The Role Of NLRP3 Inflammasome In Mouse Inflammation-related Lung Tumorigenesis

ObjectiveInflammatory micro-environment has been proposed to play a critical role in lung tumorigenesis.NLRP3 inflammasome is known as an intracellular receptor involving inflammation and has been reported which is increasingly associated with tumor development,but the role in lung cancer,especially in inflammation-related lung cancer,remains unclear.In this study,we aim to explore the role of NLRP3 inflammasome in benzo(a)pyrene [B(a)p] and lipopolysaccharide(LPS)-induced inflammation-related lung tumorigenesis in mice,which will provide a novel preventive and therapeutic strategy for lung cancer.Methods1.Wild-type(WT)mice and NLRP3-/-mice in SPF grade 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?L 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?L saline,2.5?g/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 using a straightedge.The morphological changes of alveolar cells were evaluated using transmission electron microscopy.The pathological alterations and pathological types of lung tumors were evaluated by HE staining.2.The expression of NLRP3,interleukin-1?(IL-1?)and IL-18 protein were detected using immunohistochemistry.The co-localization of NLRP3 with caspase-1 was determined using immunofluorescence staining.Western blot was used to evaluate the levels of caspase-1 p10 and cleaved-IL-1? protein.The expression of IL-18 protein in bronchoalveolar lavage fluid(BALF)was evaluated using ELISA kit.Results1.Mice exposed to B(a)p or B(a)p plus LPS could induce lung tumors,whereas LPS or vehicles exposure could not induce lung tumorigenesis.In WT mice,the tumor incidence of mice exposed to B(a)p and B(a)p plus LPS was 82.05% and 96.97%,respectively,but the incidence of NLRP3-/-mice were 64.71% and 84.62%,respectively.WT mice treated with B(a)p plus LPS developed 13.0±12.4 visible tumors/mouse on the surface of the lung,which was significantly higher compared with that in mice treated with B(a)p alone(4.7±5.7 tumors/mouse)(P<0.05).Similarly,the visible tumors/mouse on the surface of the lung in B(a)p plus LPS group(5.7±6.1)were also higher than that in B(a)p group(2.0±2.4)in NLRP3-/-mice(P<0.05).Moreover,NLRP3 deletion decreased the visible tumors/mouse on the surface of the lung induced by B(a)p or B(a)p plus LPS(P<0.05,respectively).In addition,smaller tumors(?1mm)were more abundant in B(a)p plus LPS treatment than that in B(a)p treatment alone in both WT mice and NLRP3-/-mice(P<0.05,respectively).Also,the frequency of larger tumors(>1mm)was significantly higher in WT mice exposed to B(a)p or B(a)p plus LPS than those in NLRP3-/-mice(P<0.05,respectively).B(a)p plus LPS exposure could induce the formation of heterochromatin in WT mice,which was detected using transmission electron microscopy.Histopathological examination found the significantly inflammatory changes in WT mice exposed to LPS or vehicles,and NLRP3 deletion could improve the inflammatory changes induced by LPS,but vehicles-induced inflammatory changes still persist.B(a)p or B(a)p plus LPS treatment induced the formation of pathological tumor nests in both WT mice and NLRP3-/-mice,and NLRP3 deletion decreased the pathological tumor nests induced by B(a)p or B(a)p plus LPS.WT mice and NLRP3-/-mice exposed to B(a)p or B(a)p plus LPS could induce the development of lung adenoma and squamous cell carcinoma.In WT mice,the incidence of adenoma in mice exposed to B(a)p(91.7%)or B(a)p plus LPS(85.7%)were significantly increased compared with the development of lung squamous cell carcinoma in respective treatment(P<0.05,respectively).Moreover,NLRP3-/-mice treated with B(a)p or B(a)p plus LPS also mainly developed lung adenoma with 92.9% and 87.5%,respectively.2.The levels of NLRP3 and IL-18 protein were increased in WT mice treated with B(a)p or B(a)p plus LPS compared with those in Vehicle control group,and the IL-18 protein in lung tissues of mice treated with B(a)p plus LPS was increased than that in mice treated with B(a)p alone(P<0.05,respectively).The results of immunofluorescence staining indicated the co-localization of NLRP3 with caspase-1 was increased in LPS-,B(a)p-and B(a)p plus LPS-treated mice than that in Vehicle control group(P<0.05,respectively).In addition,mice exposed to B(a)p plus LPS could induce caspase-1 activation(p10),and the increase of cleaved-IL-1? in lung tissues and IL-18 protein in BALF compared with those in B(a)p group(P<0.05,respectively).Conclusions1.Pulmonary inflammation induced by LPS could enhance B(a)p-induced lung tumorigenesis,suggesting that this study successfully developed a mouse model of inflammation-related lung tumorigenesis,and NLRP3 deletion inhibited lung tumorigenesis induced by B(a)p or B(a)p plus LPS.2.NLRP3 inflammasome activation may be involved in B(a)p plus LPS-induced inflammation-related lung tumorigenesis in mice,but the mechanisms of NLRP3 activation should be further investigated.