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Is NLRP3 or NLRP6 inflammasome activation associated with inflammation-related lung tumorigenesis induced by benzo(a)pyrene and lipopolysaccharide?
Ecotoxicology and Environmental Safety 185 (2019) 109687
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Is NLRP3 or NLRP6 inflammasome activation associated with inflammationrelated lung tumorigenesis induced by benzo(a)pyrene and lipopolysaccharide?
T
Min Gaoa,1, Peng Zhangb,1, Li Huanga, Hua Shaoa, Shuyin Duanc, Chunyang Lia, Qiao Zhanga, Wei Wangc, Yongjun Wuc, Jing Wangd, Hong Liud, Feifei Fenga,∗ a
Department of Toxicology, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China Department of Bone and Soft Tissue Cancer, The Affiliated Cancer Hospital of Zhengzhou University (Henan Cancer Hospital), Zhengzhou, Henan, China Department of Occupational and Environmental Health, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China d Department of Pulmonary Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Inflammasome NLRP3 NLRP6 Lung inflammation Benzo(a)pyrene Lipopolysaccharide
Chronic inflammation has been shown to play a vital role in lung tumorigenesis. Recently, we have successfully developed a C57BL/6 mouse model of inflammation-related lung tumorigenesis induced by benzo(a)pyrene [B (a)p] and lipopolysaccharide (LPS), which will contribute to better understand the association between pulmonary inflammation and cancer. In this study, we aim to explore the role of NLRP3 and NLRP6 inflammasome in lung tumorigenesis in the animal model that we set up previously. Levels of NLRP3, NLRP6, interleukin-1β (IL-1β) and IL-18 protein in lung tissues were detected by using immunohistochemistry. The co-localization of NLRP3 or NLRP6 with caspase-1 was examined using immunofluorescence and confocal. Western blotting was used to evaluate the levels of caspase-1 p10 and cleaved-IL-1β protein. The expression of IL-18 in bronchoalveolar lavage fluid (BALF) was measured using ELISA kit. The expression of NLRP3, NLRP6 and IL-18 protein in the lung tissues of mice exposed to B(a)p plus LPS was upregulated significantly compared with those in Vehicle control group. Immunofluorescent results indicated the co-localization of NLRP3 with caspase-1 was increased in the lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice than that in Vehicle control group, but no co-localization of NLRP6 with caspase-1. Additionally, caspase-1 activation was induced, cleaved-IL-1β in lung tissues and IL-18 protein in BALF were increased in B(a)p plus LPS-exposed mice compared with those in B (a)p group. In conclusion, our results from this study demonstrate that NLRP3 inflammasome, not NLRP6 inflammasome, activation is involved in B(a)p plus LPS-induced inflammation-related lung tumorigenesis in mice, but the mechanisms of NLRP6 participate in the development of lung cancer should be further investigated.
1. Introduction Nod-like receptors (NLRs), a major form of innate immune sensors, can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to regulate inflammatory disorders (Fukata et al., 2009). Of these, NLRP1, NLRP3, NLRC4, and NLRP6 have been reported that can assemble with the adaptor apoptosis-associated speck-like protein (ASC) and caspase-1 to form inflammasomes, which regulate the maturation and secretion of inflammation-related cytokines including interleukin (IL)-1β and IL-18. More recently, there are substantial evidence demonstrate that chronic
inflammation plays a vital role in carcinogenesis (Bozinovski et al., 2016; Mantovani, 2009). Inflammasomes function as critical inflammatory regulators have been shown to involve in tumor development (Janowski et al., 2013; Karki et al., 2017). NLRP3 inflammasome, as the most well investigated inflammasome, also have been reported to be involved in tumorigenesis (Moossavi et al., 2018). It has been showed that NLRP3 inflammasome was associated with gastric cancer (Li et al., 2018), colorectal cancer (Allen et al., 2010; Zaki et al., 2010), prostate cancer (Panchanathan et al., 2016) and so on. However, the evidence regarding to the association between NLRP3 inflammasome and lung tumorigenesis was insufficient. In our previous study, we
∗
Corresponding author. Department of Toxicology, College of Public Health, Zhengzhou University, No.100 Kexue Avenue, Zhengzhou, Henan province, 450001, China. E-mail address: [email protected] (F. Feng). 1 Equal contribution. https://doi.org/10.1016/j.ecoenv.2019.109687 Received 28 July 2019; Received in revised form 7 September 2019; Accepted 15 September 2019 Available online 24 September 2019 0147-6513/ © 2019 Published by Elsevier Inc.
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using a straightedge. Lung tissues were stored at −80 °C for subsequent assays.
explored the role of NLRP3 inflammasome in lipopolysaccharide (LPS) and coal tar pitch extract (CTPE)-induced malignant transformation of human bronchial epithelial cells (BEAS-2B), and found NLRP3 inflammasome activation was involved in the malignant transformation of BEAS-2B cells (Duan et al., 2019). In addition, we further explored its role in mouse inflammation-related lung tumorigenesis, and reported NLRP3 deletion inhibits lung tumorigenesis induced by benzo(a)pyrene [B(a)p] or B(a)p plus LPS, but the mechanisms of NLRP3 expression and NLRP3 inflammasome activation in lung tumorigenesis have not been clarified (Huang et al., 2019a). NLRP6, another subset of NLRs family, has been reported that not only is involved in the negative regulation of common NF-κB and MAPK-dependent inflammatory signaling, but also can form a functional inflammasome in a stimulus-specific manner (Levy et al., 2017; Shen et al., 2019). NLRP6 is highly expressed in the intestine, and plays crucial functions in maintenance of intestinal homeostasis by regulating the gut microbiome, epithelial repair and proliferation (Chen, 2014). Indeed, there is a body of evidence suggesting that NLRP6 plays an important role in intestinal inflammation and cancer (Chen et al., 2011; Huber et al., 2012; Normand et al., 2011). For example, Chen, et al. found that NLRP6 deletion increased susceptibility to dextran sulfate sodium (DSS)-induced colitis and colitis-induced tumorigenesis compared with wild-type mice (Chen et al., 2011). Recently, role of NLRP6 in pneumonia was investigated, and these findings indicated that NLRP6 negatively regulates neutrophil-mediated pulmonary host defense during Staphylococcus aureus infection (Ghimire et al., 2018). However, the involvement of NLRP6 in lung tumorigenesis remains unclear. In this study, we explored the role of NLRP3 and NLRP6 inflammasome in lung tumorigenesis using the animal model of inflammation-related lung tumorigenesis induced by B(a)p plus LPS reported in our previous study (Huang et al., 2019a), which will provide novel therapeutic targets for the treatment of lung cancer.
2.3. Bronchoalveolar lavage fluid (BALF) Mice were subjected to bronchoalveolar lavage (BAL) as described previously (Feng et al., 2016). In detail, the lungs were lavaged with 1 ml physiological saline. The lavage fluids were collected and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatants were collected for evaluating the level of IL-18 using ELISA kit (Cusabio Biotechnology, Wuhan, China). 2.4. Immunohistochemistry NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues were examined using immunohistochemistry (IHC). Briefly, lung tissues sections were first blocked with goat serum for 30 min at room temperature, and then incubated with rabbit anti-mouse NLRP3 (Abcam, 1:100 dilution), NLRP6 (Bioss, Beijing, China, 1:100 dilution), IL-1β (Bioss, Beijing, China, 1:100 dilution) and IL-18 (Bioss, Beijing, China, 1:100 dilution) antibody overnight at 4 °C, respectively, followed by incubation with biotinylated goat anti-rabbit (1:100 dilution) at 37 °C for 30 min. Positive IHC staining was reflected as the brown staining in the cytoplasm and estimated by average optical density in 10 high vision field using Image-Pro Plus 6.0 software [AOD = Integrated Optical Density (IOD) SUM/Area SUM]. 2.5. Immunofluorescence To confirm the formation of NLRP3 or NLRP6 inflammasome in lung tissues, the sections were first blocked with goat serum for 30 min at room temperature, and then incubated with rabbit anti-mouse NLRP3 (Abcam, 1:100 dilution), NLRP6 (Bioss, Beijing, China, 1:100 dilution), and caspase-1 (Boster, Wuhan, China, 1:100 dilution) antibody overnight at 4 °C, respectively, followed by incubation with FITC- and CY3labeled goat anti-rabbit (1:300 dilution) at 37 °C for 50 min. Then, cell nucleus was stained with blue-fluorescent DAPI. The co-localization (orange) of NLRP3 (green) or NLRP6 (green) with caspase-1 (red) was calculated in Image Pro Plus 6.0 software, and the co-localization coefficient was calculated as the Pearson's correlation coefficient (PCC).
2. Methods 2.1. Animals C57BL/6 mice were bred from the heterozygous littermates of NLRP3 ± mice in the College of Public Health of Zhengzhou University, Henan, China (Huang et al., 2019b). All C57BL/6J mice used in this study were genotyped. Mice were raised in stainless steel cages under standard conditions, and allowed food and water ad libitum. The temperature was maintained at 22 °C, and the lights began from 08:00 a.m. to 20:00 p.m. All experimental procedures were approved by the Life Science Institutional Review Board of Zhengzhou University and performed strictly in accordance with the Guideline of Zhengzhou University for Animal Experiments.
2.6. Western blotting Mouse lung tissues were lysed using RIPA lysis buffer with proteinase inhibitors, protein concentration was determined by BCA Protein Assay Kit (CWbio Company LTD., Beijing, China), 50 μg protein per sample was mixed with sample loading buffer and boiled for 5 min. Samples were separated by 12% or 15% SDS-PAGE and were transferred onto Polyvinylidene Fluoride (PVDF) membranes. Immunoblots were blocked by 5% milk in TBST for 2 h at room temperature, then incubated overnight at 4 °C with 1:1000 rabbit polyclonal antibody to mouse caspase-1 p10 (Abcam) or 1:1000 mouse polyclonal antibody to mouse IL-1β (Cell Signaling Technology) or 1:1000 rabbit polyclonal antibody to mouse β-actin (Servicebio), 1:5000 goat anti-rabbit IgG or 1:10,000 goat anti-mouse IgG as secondary antibodies were used to incubate membranes at room temperature for 1 h. Bands were detected by ECL plus western blotting detection system. The intensity of bands was quantified using Image J software.
2.2. Inflammation-related lung tumorigenesis mouse model The C57BL/6 mouse model of inflammation-related lung tumorigenesis using in this study has been set up in our previous study (Huang et al., 2019a). In detail, C57BL/6 mice were randomly divided into four treatment groups, respectively: B(a)p plus LPS group (n = 35), B(a)p group (n = 35), LPS group (n = 15) and Vehicle control group (n = 15). The mice in B(a)p plus LPS group were instilled intratracheally with B(a)p (dissolved in 50 μL glyceryl trioctanoate, 1mg/ mouse), once a week for 4 times [the week of the last dose of B(a)p treatment named Week 0], and then instilled intratracheally with LPS (dissolved in 50 μL saline, 2.5μg/mouse), once every three weeks for 5 times. The mice were instilled intratracheally with B(a)p or LPS alone in a same manner were named as B(a)p group or LPS group, respectively. Glyceryl trioctanoate and saline were used for vehicle controls. At week 30, the mice were euthanasia, and visible tumors on the surface of the lung tissues were counted and the size of lung tumors was accessed
2.7. Statistical analysis Data comparison was carried out by two-tailed Student's t-test and one-way ANOVAs using SPSS version 21.0 (IBM, NC, USA). A two-tailed P value < 0.05 was considered statistically significant. 2
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3. Results
Table 1 B(a)p or B(a)p plus LPS exposure induced lung tumorigenesis in mice. Group
Vehicle control LPS B(a)p B(a)p + LPS
N
15 15 35 35
3.1. B(a)p plus LPS exposure promoted inflammation-related lung tumorigenesis in mice
Lung tumor Tumor incidence (%)
Tumors/mouse (Mean ± SD)
0 0 82.05 96.97 *
0 0 4.7 ± 5.7 13.0 ± 12.4*
Tumor size ≤1 mm
> 1 mm
0 0 3.2 ± 4.6 9.0 ± 10.8*
0 0 1.4 ± 2.3 3.3 ± 3.7*
As shown in Table 1, no tumor was found in LPS or Vehicle control group, but B(a)p or B(a)p plus LPS exposure could induce lung tumorigenesis in mice. The tumor incidence in B(a)p plus LPS-exposed mice was increased than that in B(a)p-exposed mice (P < 0.05). Moreover, the numbers of visible tumors/mouse, smaller tumors (≤1 mm) and larger tumors (> 1 mm) were also more abundant in mice exposed to B(a)p plus LPS compared with those in B(a)p exposure alone (P < 0.05, respectively).
*P < 0.05, versus B(a)p.
Fig. 1. The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues of mice in different groups. (A) The representative images of IHC staining of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissue with amplification (200X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (B) Levels of NLRP3, NLRP6, IL-1β and IL-18 protein were quantified using AOD in 10 high vision field using Image-Pro Plus 6.0 software. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control; #P < 0.05, versus LPS; ΔP < 0.05, versus B(a)p. 3
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Fig. 2. The co-localization of NLRP3 with caspase-1 in lung tissues of mice in different groups. (A) Representative images of co-localization (orange) between NLRP3 (green) and caspase-1 (red) in lung tissues with amplification (400X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (B) The fold changes of PCC for the co-localization of NLRP3 with caspase-1 in lung tissues. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
no significant difference on the level of IL-1β protein in lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice compared with that in Vehicle control group. These results demonstrate that B(a)p or B(a)p plus LPS exposure can upregulate the expression of NLRP3, NLRP6 and IL-18 protein in lung tissues of mice.
3.2. The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues of mice in different groups The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues was examined using immunohistochemistry. As shown in Fig. 1, the levels of NLRP3, NLRP6 and IL-18 protein in lung tissues of mice exposed to B(a)p or B(a)p plus LPS were increased significantly compared with those in Vehicle control or LPS group (P < 0.05, respectively). In addition, the expression of NLRP6 and IL-18 protein in lung tissues of B(a)p plus LPS-exposed mice was higher than those in mice exposed to B(a)p alone (P < 0.05, respectively). However, there was
3.3. The role of NLRP3 and NLRP6 in lung tumorigenesis via inflammasome signaling To investigate whether NLRP3 and NLRP6 involves in lung tumorigenesis via inflammasome signaling, we examined the co-localization 4
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Fig. 3. There was no co-localization of NLRP6 with caspase-1 in lung tissues of mice in different groups. Representative images of co-localization (orange) between NLRP6 (green) with caspase-1 (red) in lung tissues with amplification (400X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
inflammation-related lung tumorigenesis.
(orange) of NLRP3 (green) or NLRP6 (green) with caspase-1 (red) in lung tissues using immunofluorescence staining. Confocal microscopy showed increased co-localization of NLRP3 with caspase-1 in lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice compared with that in Vehicle control group (P < 0.05) (Fig. 2). However, there was no combination of NLRP6 and caspase-1 in Vehicle control, LPS, B(a)p or B (a)p plus LPS group, suggesting NLRP6 could not form a functional inflammasome like NLRP3 in lung tumorigenesis induced by B(a)p or B (a)p plus LPS (Fig. 3). These results demonstrate that NLRP3 inflammasome signaling may be involved in lung tumorigenesis, but not NLRP6 inflammasome.
4. Discussion In our previous study, NLRP3 deletion inhibits inflammation-related mouse lung tumorigenesis induced by B(a)p plus LPS, but the mechanisms of NLRP3 expression and NLRP3 inflammasome activation in inflammation-related lung cancer remain unclear (Huang et al., 2019a). Herein, we first identified NLRP3 inflammasome activation involves in inflammation-related lung tumorigenesis induced by B(a)p plus LPS in mice. It has been reported that the basal protein amounts of NLRP3 is not sufficient for inflammasome activation in resting cells, and a twosignal model has been proposed for NLRP3 inflammasome activation: a priming step and a second activation (He et al., 2016). In this study, immunohistochemical results showed that levels of NLRP3 and IL-18 protein of lung tissues were elevated in B(a)p or B(a)p plus LPS-exposed mice, suggesting B(a)p or B(a)p plus LPS exposure may trigger NLRP3 activation. LPS, a major pro-inflammatory component of the gram-negative bacterial, has been reported that can mediate inflammation via activating NLRP3 inflammasome (Xiang et al., 2015; Zhang et al., 2016). However, LPS exposure did not induce the expression of NLRP3, IL-1β or IL-18 protein in this study. We next examined the formation of NLRP3 inflammasome and the caspase-1 activation, and found that LPS alone or B(a)p plus LPS not only increased the co-localization of NLRP3 with caspase-1 in lung tissues in mice compared with that in Vehicle
3.4. The effect of B(a)p plus LPS on NLRP3 inflammasome in lung tissue As shown in Fig. 4, B(a)p plus LPS exposure increased the level of pro-caspase-1 protein in lung tissues in mice than that in Vehicle control or B(a)p group (P < 0.05, respectively). Importantly, we found only LPS or B(a)p plus LPS exposure could induce caspase-1 activation (p10). Levels of cleaved-IL-1β in lung tissues and IL-18 in BALF in LPS, B(a)p and B(a)p plus LPS group were significant increased compared with those in Vehicle control group (P < 0.05, respectively). In addition, B(a)p plus LPS exposure significantly increased the expression of cleaved-IL-1β in lung tissues and IL-18 in BALF than that mice treated with B(a)p alone (P < 0.05, respectively). These results indicated that NLRP3 inflammasome activation involved in B(a)p plus LPS-induced 5
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Fig. 4. B(a)p plus LPS exposure induced NLRP3 inflammasome activation in lung tissues. (A) The expression of pro-caspase-1, caspase-1 p10 and cleaved-IL-1β protein in lung tissues was examined using Western blot. Relative density of pro-caspase-1 (B) and cleaved-IL-1β (C) in lung tissues was evaluated using Image J software. (D) The level of IL-18 in BALF was examined using ELISA kit. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control; #P < 0.05, versus LPS; ΔP < 0.05, versus B(a)p.
MAPK pathway (Yang et al., 2015). However, there are substantial evidence indicate that NLRP3 inflammasome functions as a negative regulator in the inflammation-driven colon tumorigenesis mouse model, which may be associated with upregulation of IL-1β and IL-18 in tumor lesions (Allen et al., 2010; Zaki et al., 2010). Indeed, Ghiringhelli et al. found NLRP3 inflammasome-mediated IL-1β in dendritic cells played a critical role in anticancer chemotherapy via priming IFN-γproducing CD8+ T cells (Ghiringhelli et al., 2009). In liver colorectal cancer (CRC) metastasis models, it was showed that NLRP3 inflammasome components deficient exacerbated liver CRC metastatic growth mediated by impaired IL-18 signaling (Dupaul-Chicoine et al., 2015). Taken together, IL-1β and IL-18 exerts dual functions in tumorigenesis, which may be attributed to tissue specific and should further research to provide effectively therapeutic strategies for cancer. In addition, we provided the first evidence of NLRP6 expression in lung tumorigenesis, showing that the level of NLRP6 protein was significantly upregulated in lung tissues of mice treated with B(a)p or B(a) p plus LPS compared to that in Vehicle control group. However, NLRP6 could not form a functional inflammasome like NLRP3 in lung tumorigenesis in this study. The data suggests that NLRP6 may be involved in lung tumorigenesis, but not via inflammasome signaling. Prior reports have demonstrated the protective role of NLRP6 in the development of colitis and colitis-related tumorigenesis in mice (Chen et al., 2011; Normand et al., 2011). In azoxymethane (AOM)/DSS-treated mice, NLRP6 expression was decreased in colorectal cancer tissues than that in normal tissues, and mice lacking NLRP6 enhanced the formation of colitis-induced colorectal cancer (Chen et al., 2011). It is worth noting that NLRP6-deficient mice decreased IL-18 production in AOM/DSSinduced colorectal cancer (Chen et al., 2011; Huber et al., 2012). In addition, inflammasome components, including ASC and caspase-1 deletion also have been showed that were associated with increased colitis and tumors. Although NLRP6 has been shown to can assemble
control group, but also could induce the caspase-1 activation. These results indicated that LPS may prime NLRP3 inflammasome by a nontranscriptional mechanism, which does not require new protein synthesis (Juliana et al., 2012; Lin et al., 2014). In addition, it also suggested that B(a)p plus LPS exposure could activate NLRP3 inflammasome to involve in the occurrence and development of lung cancer. Upon activation of NLRP3 inflammasome, active caspase-1 leads to the maturation and secretion of IL-1β and IL-18. Indeed, we found B(a)p plus LPS exposure could induced the increase of cleaved-IL-1β in lung tissues and IL-18 protein in BALF in mice compared with those in B(a)p group. These results suggested that NLRP3 inflammasome activationdependent on IL-1β and IL-18 enhanced inflammation-related lung tumorigenesis induced by B(a)p plus LPS, which was in line with the cell model in vitro our previous reported (Duan et al., 2019). In addition, Wang et al. also found upregulation of IL-1β and IL-18 induced by NLRP3 inflammasome activation could enhance the proliferation and migration of lung adenocarcinoma cell line A549 (Wang et al., 2016). IL-1β, as a common proinflammatory cytokine, is significantly upregulated in the serum of patients with non-small cell lung cancer (NSCLC) and exerts a tumor-promoting effect in NSCLC cells (Wang et al., 2014). Furthermore, it has been reported that overexpression of IL-1β could mobilize myeloid-derived suppressor cells (MDSCs) and induce gastric inflammation and cancer in mice (Tu et al., 2008). Besides IL-1β, activation of NLRP3 inflammasome also leads to secretion of IL-18. In gastric cancer patients, serum IL-18 concentration was significantly elevated than that in healthy controls (Tas et al., 2015). Recently, Terme M et al. defined IL-18 as an immunosuppressive cytokine in cancer, which can suppress NK cell-regulated tumor immunosurveillance in a PD-1-dependent manner (Terme et al., 2011). Additionally, IL-18 also reported to have a pro-tumor effect in breast cancer cell via downregulation of claudin-12 and induction of the p38 6
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with ASC and caspase-1 to form an inflammasome (Grenier et al., 2002; Shen et al., 2019), the directly evidence about the co-localization of NLRP6 with ASC or caspase-1 in tumorigenesis of NLRP6 inflammasome components-deficient mice was absent. Therefore, the functional role for NLRP6 and underlying mechanism in cancer should be further investigated. Recently, there have been several studies indicated that NLRP6 could significantly suppress the proliferation and migration of gastric cancer cells via regulating STAT3 signaling and P14ARF-Mdm2P53-dependent cellular senescence (Wang et al., 2018a, 2018b). The protective role of NLRP6 in gastric cancer and colorectal cancer was inconsistent with the present results. In conclusion, our results from this study demonstrate that NLRP3 inflammasome, not NLRP6 inflammasome, activation is involved in B (a)p plus LPS-induced inflammation-related lung tumorigenesis in mice, but the mechanisms of NLRP6 participate in the development of lung cancer should be further investigated.
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Conflicts of interest The authors declare that they have no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (81402712); and the training grant of Zhengzhou University (2017ZDGGJS039); and the grant from Henan Department of Education (No. 20B330004 and 20B320042); and the grants from Henan Department of Science and Technology, China (No. 162102310319); and the grant of Medical Science Research Foundation of Henan Province (No. 2018020477). References Allen, I.C., et al., 2010. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer. J. Exp. Med. 207, 1045–1056. Bozinovski, S., et al., 2016. COPD and squamous cell lung cancer: aberrant inflammation and immunity is the common link. Br. J. Pharmacol. 173, 635–648. Chen, G.Y., 2014. Role of Nlrp6 and Nlrp12 in the maintenance of intestinal homeostasis. Eur. J. Immunol. 44, 321–327. Chen, G.Y., et al., 2011. A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–7194. Duan, S., et al., 2019. NLRP3 inflammasome activation involved in LPS and coal tar pitch extract-induced malignant transformation of human bronchial epithelial cells. Environ. Toxicol. 34 (5), 585–593. Dupaul-Chicoine, J., et al., 2015. The Nlrp3 inflammasome suppresses colorectal cancer metastatic growth in the liver by promoting natural killer cell tumoricidal activity. Immunity 43, 751–763. Feng, F., et al., 2016. Regulation of ozone-induced lung inflammation by the epidermal growth factor receptor in mice. Environ. Toxicol. 31, 2016–2027. Fukata, M., et al., 2009. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders. Semin. Immunol. 21, 242–253. Ghimire, L., et al., 2018. NLRP6 negatively regulates pulmonary host defense in Grampositive bacterial infection through modulating neutrophil recruitment and function.
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Is NLRP3 or NLRP6 inflammasome activation associated with inflammationrelated lung tumorigenesis induced by benzo(a)pyrene and lipopolysaccharide?
T
Min Gaoa,1, Peng Zhangb,1, Li Huanga, Hua Shaoa, Shuyin Duanc, Chunyang Lia, Qiao Zhanga, Wei Wangc, Yongjun Wuc, Jing Wangd, Hong Liud, Feifei Fenga,∗ a
Department of Toxicology, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China Department of Bone and Soft Tissue Cancer, The Affiliated Cancer Hospital of Zhengzhou University (Henan Cancer Hospital), Zhengzhou, Henan, China Department of Occupational and Environmental Health, College of Public Health, Zhengzhou University, Zhengzhou, Henan, China d Department of Pulmonary Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Inflammasome NLRP3 NLRP6 Lung inflammation Benzo(a)pyrene Lipopolysaccharide
Chronic inflammation has been shown to play a vital role in lung tumorigenesis. Recently, we have successfully developed a C57BL/6 mouse model of inflammation-related lung tumorigenesis induced by benzo(a)pyrene [B (a)p] and lipopolysaccharide (LPS), which will contribute to better understand the association between pulmonary inflammation and cancer. In this study, we aim to explore the role of NLRP3 and NLRP6 inflammasome in lung tumorigenesis in the animal model that we set up previously. Levels of NLRP3, NLRP6, interleukin-1β (IL-1β) and IL-18 protein in lung tissues were detected by using immunohistochemistry. The co-localization of NLRP3 or NLRP6 with caspase-1 was examined using immunofluorescence and confocal. Western blotting was used to evaluate the levels of caspase-1 p10 and cleaved-IL-1β protein. The expression of IL-18 in bronchoalveolar lavage fluid (BALF) was measured using ELISA kit. The expression of NLRP3, NLRP6 and IL-18 protein in the lung tissues of mice exposed to B(a)p plus LPS was upregulated significantly compared with those in Vehicle control group. Immunofluorescent results indicated the co-localization of NLRP3 with caspase-1 was increased in the lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice than that in Vehicle control group, but no co-localization of NLRP6 with caspase-1. Additionally, caspase-1 activation was induced, cleaved-IL-1β in lung tissues and IL-18 protein in BALF were increased in B(a)p plus LPS-exposed mice compared with those in B (a)p group. In conclusion, our results from this study demonstrate that NLRP3 inflammasome, not NLRP6 inflammasome, activation is involved in B(a)p plus LPS-induced inflammation-related lung tumorigenesis in mice, but the mechanisms of NLRP6 participate in the development of lung cancer should be further investigated.
1. Introduction Nod-like receptors (NLRs), a major form of innate immune sensors, can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to regulate inflammatory disorders (Fukata et al., 2009). Of these, NLRP1, NLRP3, NLRC4, and NLRP6 have been reported that can assemble with the adaptor apoptosis-associated speck-like protein (ASC) and caspase-1 to form inflammasomes, which regulate the maturation and secretion of inflammation-related cytokines including interleukin (IL)-1β and IL-18. More recently, there are substantial evidence demonstrate that chronic
inflammation plays a vital role in carcinogenesis (Bozinovski et al., 2016; Mantovani, 2009). Inflammasomes function as critical inflammatory regulators have been shown to involve in tumor development (Janowski et al., 2013; Karki et al., 2017). NLRP3 inflammasome, as the most well investigated inflammasome, also have been reported to be involved in tumorigenesis (Moossavi et al., 2018). It has been showed that NLRP3 inflammasome was associated with gastric cancer (Li et al., 2018), colorectal cancer (Allen et al., 2010; Zaki et al., 2010), prostate cancer (Panchanathan et al., 2016) and so on. However, the evidence regarding to the association between NLRP3 inflammasome and lung tumorigenesis was insufficient. In our previous study, we
∗
Corresponding author. Department of Toxicology, College of Public Health, Zhengzhou University, No.100 Kexue Avenue, Zhengzhou, Henan province, 450001, China. E-mail address: [email protected] (F. Feng). 1 Equal contribution. https://doi.org/10.1016/j.ecoenv.2019.109687 Received 28 July 2019; Received in revised form 7 September 2019; Accepted 15 September 2019 Available online 24 September 2019 0147-6513/ © 2019 Published by Elsevier Inc.
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using a straightedge. Lung tissues were stored at −80 °C for subsequent assays.
explored the role of NLRP3 inflammasome in lipopolysaccharide (LPS) and coal tar pitch extract (CTPE)-induced malignant transformation of human bronchial epithelial cells (BEAS-2B), and found NLRP3 inflammasome activation was involved in the malignant transformation of BEAS-2B cells (Duan et al., 2019). In addition, we further explored its role in mouse inflammation-related lung tumorigenesis, and reported NLRP3 deletion inhibits lung tumorigenesis induced by benzo(a)pyrene [B(a)p] or B(a)p plus LPS, but the mechanisms of NLRP3 expression and NLRP3 inflammasome activation in lung tumorigenesis have not been clarified (Huang et al., 2019a). NLRP6, another subset of NLRs family, has been reported that not only is involved in the negative regulation of common NF-κB and MAPK-dependent inflammatory signaling, but also can form a functional inflammasome in a stimulus-specific manner (Levy et al., 2017; Shen et al., 2019). NLRP6 is highly expressed in the intestine, and plays crucial functions in maintenance of intestinal homeostasis by regulating the gut microbiome, epithelial repair and proliferation (Chen, 2014). Indeed, there is a body of evidence suggesting that NLRP6 plays an important role in intestinal inflammation and cancer (Chen et al., 2011; Huber et al., 2012; Normand et al., 2011). For example, Chen, et al. found that NLRP6 deletion increased susceptibility to dextran sulfate sodium (DSS)-induced colitis and colitis-induced tumorigenesis compared with wild-type mice (Chen et al., 2011). Recently, role of NLRP6 in pneumonia was investigated, and these findings indicated that NLRP6 negatively regulates neutrophil-mediated pulmonary host defense during Staphylococcus aureus infection (Ghimire et al., 2018). However, the involvement of NLRP6 in lung tumorigenesis remains unclear. In this study, we explored the role of NLRP3 and NLRP6 inflammasome in lung tumorigenesis using the animal model of inflammation-related lung tumorigenesis induced by B(a)p plus LPS reported in our previous study (Huang et al., 2019a), which will provide novel therapeutic targets for the treatment of lung cancer.
2.3. Bronchoalveolar lavage fluid (BALF) Mice were subjected to bronchoalveolar lavage (BAL) as described previously (Feng et al., 2016). In detail, the lungs were lavaged with 1 ml physiological saline. The lavage fluids were collected and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatants were collected for evaluating the level of IL-18 using ELISA kit (Cusabio Biotechnology, Wuhan, China). 2.4. Immunohistochemistry NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues were examined using immunohistochemistry (IHC). Briefly, lung tissues sections were first blocked with goat serum for 30 min at room temperature, and then incubated with rabbit anti-mouse NLRP3 (Abcam, 1:100 dilution), NLRP6 (Bioss, Beijing, China, 1:100 dilution), IL-1β (Bioss, Beijing, China, 1:100 dilution) and IL-18 (Bioss, Beijing, China, 1:100 dilution) antibody overnight at 4 °C, respectively, followed by incubation with biotinylated goat anti-rabbit (1:100 dilution) at 37 °C for 30 min. Positive IHC staining was reflected as the brown staining in the cytoplasm and estimated by average optical density in 10 high vision field using Image-Pro Plus 6.0 software [AOD = Integrated Optical Density (IOD) SUM/Area SUM]. 2.5. Immunofluorescence To confirm the formation of NLRP3 or NLRP6 inflammasome in lung tissues, the sections were first blocked with goat serum for 30 min at room temperature, and then incubated with rabbit anti-mouse NLRP3 (Abcam, 1:100 dilution), NLRP6 (Bioss, Beijing, China, 1:100 dilution), and caspase-1 (Boster, Wuhan, China, 1:100 dilution) antibody overnight at 4 °C, respectively, followed by incubation with FITC- and CY3labeled goat anti-rabbit (1:300 dilution) at 37 °C for 50 min. Then, cell nucleus was stained with blue-fluorescent DAPI. The co-localization (orange) of NLRP3 (green) or NLRP6 (green) with caspase-1 (red) was calculated in Image Pro Plus 6.0 software, and the co-localization coefficient was calculated as the Pearson's correlation coefficient (PCC).
2. Methods 2.1. Animals C57BL/6 mice were bred from the heterozygous littermates of NLRP3 ± mice in the College of Public Health of Zhengzhou University, Henan, China (Huang et al., 2019b). All C57BL/6J mice used in this study were genotyped. Mice were raised in stainless steel cages under standard conditions, and allowed food and water ad libitum. The temperature was maintained at 22 °C, and the lights began from 08:00 a.m. to 20:00 p.m. All experimental procedures were approved by the Life Science Institutional Review Board of Zhengzhou University and performed strictly in accordance with the Guideline of Zhengzhou University for Animal Experiments.
2.6. Western blotting Mouse lung tissues were lysed using RIPA lysis buffer with proteinase inhibitors, protein concentration was determined by BCA Protein Assay Kit (CWbio Company LTD., Beijing, China), 50 μg protein per sample was mixed with sample loading buffer and boiled for 5 min. Samples were separated by 12% or 15% SDS-PAGE and were transferred onto Polyvinylidene Fluoride (PVDF) membranes. Immunoblots were blocked by 5% milk in TBST for 2 h at room temperature, then incubated overnight at 4 °C with 1:1000 rabbit polyclonal antibody to mouse caspase-1 p10 (Abcam) or 1:1000 mouse polyclonal antibody to mouse IL-1β (Cell Signaling Technology) or 1:1000 rabbit polyclonal antibody to mouse β-actin (Servicebio), 1:5000 goat anti-rabbit IgG or 1:10,000 goat anti-mouse IgG as secondary antibodies were used to incubate membranes at room temperature for 1 h. Bands were detected by ECL plus western blotting detection system. The intensity of bands was quantified using Image J software.
2.2. Inflammation-related lung tumorigenesis mouse model The C57BL/6 mouse model of inflammation-related lung tumorigenesis using in this study has been set up in our previous study (Huang et al., 2019a). In detail, C57BL/6 mice were randomly divided into four treatment groups, respectively: B(a)p plus LPS group (n = 35), B(a)p group (n = 35), LPS group (n = 15) and Vehicle control group (n = 15). The mice in B(a)p plus LPS group were instilled intratracheally with B(a)p (dissolved in 50 μL glyceryl trioctanoate, 1mg/ mouse), once a week for 4 times [the week of the last dose of B(a)p treatment named Week 0], and then instilled intratracheally with LPS (dissolved in 50 μL saline, 2.5μg/mouse), once every three weeks for 5 times. The mice were instilled intratracheally with B(a)p or LPS alone in a same manner were named as B(a)p group or LPS group, respectively. Glyceryl trioctanoate and saline were used for vehicle controls. At week 30, the mice were euthanasia, and visible tumors on the surface of the lung tissues were counted and the size of lung tumors was accessed
2.7. Statistical analysis Data comparison was carried out by two-tailed Student's t-test and one-way ANOVAs using SPSS version 21.0 (IBM, NC, USA). A two-tailed P value < 0.05 was considered statistically significant. 2
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3. Results
Table 1 B(a)p or B(a)p plus LPS exposure induced lung tumorigenesis in mice. Group
Vehicle control LPS B(a)p B(a)p + LPS
N
15 15 35 35
3.1. B(a)p plus LPS exposure promoted inflammation-related lung tumorigenesis in mice
Lung tumor Tumor incidence (%)
Tumors/mouse (Mean ± SD)
0 0 82.05 96.97 *
0 0 4.7 ± 5.7 13.0 ± 12.4*
Tumor size ≤1 mm
> 1 mm
0 0 3.2 ± 4.6 9.0 ± 10.8*
0 0 1.4 ± 2.3 3.3 ± 3.7*
As shown in Table 1, no tumor was found in LPS or Vehicle control group, but B(a)p or B(a)p plus LPS exposure could induce lung tumorigenesis in mice. The tumor incidence in B(a)p plus LPS-exposed mice was increased than that in B(a)p-exposed mice (P < 0.05). Moreover, the numbers of visible tumors/mouse, smaller tumors (≤1 mm) and larger tumors (> 1 mm) were also more abundant in mice exposed to B(a)p plus LPS compared with those in B(a)p exposure alone (P < 0.05, respectively).
*P < 0.05, versus B(a)p.
Fig. 1. The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues of mice in different groups. (A) The representative images of IHC staining of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissue with amplification (200X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (B) Levels of NLRP3, NLRP6, IL-1β and IL-18 protein were quantified using AOD in 10 high vision field using Image-Pro Plus 6.0 software. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control; #P < 0.05, versus LPS; ΔP < 0.05, versus B(a)p. 3
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Fig. 2. The co-localization of NLRP3 with caspase-1 in lung tissues of mice in different groups. (A) Representative images of co-localization (orange) between NLRP3 (green) and caspase-1 (red) in lung tissues with amplification (400X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (B) The fold changes of PCC for the co-localization of NLRP3 with caspase-1 in lung tissues. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
no significant difference on the level of IL-1β protein in lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice compared with that in Vehicle control group. These results demonstrate that B(a)p or B(a)p plus LPS exposure can upregulate the expression of NLRP3, NLRP6 and IL-18 protein in lung tissues of mice.
3.2. The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues of mice in different groups The expression of NLRP3, NLRP6, IL-1β and IL-18 protein in lung tissues was examined using immunohistochemistry. As shown in Fig. 1, the levels of NLRP3, NLRP6 and IL-18 protein in lung tissues of mice exposed to B(a)p or B(a)p plus LPS were increased significantly compared with those in Vehicle control or LPS group (P < 0.05, respectively). In addition, the expression of NLRP6 and IL-18 protein in lung tissues of B(a)p plus LPS-exposed mice was higher than those in mice exposed to B(a)p alone (P < 0.05, respectively). However, there was
3.3. The role of NLRP3 and NLRP6 in lung tumorigenesis via inflammasome signaling To investigate whether NLRP3 and NLRP6 involves in lung tumorigenesis via inflammasome signaling, we examined the co-localization 4
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Fig. 3. There was no co-localization of NLRP6 with caspase-1 in lung tissues of mice in different groups. Representative images of co-localization (orange) between NLRP6 (green) with caspase-1 (red) in lung tissues with amplification (400X) from mice exposed to Vehicle control, LPS, B(a)p and B(a)p plus LPS, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
inflammation-related lung tumorigenesis.
(orange) of NLRP3 (green) or NLRP6 (green) with caspase-1 (red) in lung tissues using immunofluorescence staining. Confocal microscopy showed increased co-localization of NLRP3 with caspase-1 in lung tissues of LPS-, B(a)p- or B(a)p plus LPS-exposed mice compared with that in Vehicle control group (P < 0.05) (Fig. 2). However, there was no combination of NLRP6 and caspase-1 in Vehicle control, LPS, B(a)p or B (a)p plus LPS group, suggesting NLRP6 could not form a functional inflammasome like NLRP3 in lung tumorigenesis induced by B(a)p or B (a)p plus LPS (Fig. 3). These results demonstrate that NLRP3 inflammasome signaling may be involved in lung tumorigenesis, but not NLRP6 inflammasome.
4. Discussion In our previous study, NLRP3 deletion inhibits inflammation-related mouse lung tumorigenesis induced by B(a)p plus LPS, but the mechanisms of NLRP3 expression and NLRP3 inflammasome activation in inflammation-related lung cancer remain unclear (Huang et al., 2019a). Herein, we first identified NLRP3 inflammasome activation involves in inflammation-related lung tumorigenesis induced by B(a)p plus LPS in mice. It has been reported that the basal protein amounts of NLRP3 is not sufficient for inflammasome activation in resting cells, and a twosignal model has been proposed for NLRP3 inflammasome activation: a priming step and a second activation (He et al., 2016). In this study, immunohistochemical results showed that levels of NLRP3 and IL-18 protein of lung tissues were elevated in B(a)p or B(a)p plus LPS-exposed mice, suggesting B(a)p or B(a)p plus LPS exposure may trigger NLRP3 activation. LPS, a major pro-inflammatory component of the gram-negative bacterial, has been reported that can mediate inflammation via activating NLRP3 inflammasome (Xiang et al., 2015; Zhang et al., 2016). However, LPS exposure did not induce the expression of NLRP3, IL-1β or IL-18 protein in this study. We next examined the formation of NLRP3 inflammasome and the caspase-1 activation, and found that LPS alone or B(a)p plus LPS not only increased the co-localization of NLRP3 with caspase-1 in lung tissues in mice compared with that in Vehicle
3.4. The effect of B(a)p plus LPS on NLRP3 inflammasome in lung tissue As shown in Fig. 4, B(a)p plus LPS exposure increased the level of pro-caspase-1 protein in lung tissues in mice than that in Vehicle control or B(a)p group (P < 0.05, respectively). Importantly, we found only LPS or B(a)p plus LPS exposure could induce caspase-1 activation (p10). Levels of cleaved-IL-1β in lung tissues and IL-18 in BALF in LPS, B(a)p and B(a)p plus LPS group were significant increased compared with those in Vehicle control group (P < 0.05, respectively). In addition, B(a)p plus LPS exposure significantly increased the expression of cleaved-IL-1β in lung tissues and IL-18 in BALF than that mice treated with B(a)p alone (P < 0.05, respectively). These results indicated that NLRP3 inflammasome activation involved in B(a)p plus LPS-induced 5
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Fig. 4. B(a)p plus LPS exposure induced NLRP3 inflammasome activation in lung tissues. (A) The expression of pro-caspase-1, caspase-1 p10 and cleaved-IL-1β protein in lung tissues was examined using Western blot. Relative density of pro-caspase-1 (B) and cleaved-IL-1β (C) in lung tissues was evaluated using Image J software. (D) The level of IL-18 in BALF was examined using ELISA kit. Data were expressed as mean ± SEM. *P < 0.05, versus Vehicle control; #P < 0.05, versus LPS; ΔP < 0.05, versus B(a)p.
MAPK pathway (Yang et al., 2015). However, there are substantial evidence indicate that NLRP3 inflammasome functions as a negative regulator in the inflammation-driven colon tumorigenesis mouse model, which may be associated with upregulation of IL-1β and IL-18 in tumor lesions (Allen et al., 2010; Zaki et al., 2010). Indeed, Ghiringhelli et al. found NLRP3 inflammasome-mediated IL-1β in dendritic cells played a critical role in anticancer chemotherapy via priming IFN-γproducing CD8+ T cells (Ghiringhelli et al., 2009). In liver colorectal cancer (CRC) metastasis models, it was showed that NLRP3 inflammasome components deficient exacerbated liver CRC metastatic growth mediated by impaired IL-18 signaling (Dupaul-Chicoine et al., 2015). Taken together, IL-1β and IL-18 exerts dual functions in tumorigenesis, which may be attributed to tissue specific and should further research to provide effectively therapeutic strategies for cancer. In addition, we provided the first evidence of NLRP6 expression in lung tumorigenesis, showing that the level of NLRP6 protein was significantly upregulated in lung tissues of mice treated with B(a)p or B(a) p plus LPS compared to that in Vehicle control group. However, NLRP6 could not form a functional inflammasome like NLRP3 in lung tumorigenesis in this study. The data suggests that NLRP6 may be involved in lung tumorigenesis, but not via inflammasome signaling. Prior reports have demonstrated the protective role of NLRP6 in the development of colitis and colitis-related tumorigenesis in mice (Chen et al., 2011; Normand et al., 2011). In azoxymethane (AOM)/DSS-treated mice, NLRP6 expression was decreased in colorectal cancer tissues than that in normal tissues, and mice lacking NLRP6 enhanced the formation of colitis-induced colorectal cancer (Chen et al., 2011). It is worth noting that NLRP6-deficient mice decreased IL-18 production in AOM/DSSinduced colorectal cancer (Chen et al., 2011; Huber et al., 2012). In addition, inflammasome components, including ASC and caspase-1 deletion also have been showed that were associated with increased colitis and tumors. Although NLRP6 has been shown to can assemble
control group, but also could induce the caspase-1 activation. These results indicated that LPS may prime NLRP3 inflammasome by a nontranscriptional mechanism, which does not require new protein synthesis (Juliana et al., 2012; Lin et al., 2014). In addition, it also suggested that B(a)p plus LPS exposure could activate NLRP3 inflammasome to involve in the occurrence and development of lung cancer. Upon activation of NLRP3 inflammasome, active caspase-1 leads to the maturation and secretion of IL-1β and IL-18. Indeed, we found B(a)p plus LPS exposure could induced the increase of cleaved-IL-1β in lung tissues and IL-18 protein in BALF in mice compared with those in B(a)p group. These results suggested that NLRP3 inflammasome activationdependent on IL-1β and IL-18 enhanced inflammation-related lung tumorigenesis induced by B(a)p plus LPS, which was in line with the cell model in vitro our previous reported (Duan et al., 2019). In addition, Wang et al. also found upregulation of IL-1β and IL-18 induced by NLRP3 inflammasome activation could enhance the proliferation and migration of lung adenocarcinoma cell line A549 (Wang et al., 2016). IL-1β, as a common proinflammatory cytokine, is significantly upregulated in the serum of patients with non-small cell lung cancer (NSCLC) and exerts a tumor-promoting effect in NSCLC cells (Wang et al., 2014). Furthermore, it has been reported that overexpression of IL-1β could mobilize myeloid-derived suppressor cells (MDSCs) and induce gastric inflammation and cancer in mice (Tu et al., 2008). Besides IL-1β, activation of NLRP3 inflammasome also leads to secretion of IL-18. In gastric cancer patients, serum IL-18 concentration was significantly elevated than that in healthy controls (Tas et al., 2015). Recently, Terme M et al. defined IL-18 as an immunosuppressive cytokine in cancer, which can suppress NK cell-regulated tumor immunosurveillance in a PD-1-dependent manner (Terme et al., 2011). Additionally, IL-18 also reported to have a pro-tumor effect in breast cancer cell via downregulation of claudin-12 and induction of the p38 6
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with ASC and caspase-1 to form an inflammasome (Grenier et al., 2002; Shen et al., 2019), the directly evidence about the co-localization of NLRP6 with ASC or caspase-1 in tumorigenesis of NLRP6 inflammasome components-deficient mice was absent. Therefore, the functional role for NLRP6 and underlying mechanism in cancer should be further investigated. Recently, there have been several studies indicated that NLRP6 could significantly suppress the proliferation and migration of gastric cancer cells via regulating STAT3 signaling and P14ARF-Mdm2P53-dependent cellular senescence (Wang et al., 2018a, 2018b). The protective role of NLRP6 in gastric cancer and colorectal cancer was inconsistent with the present results. In conclusion, our results from this study demonstrate that NLRP3 inflammasome, not NLRP6 inflammasome, activation is involved in B (a)p plus LPS-induced inflammation-related lung tumorigenesis in mice, but the mechanisms of NLRP6 participate in the development of lung cancer should be further investigated.
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Conflicts of interest The authors declare that they have no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (81402712); and the training grant of Zhengzhou University (2017ZDGGJS039); and the grant from Henan Department of Education (No. 20B330004 and 20B320042); and the grants from Henan Department of Science and Technology, China (No. 162102310319); and the grant of Medical Science Research Foundation of Henan Province (No. 2018020477). References Allen, I.C., et al., 2010. The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer. J. Exp. Med. 207, 1045–1056. Bozinovski, S., et al., 2016. COPD and squamous cell lung cancer: aberrant inflammation and immunity is the common link. Br. J. Pharmacol. 173, 635–648. Chen, G.Y., 2014. Role of Nlrp6 and Nlrp12 in the maintenance of intestinal homeostasis. Eur. J. Immunol. 44, 321–327. Chen, G.Y., et al., 2011. A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–7194. Duan, S., et al., 2019. NLRP3 inflammasome activation involved in LPS and coal tar pitch extract-induced malignant transformation of human bronchial epithelial cells. Environ. Toxicol. 34 (5), 585–593. Dupaul-Chicoine, J., et al., 2015. The Nlrp3 inflammasome suppresses colorectal cancer metastatic growth in the liver by promoting natural killer cell tumoricidal activity. Immunity 43, 751–763. Feng, F., et al., 2016. Regulation of ozone-induced lung inflammation by the epidermal growth factor receptor in mice. Environ. Toxicol. 31, 2016–2027. Fukata, M., et al., 2009. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders. Semin. Immunol. 21, 242–253. Ghimire, L., et al., 2018. NLRP6 negatively regulates pulmonary host defense in Grampositive bacterial infection through modulating neutrophil recruitment and function.
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