Myocardial dysfunction in sepsis has been linked to inflammation caused by pathogen-associated molecular patterns (PAMPs) as well as by host danger-associated molecular patterns (DAMPs). These include soluble heparan sulfate (HS), which triggers the devastating consequences of the pro-inflammatory cascades in severe sepsis and septic shock. Thus, there is increasing interest in the development of anti-infective agents, with effectiveness against both PAMPs and DAMPs. We hypothesized that a synthetic antimicrobial peptide (peptide 19-2.5) inhibits inflammatory response in murine cardiomyocytes (HL-1 cells) stimulated with PAMPs, DAMPs or serum from patients with septic shock by reduction and/or neutralization of soluble HS. In the current study, our data indicate that the treatment with peptide 19-2.5 decreases the inflammatory response in HL-1 cells stimulated with either PAMPs or DAMPs. Furthermore, our work shows that soluble HS in serum from patients with Gram-negative or Gram-positive septic shock induces a strong pro-inflammatory response in HL-1 cells, which can be effectively blocked by peptide 19-2.5. Based on these findings, peptide 19-2.5 is a novel anti-inflammatory agent interacting with both PAMPs and DAMPs, suggesting peptide 19-2.5 may have the potential for further development as a broad-spectrum anti-inflammatory agent in sepsis-induced myocardial inflammation and dysfunction.
Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signaling molecules modulates the expression of pro-and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous nuclear ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating hematopoietic cells, was studied in noninduced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed several mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor-β-activated kinase 1 (TAK1) a central player in TLR4 signaling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3 ′ UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis or stability but enhances TAK1 mRNA translation, resulting in elevated TNF-α, IL-1β, and IL-10 mRNA expression. Our data suggest that the hnRNP K-3 ′ UTR complex inhibits TAK1 mRNA translation in noninduced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesized TAK1 boosts macrophage inflammatory response.
Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments) and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19–2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19–2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19–2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19–2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19–2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19–2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19–2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19–2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19–2.5 seems to be a potential anti-inflammatory agent in sepsis.
To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPSactivated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan TM we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPSinduced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.
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