Full
Outline
To investigate the role and underlying mechanism of activating transcription factor 3 (ATF3) in suppressing lipopolysaccharide (LPS)-induced autophagy and inflammatory responses in macrophages.
Firstly, the gene expression omnibus (GEO) database was used to analyze ATF3 expression in peripheral blood mononuclear cells (PBMCs) from sepsis patients, and gene set enrichment analysis (GSEA) was performed to identify enriched signaling pathways. Secondly, RAW264.7 macrophages were divided into a blank control group and an LPS-stimulated group (100 ng/mL LPS). Western blotting and immunofluorescence assay were used to detect ATF3 protein expression and observe its subcellular localization, respectively. Lentiviral transduction was used to generate ATF3 knockdown and overexpression cell lines to evaluate their effects on cytokine release and bacterial clearance. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing was employed to identify downstream target genes transcriptionally regulated by ATF3. Furthermore, the impact of ATF3 knockdown or overexpression on autophagy-related gene 5 (ATG5), autophagy-related gene 16-like 1 (ATG16L1), and autophagy levels was evaluated.
GEO analysis revealed that ATF3 expression was significantly elevated in PBMCs from sepsis patients (P<0.01), and GSEA showed significant enrichment of autophagy-related and inflammation-related pathways (P<0.01). In RAW264.7 cells, 100 ng/mL LPS stimulation significantly increased ATF3 expression in the nucleus than the blank control group (P<0.01). ATF3 knockdown led to increased secretions of TNF-α and IL-6 and enhanced bacterial clearance of macrophages (P<0.01), whereas ATF3 overexpression significantly suppressed TNF-α and IL-6 releases, and remained bacterial clearance at a low level when compared with the conditions in the negative control (NC) group (P<0.01). CUT&Tag results demonstrated that ATF3 was enriched at the promoter regions of key autophagy genes Atg5 and Atg16l1. Compared with the NC group, ATF3 knockdown significantly up-regulated the protein levels of LC3-II/I, ATG5, and ATG16L1 while decreased p62 expression (P<0.01). Conversely, ATF3 overexpression inhibited the expression of LC3-II/I, ATG5, and ATG16L1 (P<0.01), but had no significant effect on p62 level.
Sepsis induces elevated ATF3 expression in macrophages, and suppresses autophagic activity and down-regulates pro-inflammatory cytokines TNF-α and IL-6, which probably mediated by ATF3 regulating transcription of ATG5 and ATG16L1, suggesting ATF3 as a potential therapeutic target for autophagy-inflammation imbalance.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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