Perfluorodecanoic acid stimulates NLRP3 inflammasome assembly in gastric cells

Perfluorodecanoic acid (PFDA), a perfluorinated carboxylic acid, presents in the environment and accumulates in human blood and organs, but its association with tumor promotion are not clear. Given that inflammation plays a significant role in the development of gastric malignancies, we evaluated the effects of PFDA on activation of the inflammasome and inflammation regulation in the gastric cell line AGS. When added to cell cultures, PFDA significantly stimulated IL-1β and IL18 secretion and their mRNA levels compared with control cells. By RT-PCR and western-blot we found that up-regulation of NLRP3 were associated with promotion of IL-1β and IL-18 production. Then expression variation of cIAP1/2, c-Rel and p52 were analyzed, the results demonstrated raised mRNA expression in all the tested genes concomitant with enhanced inflammasome activity after exposure to PFDA. Assays with cIAP2 siRNA and NFκB reporter provided additional evidence that these genes were involved in PFDA-induced inflammasome assembly. Furthermore, increased secretion of IL-1β and IL-18 were detected in stomach of PFDA-treated mice, disorganized alignment of epithelial cells and inflammatory cell infiltration were also observed in the stomach tissues upon PFDA treatment. This study reports for the first time that PFDA regulates inflammasome assembly in human cells and mice tissues.

have been described, i.e. NLRP1, NLRP3 and NLRC4. Inflammasomes assembly generally requires NFκ B activation and danger signals, generating active caspase-1 that cleaves the precursor forms of IL-1β and IL-18 into the mature and active forms. Some evidence suggested that NLRs are closely correlated to cancer occurrence, the level of IL-1β and IL-18 were found to be significantly elevated in various types of malignancies 12,14,15 . Therefore, elucidating the mechanisms that involve activation of the inflammasomes and their modulatory effects has become a novel strategy for inflammation regulation and cancer prevention research.
In this study, we found that PFDA induced IL-1β and IL-18 secretion in culture solution of cell line AGS and mice stomachs. Our results demonstrated that the IL-1β and IL-18 inductions were associated with activation of NLRP3 inflammasome. This study reports for the first time that PFDA activates inflammasome and promotes gastric inflammation in gastric cells and mice tissues.

Material & methods
Cell line and animal treatment. Human gastric adenocarcinoma cell line AGS, huaman hepatocellular carcinoma cell lines BEL7402 and human leukemia macrophage cell line THP-1 were all from American type culture collection (ATCC) and maintained in our laboratory. They were cultured in Ham's F-12 medium (HyClone, Utah, USA) containing 10% FCS and 1% penicillin-streptomycin. PFDA was purchased from Sigma Chemical Company (CAS number 335-76-2, St. Louis, MO, USA). Different volumes of PFDA were added into cell culture media for treatments. Six-week-old female Balb/c mice were acclimated and caged in groups of 5 or less. NLRP3 knockout mice were kindly offered by Professor Rongbin Zhou (University of Science and Technology of China). Mice were administered with PFDA at a dose of 25 mg/kg/d in drinking water, 0.014 M dimethyl sulfoxide (DMSO) was utilized as the control. Mice treatment and dissection were performed according to the protocol approved by the Ethics Committee on Animal Experiments of Medical School of Shandong University. Stomachs were harvested 18d after PFDA dosing. Some stomachs were cut into small pieces and mixed with Phosphate-buffered saline in a certain percentage for tissue grinding. The obtained tissue homogenates were used for subsequent Enzyme-linked immunosorbent assay. Some stomachs were Formalin-fixed and paraffin-embedded for Hematoxylin and eosin staining. The rest of stomachs were snap-frozen in liquid nitrogen and stored at − 80 °C for RNA extraction and quantitative RT-PCR. FuGENEs HD Transfection Reagent (Roche Applied Science, Basel, Switzerland) was used for transfection. All transfections were performed according to the manufacturer's instructions.
Enzyme-linked immunosorbent assay (ELISA). IL-1β and IL-18 in culture solution or mouse gastric tissues were detected with human/mouse IL-1β and IL-18 ELISA Kit (4 A Biotech Co., Ltd, Beijing, China) according to the manufacturer's instructions. Mice treatment and dissection were performed according to the protocol approved by the Ethics Committee on Animal Experiments of Medical School of Shandong University. Briefly, add 100 μ l diluted Standard in triplicate or 100 μ l sample to each well, then add 50 μ l biotin labeled Antibody Working Solution per well. After aspiration and wash for 4 times, incubate for 2 hours at room temperature. Next, add 100 μ l Enzyme Binding Working Solution to each well, aspirate and wash 4 times. And then add 100 μ l Substrate Solution to each well. Protect form light, incubate for 10-20 minutes at RT. Finally, add 100 μ l Stop Solution to each well and read at 450 nm within 30 minutes.
Western Blot Analysis. Western Blot Analysis was performed as described previously 16 . Briefly, cell lysates (20 μ g/lane) were separated on 10% SDS polyacrylamide gel and then were transferred to a poly (vinylidene fluoride) membrane. NLRP3 (Cat No.TA336883), cIAP1 (sc-271419) and 2 (Cat No.TA800041), c-Rel (Cat No.TA324346) and p52 (sc-7386) protein was detected by a mouse monoclonal IgG (OriGene Co. Ltd, Beijing, China and Santa Cruz Biotechnology Inc., USA) and visualized by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The density of the bands was quantitated using the NIH image software package. The intensity of gene expression was judged by the ratio of their expression in PFDA treated groups to their corresponding expression in DMSO groups, and a ratio of more than 1.0 was considered to be an indication of over-expression. and diaminobenzidine (DAB, Code No. ZLI9632) staining kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Mice treatment and dissection were performed according to the protocol approved by the Ethics Committee on Animal Experiments of Medical School of Shandong University. Formalin-fixed, paraffin-embedded mice stomach tissues were cut into 4-μ m thick tissue sections and then stained with hematoxylin and eosin (HE) according to the manufacturer's instructions. The staining images were acquired using a light microscope to observe the pathological alterations of the stomach tissues.
Statistical Data analysis. Data were expressed as mean ± standard deviation (SD). Differences between three groups were compared using the Student's t-tests and ANOVA. All experiments were repeated at least three times and P < 0.05 (*) was considered statistically significant.

PFDA exposure increases the secretion and mRNA levels of IL-1β and IL-18 in AGS cells.
To assess effects of PFDA on the secretion of the cytokines that may be regulated by the inflammasome mechanism, we treated the gastric epithelial cell line AGS with PFDA and monitored IL-1β and IL-18 productions by enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 1a and b, cells incubated with PFDA had significantly promoted IL-1β and IL-18 secretion in culture media compared with DMSO-treated control cells. PFDA enhanced IL-1β production in culture media by more than 90% compared with control cells within 24 h; this ratio increased to 220% for IL-18. This increased yield of IL-1β and IL-18 was verified by RT-qPCR of AGS cells (Fig. 1c  and d). The results confirm that PFDA increased IL-1β and IL-18 production in human gastric cells.
Moreover, the human hepatic cell line Bel7402, macrophage cell line THP-1 and normal murine hepatic cells were also used for testing IL-1β and IL-18 production by ELISA after treatment with PFDA. As demonstrated in Fig. 1e,f,g,h,i and j, Bel7402, THP-1 and normal murine hepatic cells treated with PFDA raised IL-1β and IL-18 production compared with DMSO-treated control cells. These results support the hypothesis that PFDA has an effect on IL-1β and IL-18 production in human cells.

PFDA promotes caspase-1 activation in gastric epithelial cells via NLRP3 inflammasome.
To determine if PFDA-promoted secretion of IL-1β and IL-18 was a consequence of increased inflammasome activity, the level of active caspase-1 was examined in AGS concomitant with PFDA treatment. As shown in Fig. 2a, PFDA stimulated production of activate caspase-1.
To find out which inflammasome(s) were involved in PFDA's action, we performed real-time PCR to analyze the changes of mRNA levels of NLRP1, NLRP3 and NLRC4. The mRNA level of NLRP3 was increased by PFDA treatment and in a time-dependent manner (Fig. 2b). On the contrary, NLRP1 had no significant change in mRNA level in response to PFDA treatments (Fig. 2c), whereas NLRC4 could not be detected (no visible peak). This observation was consistent with an increase of NLRP3 protein level upon PFDA exposure as detected by Western blot (Fig. 2d). The dependence of cytokine secretion on NLRP3 was further verified by NLRP3 knockout mice, the promotions in IL-1β and IL-18 production were abrogated in their stomach ( Fig. 2e and f). cIAP1 and -2, which are required for efficient caspase-1 activation by the inflammasomes, were involved in PFDA induced NLRP3 inflammasome activation. cIAP1 and cIAP2 are critical effectors of the inflammasomes and are required for inflammasome assembly and Caspase-1 activation. siRNA-mediated depletion of endogenous cIAP1 or cIAP2, alone or in combination, dramatically blunted caspase-1 activity 17 . Therefore, we thought to determine if cIAP1 and cIAP2 were involved in PFDA-promoted inflammasome activation. As shown in Fig. 3a and b, cIAP1 and cIAP2 mRNA levels were increased by PFDA, as shown by western blot at protein level ( Fig. 3c and d). To confirm the role of cIAPs in PFDA-induced inflammasome activation, we investigated the effect of inhibition of cIAP2, by cIAP2 siRNA, on activation of caspase-1 and secretion of IL-1β and IL-18. As shown in Fig. 3e,f and g, inhibition of cIAP2 abrogated PFDA-induced cIAP2 production and IL-1β /IL-18 secretion.

NFκB activity was enhanced in PFDA-treated gastric epithelial cells. NFκ B plays an important
role in activating the priming step of inflammasomes and NFκ B inhibition results in a significant reduction of NLRP3 expression. To assess effects of PFDA on NFκ B activation, we treated AGS gastric epithelial cells with PFDA and monitored NFκ B activity with NFκ B reporter plasmid and Duo-Luciferase Assay Kit. As shown in Fig. 4a, cells incubated with PFDA had significantly increased NFκ B activity by 240% after PFDA treatment compared with DMSO-treated control cells. The enhanced NFκ B activity was verified by enhanced expression of c-Rel and p52 (Fig. 4b).

Promotion in secretion of IL-1β and IL-18 and inflammation were detected in the stomach of PFDA-treated mice. To investigate the effects of PFDA on inflammasome activation and inflammation
in vivo, we treated wild type C57BL/6 mice with PFDA in drinking water for 2 weeks and tested mice stomachs with ELISA. As shown in Fig. 5a and b, IL-1β and IL-18 were increased in the stomach tissues of PFDA treated mice. Moreover, hematoxylin and eosin (HE) staining of stomach tissues of PFDA-or DMSO-treated C57BL/6 mice, showed disorganized alignment of cells and increased inflammatory cells infiltration ( Fig. 5c and d). Therefore, these results demonstrate for the first time that PFDA regulates inflammasome assembly and inflammation in human cells and mice tissues.

Discussion
Perfluorinated carboxylic acids or perfluorinated fatty acids (PFCAs) have been used for decades to make products that resist heat, oil, and water. Because they are used in the manufacture of nonstick cookware, fire-fighting foam, and many other industrial products 18,19 , perfluorinated compounds can be detected globally in the environment 6,20 , wildlife [21][22][23][24] and humans [25][26][27][28][29][30] . Continued monitoring of PFCAs is necessary since these chemicals persist in the environment and accumulate at much higher concentrations in human blood and organs because of their nondegradable nature and extremely long biological half-lives 31,32 .
The reported cellular and physiological effects of PFCAs are associated with almost all systems of the human body; reports on the immunotoxicity of PFCs in humans are limited and controversial. On one hand, some reports suggested that exposure to PFCAs might be associated with immunosuppressive effects. Brieger et al. reported that Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonate (PFOS) were associated with reduced Natural killer (NK) cell activity and reduced the release of the pro-inflammatory cytokine Tumor necrosis factor (TNF)-α following lipopolysaccharide (LPS)-stimulation 33 . Two recently published studies indicate that PFCAs may lower vaccine protection in children 34  Despite recent advances in understanding the molecular mechanisms of perfluorinated environmental pollutants, many unanswered questions remain. PFDA is a straight chain ten-carbon PFCA that is structurally similar to fatty acids. In vivo, PFDA has highly potent and persistent toxicity 5 ; it is several times as toxic as PFOA, so PFDA may be important for understanding the molecular mechanisms of toxicity. It is thus hoped that PFDA can assist in dissecting the sequence of events that ultimately results in tumor formation 5 . However, despite the evidence of PFDA toxicity, so far little is known of its effect on malignancy promotion and how it acts in inflammation. In this study, we evaluated the effects of PFDA on activation of the inflammasomes and inflammation regulation in the gastric cell line AGS. When added to cell cultures, PFDA significantly increased NLRP3 inflammasome activation compared with control cells. Moreover, the promotion in synthesis of IL-1β and IL-18 were detected in the stomach of PFDA-treated mice. This report describes for the first time that PFDA regulates inflammasome assembly in gastric cells.  a and b), cIAP1 and -2 were activated by PFDA at mRNA level, this was evidenced by western blot at protein level ( Fig. 3c and d). (e), inhibition of cIAP2 by siRNA abrogated PFDA-induced cIAP2 production; (f and g), inhibition of cIAP2 reduced IL-1β and IL-18 secretion. ELISA, Western blot and RT-qPCR were performed as described in Materials and Methods.
Proteins of the inhibitor of apoptosis protein (IAP) gene family have emerged as among the most important intrinsic inhibitors of apoptosis and could exert important roles in inflammation and innate immunity. They are characterized by the presence of a baculovirus IAP repeat (BIR) domain in one to three copies. cIAP1 and -2  function as ubiquitin ligases 41,42 and were recently identified as important effectors of innate immunity, mediating critical steps in Nucleotide-binding oligomerization domain-containing protein (NOD) signaling 43 . Moreover, cIAP1 and -2 were shown to mediate Mitogen-activated protein kinases (MAPK) activation downstream of Toll-like receptor 4 (TLR4) signaling by catalyzing degradative K48-linked ubiquitination of TNF Receptor Associated Factor 3 (TRAF3) 44 . cIAP2-deficient mice have reduced cytokine amounts in response to LPS and are resistant to LPS endotoxemia 45 . Labbe et al. reported that the cIAPs were critical effectors of the inflammasomes and were required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1 17 . Dagenais et al. reported that cIAP2-deficient mice had impaired activation of the regenerative inflammasome-interleukin-18 (IL-18) pathway 46 . Moreover, the results of Mayer et al. revealed IAP antagonism as a profound anti-inflammatory principle in vivo and highlighted IAPs as important regulators of inflammatory processes in endothelial cells 47 . In this study, we discovered that PFDA activated NLRP3 inflammasome through cIAP1/2, as a result of knockdown with cIAP2 siRNA, providing additional evidence that cIAP1/2 was involved in PFDA-induced inflammasome assembly.
NFκ B transcription factors play an important role in many physiological processes and diseases 48,49 . IKKβ preferentially phosphorylates the Iκ B proteins, resulting in the release and activation of canonical NFκ B p50/p65 (RelA) and c-Rel/p65 complexes. IKKα homodimer-mediated phosphorylation of cytoplasmic NFκ B2/p100 results in partial proteasomal processing of p100 to produce mature p52, thereby activating the alternative pathway 50 . NFκ B is thought to serve as the first signal that primes NLR and pro-IL-1β expression 51-53 . Qiao et al. demonstrated that NLRP3 promoter contains a putative NFκ B binding site and NFκ B inhibition resulted in a significant reduction of NLRP3 expression 54 . Meanwhile, mounting evidence suggests that NLRP3 inflammasome formation is positively associated with NFκ B activity 55,56 . All these findings implied that the pro-tumorigenic ability of NFκ B might be attributed to inflammasome activation. cIAP1 and -2 are required for NFκ B signaling. Bertrand et al. revealed that cIAP1 and -2 were responsible for K63-linked polyubiquitination of receptor interacting protein 1 (RIP1), promoting NFκ B activation signaling 57 . cIAP1 and cIAP2 also direct the ubiquitination of NFκ B-inducing kinase (NIK) to maintain the noncanonical NFκ B pathway 58,59 . In this study, we discovered that PFDA promoted NFκ B reporter activity as well as enhanced c-Rel and p52 expression; thereby confirming activation of NFκ B signaling. Conclusively, this report describes for the first time that PFDA regulates inflammasome activation and inflammation in gastric cells.