TET2 promotes IL-1ß expression in J774.1 cell through TLR4/MAPK signaling pathway with demethylation of TAB2 promoter.

Interleukin (IL)-1ß produced by macrophages plays an important role in inflammation development. However, the underlying mechanism in epigenetic regulation of IL-1ß production is not fully addressed. Though DNA methylcytosine dioxygenase ten-eleven translocation 2 (TET2) is known to be involved in the regulation of inflammatory factors by oxidizing 5-methylcytosine (5mC), the underlying molecular mechanism is largely unknown. In this study, we found that the expression of both IL-1ß and TET2 is upregulated by lipopolysaccharide (LPS)-stimulated mononuclear macrophage. We then knocked down TET2 in mouse macrophagelike cell line (J774.1) and found that LPS-induced IL-1ß is also downregulated. In addition, LPS-stimulated phosphorylation of the mitogen-activated protein kinase (MAPK) signaling pathway and intracellular effectors of the toll-like receptor 4 (TLR4) signaling pathway were also suppressed in TET2-knockdown cells. The methylation status in the promoter regions of myeloid differentiation primary response gene (MyD)88 and TAK1 binding protein 2 (TAB2) were estimated by bisulfite polymerase chain reaction. Compared with that of the control, the 5mC level on the TAB2 promoter is downregulated in the LPS-stimulated cells which can be reversed by TET2-knockdown. These findings altogether suggest that LPS-upregulated TET2 enhances IL-1ß expression through demethylating the promoter region of TAB2, the key member of the TLR4/MAPK signaling pathway, a previously unreported molecular mechanism in TET2-regulated expression of inflammatory factors.

Identification of the key differentially expressed genes and pathways involved in neutrophilia.

Polymorphonuclear neutrophils (PMNs) are the most important determinants in the acute inflammatory response. Pathologically increased numbers of PMNs in the circulation or specific tissues (or both) lead to neutrophilia. However, the genes expressed and pathways involved in neutrophilia have yet to be elucidated. By analysis of three public microarray datasets related to neutrophilia (GSE64457, GSE54644, and GSE94923) and evaluation by gene ontology, pathway enrichment, protein-protein interaction networks, and hub genes analysis using multiple methods (DAVID, PATHER, Reactome, STRING, Reactome FI Plugin, and CytoHubba in Cytoscape), we identified the commonly up-regulated and down-regulated different expressed genes. We also discovered that multiple signaling pathways (IL-mediated, LPS-mediated, TNF-α, TLR cascades, MAPK, and PI3K-Akt) were involved in PMN regulation. Our findings suggest that the commonly expressed genes involved in regulation of multiple pathways were the underlying molecular mechanisms in the development of inflammatory, autoimmune, and hematologic diseases that share the common phenotypic characteristics of increased numbers of PMNs. Taken together, these data suggest that these genes are involved in the regulation of neutrophilia and that the corresponding gene products could serve as potential biomarkers and/or therapeutic targets for neutrophilia.

Identification of key protein-coding genes and lncRNAs in spontaneous neutrophil apoptosis.

Polymorphonuclear leukocytes (PMNs) are the most abundant cells of the innate immune system in humans, and spontaneous PMN apoptosis plays crucial roles in maintaining neutrophil homeostasis and resolving inflammation. However, the detailed mechanisms of spontaneous PMN apoptosis remain to be elucidated. By analysis of the public microarray dataset GSE37416, we identified a total of 3050 mRNAs and 220 long non-coding RNAs (lncRNAs) specifically expressed during PMN apoptosis in a time-dependent manner. By short time-series expression miner (STEM) analysis, Gene Ontology analysis, and lncRNA-mRNA co-expression network analyses, we identified some key molecules specifically related to PMN apoptosis. STEM analysis identified 12 gene profiles with statistically significance, including 2 associated with apoptosis. Protein-protein interaction (PPI) network analysis of the genes from 2 profiles and lncRNA-mRNA co-expression network analysis identified a 12-gene hub (including NFκB1 and BIRC3) associated with apoptosis, as well as 2 highly correlated lncRNAs (THAP9-AS1, and AL021707.6). We experimentally examined the expression profiles of two mRNA (NFκB1 and BIRC3) and two lncRNAs (THAP9-AS1 andAL021707.6) by quantitative real-time polymerase chain reaction to confirm their time-dependent expressions. These data altogether demonstrated that these genes are involved in the regulation of spontaneous neutrophil apoptosis and the corresponding gene products could also serve as potential key regulatory molecules for PMN apoptosis and/or therapeutic targets for over-reactive inflammatory response caused by the abnormality in PMN apoptosis.

[Identification of differentially expressed genes and pathways changing in neutrophils of patients with sepsis by bioinformatics analysis].

Objective To identify key pathogenic differentially expressed genes and pathways in neutrophils of patients with sepsis. Methods Firstly, we screened and downloaded a total of 143 experimental and 65 control neutrophil samples from Gene Expression Omnibus (GEO) gene expression profile datasets GSE6535, GSE49755, GSE49756, GSE49757. Secondly, we identified differentially expressed genes (DEGs) via corresponding packages in R software. Finally, through intersecting DEGs from every two datasets of those four GEO datasets, we had got 93 DEGs as candidate DEGs, and subsequently conducted gene ontology and pathway enrichment analysis, PPI network analysis and hub gene analysis, using multiple methods containing DAVID, STRING, Cytoscape Apps such as ReactomeFIPlugIn and Cytohubba. Results We had identified most significant hub DEGs, including TLR2, SRC, MMP9, IL1R2, ARRB1, IRAK3, IL18R1, IL18RAP, and STK17B. Besides, we found that some pathogenicity-related pathways and immune-related biological processes were involved in sepsis. The hub genes found by this study might cause sepsis through a variety of signaling pathways and be implicate in the pathogenesis of sepsis. Conclusion These genes may cause the onset and development of sepsis through a variety of signaling pathways.