each well was presented as a percentage of the control (vehicle-treated group).Total proteins were extracted using the RIPA kit (Pierce Biotechnology, Rockford, IL). Protein concen- trations were determined using the Pierce BCA Protein Assay Kit (Thermo, Waltham, MA). Equal amounts of protein (25 μg) were separated on SDS-PAGE through 10% running gel and transferred to nitrocellulose mem- brane. The membranes were immediately blocked with 5% nonfat milk for 1 h, immunoreacted with primary antibodies against human NLRP3, Caspase-1, IL-1β, and β-actin, and then incubated with appropriate sec- ondary antibodies. The protein bands were visual- ized with the Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate (Millipore, Bil- lerica, MA) and visualized by the ECL chemilumines- cence system. Protein band densities were analyzed using the ImageJ software. Values were normalized by β-actin.
Extracellular ATP in cell culture supernatant was quantified using the ATP Assay Kit (Beyotime, Haimen, People’s Republic of China). Quick centrifugation of the supernatant was performed to avoid cell debris contamination. Luminescence was measured with a luminometer, and the ATP concentration was deter- mined using a standard curve allowing samples quan- tification and standardized by samples protein density.
Quantitative data are expressed as mean SEM. Differences between two groups were analyzed by the t test. Comparisons among three or more groups were tested by ANOVA plus a post hoc S-N-K or Dunnett t method, and only when all the other treatments were compared with a simple control was the Dunnett t method used. SPSS16.0 (SPSS software, Chicago, IL) was used for statistical analysis. P < 0.05 was considered to be significant. Nonquantitative results were representative of at least three independent experiments. RESULTS First, by using the MTT assay, we evaluated the cytotoxicity of DBP on both HepG2 and L02 cells. HepG2 and L02 cells were treated with the indicated concentrations (1–100 μM) of DBP for 36 h. As shown in Figures 1A and 1B, over 80% of the cells remained viable and there were no significant difference in cell viability among the groups. These results indicated that there was no significant cytotoxic effect of DBP on HepG2 and L02 cells.The cleavage and maturation of Caspase-1 and IL-1β are the hallmarks of inflammasome activa- tion. Thus, we examined whether DBP could activate Caspase-1 cleavage and IL-1β production. As shown in Figures 1C and 1E, DBP significantly increased the cleavage of Caspase-1 and IL-1β proteins in both cell lines. DBP also increased the protein expression of NLRP3. Figures 1D and 1F are the quantification of NLRP3 and the cleaved Caspase-1 and IL-1β as fold in- duction. Together, these results suggested that DBP ac- tivated NLRP3 inflammasome in HepG2 and L02 cells.Ca-074-Me and NAC Have No Effects on DBP-Induced Activation of NLRP3 Inflammasome in HepG2 and L02 CellsNext, we examined potential mechanisms by which DBP induced activation of NLRP3 inflamma- some. As mentioned above, there are three models (cathepsin B, ROS, and P2X7 receptor pathway) leading to the activation of NLRP3 inflammasome. First, we ex- amined whether DBP activated NLRP3 inflammasome by inducing lysosomal rupture and ROS generation. We pretreated HepG2 and L02 cells with Ca-074-Me or NAC, which are two inhibitors for cathepsin B and ROS, respectively, for 2 h, and then exposure the cells to DBP for 36 h. As shown in Figures 2 and 3, neither Ca-074-Me nor NAC abrogated the activation of NLRP3 inflammsome induced by DBP significantly. Then, by using KN-62, a P2X7 receptor inhibitor, we studied the role of the P2X7 receptor pathway in DBP-induced NLRP3 inflammasome activation. We pretreated HepG2 and L02 cells with KN-62 (10 μM) for 2 h before stimulation with DBP. As shown in Figure 4, KN-62 attenuated the DBP-induced elevation of NLRP3 and cleavage of Caspase-1 and IL-1β in HepG2 and L02 cells. Since LPS/ATP stimulation also activates NLRP3 inflammasome through the P2X7 receptor, KN-62 showed an inhibitory effect in LPS/ATP-treated cells. Taken together, these results suggested that DBP activated NLRP3 inflammasome in hepatocytes via the P2X7 receptor-mediated pathway.DBP Triggers ATP Release to the Medium in HepG2 and L02 Cells.To determine whether DBP activated NLRP3 in- flammasome through ATP leakage, we measured the levels of extracellular ATP in HepG2 and L02 cell cul- ture medium. As shown in Figures 5A and 5C, ATP levels in the culture medium increased significantly by DBP stimulation in both cell lines. However, the ATP level in cytoplasm did not changed significantly as shown in Figures 5B and 5D. DISCUSSION Inflammasomes respond to cellular danger signals in parenchymal or nonparenchymal cells, and their ac- tivation is implicated in various liver diseases, such as steatosis, inflammation, and fibrosis [29]. Previous studies have shown that plasticizers, including DBP and DEHP, stimulated inflammatory response with liver pathological changes [28]. DBP exposure is a major hazard in particular subjects with occupational expo- sure to the manufacture process and the patients re- ceiving blood transfusions. Our data provided novel evidence indicating that activation of inflammasome may mediate such DBP-caused liver damage (Figure 1).Three distinct mechanisms have been shown to mediate NLRP3 inflammasome activation. For the first model, engulfment of crystalline results in cytosolic release of lysosomal contents, which can be sensed by NLRP3 inflammasome [13]. DBP as small soluble molecules may pass through cell membranes to cause lysosomal rupture and the release of cathepsin. However, this mode of action is unlikely because Ca-074-Me did not attenuate the activation of NLRP3 inflammasome induced by DBP (Figure 2). Some agonists triggered the generation of ROS to induce the NLRP3 inflammasome activation [31]. It was previously described that DBP and its metabolites could bind to the active site of superoxide dismutase by forming hydrogen bonds with the active site residue R143, thus inhibiting its catalytic activity and causing the accumulation of ROS [32]. However, our data revealed that the activation of NLRP3 inflammasome by DBP could not be attributed to the ROS generation because the ROS scavenger NAC failed to abrogate the inflammasome-activating effect of DBP (Figure 3). Indeed, some studies reported that ROS alone was insufficient for triggering NLRP3 inflammasome activity [13]. For the third model, the P2X7 receptor is ubiquitously expressed in almost all tissues and organs of the body. As ATP-gated channels, P2X7 receptors can serve the nonselective macropores permeable to large ( 800 Da) inorganic or organic molecules and allow some cations to pass through the cell membrane [17, 33]. Cellular stimulation triggers ATP release and subsequent activation of P2X7 receptors at the cell surface and/or in adjacent cells, thereby modulating cellular functions in innate immunity [34]. Previous studies have demonstrated that P2X7 receptor-induced intracellular K+ outflow and decrease coupled with the activation of Caspase-1 [18]. In light of the results that the DBP-induced NLRP3 inflammasome activation was inhibited in cells pretreated with KN-62 (Figure 4) and that DBP treatment increased the ATP level in the culture medium (Figure 5), it is plausible that DBP activated the NLRP3 inflammasome via a mechanism involving the P2X7 receptor pathway and extracellular ATP, which acts as a danger signal to prime the HepG2 and L02 cells for the activation. Nevertheless, future work is needed to examine the in vivo effect of DBP on the NLRP3 inflammasome activation. In conclusion, our data demonstrated that plas- ticizer DBP activated the NLRP3 inflammasome and revealed a role of the P2X7 receptor in the DBP action. These findings may provide new insight into the molecular mechanisms underlying the liver damage caused by KN-62 plasticizers.