miR-23b Negatively Regulates Sepsis-Induced Inflammatory Responses by Targeting ADAM10 in Human THP-1 Monocytes.

BACKGROUND: Previous studies have demonstrated pivotal roles of disintegrin and metalloproteinase 10 (ADAM10) in the pathogenesis of sepsis. MicroRNA- (miR-) 23b has emerged as an anti-inflammatory factor that prevents multiple autoimmune diseases. However, the underlying mechanisms of miR-23b in the regulation of ADAM10 and sepsis remain uncharacterized. METHODS: The expression levels of ADAM10 and miR-23b were detected by quantitative RT-PCR and western blot analysis. Cytokine production and THP-1 cell apoptosis were measured by enzyme-linked immunosorbent and annexin V apoptosis assays. Bioinformatics analyses and qRT-PCR, western blot, and luciferase reporter assays were performed to identify ADAM10 as the target gene of miR-23b. RESULTS: miR-23b expression was downregulated in the peripheral blood mononuclear cells of sepsis patients and LPS-induced THP-1 cells and was negatively correlated with the expression of ADAM10 and inflammatory cytokines. miR-23b regulated ADAM10 expression by directly binding to the 3'-UTR of ADAM10 mRNA. The overexpression of miR-23b alleviated the LPS-stimulated production of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and apoptosis by targeting ADAM10 in THP-1 cells. The inhibitor or knockdown of ADAM10 elicited effects similar to those of miR-23b on THP-1 cells upon LPS stimulation. CONCLUSIONS: The present study demonstrated that miR-23b negatively regulated LPS-induced inflammatory responses by targeting ADAM10. The molecular regulatory mechanism of miR-23b in ADAM10 expression and sepsis-induced inflammatory consequences may provide potential therapeutic targets for sepsis.

A Functional Polymorphism-Mediated Disruption of EGR1/ADAM10 Pathway Confers the Risk of Sepsis Progression.

Increasing evidence has indicated that single nucleotide polymorphisms (SNPs) are related to the susceptibility of sepsis and might provide potential evidence for the mechanisms of sepsis. Our recent preliminary study showed that the ADAM10 genetic polymorphism was clinically associated with the development of sepsis, and little is known about the underlying mechanism. The aim of this study was to confirm the association between the ADAM10 promoter rs653765 G→A polymorphism and the progression of sepsis and to discover the underlying mechanism. Clinical data showed that the rs653765 G→A polymorphism was positively correlated with the development of sepsis, as evidenced by a multiple-center case-control association study with a large sample size, and showed that EGR1 and ADAM10 levels were associated well with the different subtypes of sepsis patients. In vitro results demonstrated that the rs653765 G→A variants could functionally modulate ADAM10 promoter activity by altering the binding of the EGR1 transcription factor (TF) to the ADAM10 promoter, affecting the transcription and translation of the ADAM10 gene. Electrophoretic mobility shift assay (EMSA) followed by chromatin immunoprecipitation (ChIP) assay indicated the direct interaction. Functional studies further identified that the EGR1/ADAM10 pathway is important for the inflammatory response. EGR1 intervention in vivo decreased host proinflammatory cytokine secretion and rescued the survival and tissue injury of the mouse endotoxemia model.IMPORTANCE Sepsis is characterized as life-threatening organ dysfunction, with unacceptably high mortality. Evidence has indicated that functional SNPs within inflammatory genes are associated with susceptibility, progression, and prognosis of sepsis. These mechanisms on which these susceptible sites depended often suggest the key pathogenesis and potential targets in sepsis. In the present study, we confirmed that a functional variant acts as an important genetic factor that confers the progression of sepsis in a large sample size and in multiple centers and revealed that the variants modulate the EGR1/ADAM10 pathway and influence the severity of sepsis. We believe that we provide an important insight into this new pathway involving the regulation of inflammatory process of sepsis based on the clinical genetic evidence, which will enhance the understanding of nosogenesis of sepsis and provide the potential target for inflammation-related diseases.

Association between genetic polymorphisms in the autophagy-related 5 gene promoter and the risk of sepsis.

Previous studies demonstrated significant roles of autophagy in the pathogenesis of sepsis, but few studies focused on the effect of autophagy-related SNPs on sepsis susceptibility. In this present study, five polymorphisms of ATG5/ATG16L1 were investigated for the possible risk on sepsis in a Chinese Han population. Our results showed that ATG5 expression levels decreased with the severity of sepsis, and rs506027 T > C and rs510432 G > A were associated with sepsis progression and mortality. Moreover, the rs506027 TT and rs510432 GG carriers also exhibited increased expression levels of ATG5. Functional assays showed that ATG5 knockdown elevated the secretion of pro-inflammatory cytokines in THP-1 cells, and the extracted mononuclear cell of the risk C-A carriers exhibited decreased ATG5 expression levels, leading to enhanced releases of TNF-α and IL-1ß under LPS stimulation in vitro. Furthermore, ATG5 T-G haplotype mutation showed higher promoter activities compared to C-A haplotype mutation, suggesting the effect of these SNPs on ATG5 gene transcription. Taken together, these results above indicated that these two ATG5 promoter polymorphisms may be functional and clinically significant for sepsis progression, underscoring its potentially therapeutic implications for sepsis and other inflammatory diseases.

Association study of MCP-1 promoter polymorphisms with the susceptibility and progression of sepsis.

Previous studies have indicated that the monocyte chemo-attractant protein 1 (MCP-1), also referred to as C-C motif chemokine ligand 2 (CCL2), plays a significant role in the pathogenesis of sepsis, and this study investigated the clinical relevance of two MCP-1 gene polymorphisms on sepsis onset and progression. The Multiplex SNaPshot genotyping method was used to detect MCP-1 gene polymorphisms in the Chinese Han population (403 sepsis patients and 400 controls). MCP-1 mRNA expression levels were measured using real-time quantitative PCR, and enzyme-linked immunosorbent assays were used to analyze MCP-1, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and interleukin-1 beta (IL-1ß) plasma concentrations. The rs1024611 polymorphism analysis showed lower frequencies of minor homozygous genotype (AA) and allele (A) in sepsis patients compared to the healthy controls (19.4% vs. 31.5%, P = 0.0001 and 45.9% vs. 54.8%, P = 0.0004, respectively). And the frequencies of GG genotype and G allele were lower in sepsis patients compared to the controls (19.6% vs. 31.3%, P = 0.0002 and 46.0% vs. 54.5%, P = 0.0007, respectively). The rs1024611 AG/GG and rs2857656 GC/CC genotypes were both overrepresented in patients with severe sepsis (both P = 0.0005) and septic shock (P = 0.010 and P = 0.015, respectively) compared to the patients with mild sepsis. Moreover, among sepsis patients, the rs1024611 AG/GG and rs2857656 GC/CC carriers exhibited significant increases in expression levels of MCP-1 (P = 0.025), TNF-α (P = 0.034) and IL-6 (P = 0.043) compared with the rs1024611 AA or rs2857656 GG carriers. This study provides valuable clinical evidence that the MCP-1/CCL2 polymorphisms rs1024611 and rs2857656 are associated with sepsis susceptibility and development. We conclude that MCP-1/CCL2 plays a significant role in the pathogenesis of sepsis, which has potentially important therapeutic implications.