Cytosolic DNA-STING-NLRP3 axis is involved in murine acute lung injury induced by lipopolysaccharide

Abstract

The role of NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis in acute lung injury (ALI) has been well identified previously. Stimulator of interferon genes (STING) is an indispensable adaptor protein, which could regulate inflammation and pyroptosis during infection; however, its role in lipopolysaccharide (LPS)-induced ALI remains obscure. This study aimed to explore whether STING participated in the development of LPS-induced ALI as well as the underlying mechanism. We confirmed that LPS significantly enhanced the expression and phosphorylation of STING in lung tissue and primary macrophages from mice. STING deficiency relieved inflammation and oxidative stress in LPS-treated murine lungs and macrophages. Meanwhile, STING deficiency also abolished the activation of NLRP3 inflammasome and pyroptosis; however, NLRP3 overexpression by adenovirus offset the beneficial effects of STING deficiency in macrophages treated with LPS. Additionally, the level of mitochondrial DNA (mt-DNA) significantly increased in macrophages after LPS treatment. Intriguingly, although exogenous mt-DNA stimulation did not influence the level of STING, it could still trigger the phosphorylation of STING as well as pyroptosis, inflammation, and oxidative stress of macrophages. And the adverse effects induced by mt-DNA could be offset after STING was knocked out. Furthermore, the inhibition of the sensory receptor of cytosolic DNA (cyclic GMP-AMP synthase, cGAS) also blocked the activation of STING and NLRP3 inflammasome, meanwhile, it alleviated ALI without affecting the expression of STING after LPS challenge. Furthermore, cGAS inhibition also blocked the production of cGAMP induced by LPS, indicating that mt-DNA and cGAS could activate STING-NLRP3-mediated pyroptosis independent of the expression of STING. Finally, we found that LPS upregulated the expression of transcription factor c-Myc, which subsequently enhanced the activity of STING promoter and promoted its expression without affecting its phosphorylation. Collectively, our study disclosed that LPS could activate STING in a cytosolic DNA-dependent manner and upregulate the expression of STING in a c-Myc-dependent manner, which cooperatively contribute to ALI.

Keywords: NLRP3; STING; acute lung injury; cytosolic DNA; sepsis.

FIGURE 1

LPS significantly increased the expression of STING in vivo and in vitro during ALI. A, Western blots and statistical results in murine lung tissues. B, Relative mRNA level of STING in murine lung tissues after LPS instillation. (n = 6, *P<.05 vs 0 h group). C, Western blots and statistical results in peritoneal macrophages. D, Relative mRNA level of STING in primary macrophages after LPS stimulation (n = 6, *P<.05 vs 0 h group)

FIGURE 2

STING deficiency attenuated ALI induced by LPS for 6 h in mice. A, Western blots in murine lung from wild‐type (WT) mice and STING knock out (KO) mice. B‐D, The count of the total cells, macrophages, and neutrophil in BALF. E and F, Relative mRNA levels of TNF‐α and IL‐1β. G, H&E staining for lung tissues. H, MPO activity in lung tissues. I, LDH activity in BALF. J, Wet to dry ratio of lung. K, Immunohistochemistry staining for 4‐HNE protein. L and M, The markers of oxidative stress, including MDA and SOD. N, Representative images of fluorescence probe for ROS in lung tissues (n = 6, *P<.05 vs control+WT group, #P<.05 vs LPS+WT group). O, The 7‐day survival rate after LPS instillation (n = 10, *P<.05 vs control+WT group, #P<.05 vs LPS+WT group)

FIGURE 3

STING deficiency inhibited NLRP3 inflammasome and pyroptosis in lung stimulated by LPS for 6 h. A, Western blots and statistical results for NLRP3, ASC, and Pro‐caspase‐1 in murine lung tissues. B, Western blots and statistical results for IL‐1β, IL‐18, and Cle‐GSDMD in murine lung tissues. C and D, ELISA for IL‐1β and IL‐18 in murine lung tissues. E, Immunohistochemistry staining for Caspase‐1 protein (n = 6, *P<.05 vs control+WT group, #P<.05 vs LPS+WT group)

FIGURE 4

NLRP3 overexpression abolished the protective roles of STING deficiency in macrophages stimulated by LPS for 6 h. A, Western blots and statistical results for NLRP3 in peritoneal macrophages from WT mice and STING KO mice. B, LDH content in medium in indicated group. C, Cell viability was detected by a CCK8 assay in indicated group. D‐F, Relative mRNA levels of TNF‐α, IL‐6, and IL‐1β in indicated group. G and H, The markers of oxidative stress, including MDA and SOD (n = 6, *P<.05 vs indicated group, NS means no significance)

FIGURE 5

NLRP3 overexpression is involved in STING‐mediated pyroptosis in macrophages stimulated by LPS for 6 h. A, Western blots and statistical results for IL‐1β, IL‐18, and Cle‐GSDMD in peritoneal macrophages. B and C, ELISA for IL‐1β and IL‐18 in peritoneal macrophages (n = 6, *P<.05 vs indicated group, NS means no significance)

FIGURE 6

mt‐DNA in cytoplasm triggered NLRP3 activation and pyroptosis in a STING‐dependent manner. A, mt‐DNA in cytosol in peritoneal macrophages. B, Western blots and statistical results for STING and phosphorylated STING in peritoneal macrophages. C, Relative mRNA level of STING in peritoneal macrophages with or without mt‐DNA transfection. D, Relative mRNA levels of STING with or without CCCP pretreatment. E, Western blots and statistical results for NLRP3, IL‐1β, and IL‐18 in peritoneal macrophages from WT mice and STING KO mice with or without mt‐DNA transfection. F and G, Expression levels of pyroptosis markers in culture medium as detected by ELISA. H, LDH content in medium. I, Relative mRNA levels of TNF‐α. J, SOD activity (n = 6, *P<.05 vs indicated group, NS means no significance)

FIGURE 7

cGAS inhibition alleviated ALI in vitro and in vivo. A, Relative mRNA levels of STING, NLRP3, IL‐1β, and IL‐18 in peritoneal macrophages. B, Relative mRNA level of TNF‐α. C, LDH content in medium. D, MDA content in lung tissues (n = 6, *P<.05 vs si NC+PBS group, #P<.05 vs si NC+LPS group). E, Western blots and statistical results for cGAS in lung tissues from WT mice and cGAS KO mice. F and G, Relative mRNA levels of TNF‐α and IL‐1β. H, MPO activity in lung tissues. I, Wet to dry ratio of lung. J, H&E staining for lung tissues. K, Representative images of fluorescence probe for ROS in lung tissues. L, Western blots and statistical results for phosphorylated STING, STING, and NLRP3 (n = 6, ^& P<.05 vs control+WT group,^$ P<.05 vs LPS+WT group)

FIGURE 8

LPS stimulation for 6 h enhanced the transcriptional activation of STING via c‐Myc. A, Promoter sequence of STING in Homo sapiens. B, Mutation analysis of candidate transcription factor for binding sites in promoter region of STING in HEK 293 cells. C, Chromatin immunoprecipitation assay of c‐Myc and STING promoter with or without c‐Myc overexpression in HEK 293 cells. D, Relative promoter activity of STING with or without c‐Myc overexpression in HEK 293 cells. E and F, Western blots and statistical results for phosphorylated STING and STING after c‐Myc was upregulated or inhibited. G, Western blots and statistical results for c‐Myc with or without LPS stimulation in peritoneal macrophages. H, Chromatin immunoprecipitation assay of c‐Myc and STING promoter. I, Relative promoter activity of STING. J, Relative mRNA levels of STING, NLRP3, IL‐1β, and IL‐18 (n = 6, *P<.05 vs indicated group, NS means no significance)