SUMMARY MicroRNAs regulated by LPS target genes that contribute to the inflammatory phenotype. Here we show that Akt1, which is activated by LPS, differentially regulates miRNAs including let-7e, miR-155, miR-181c and miR125b. In silico analyses and transfection studies revealed that let-7e represses TLR4 while miR-155 represses SOCS1, two genes critical for LPS-driven TLR signalling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1−/− macrophages exhibited increased responsiveness to LPS in culture and Akt1−/− mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1−/− macrophages restores sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.
High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M−/− mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M−/− macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2−/− macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1−/− macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.
During infections, pathogens bind to toll-like receptor (TLR)4 and CD14 receptors and induce cytokine release, leading to inflammation. Here, we investigated TLR4 and CD14 expression on peripheral blood leukocytes (PBLs) and their roles in lipopolysaccharide (LPS)-induced cytokine and chemokine release. Full-term and preterm neonates and adults were studied. PBLs were pretreated with anti-TLR4 -and anti-CD14 -blocking antibodies and stimulated with LPS. Cytokine and chemokine levels were measured in supernatants. TLR4, CD14 expression, and LPS-induced CXCL8 release were higher in neonates, possibly contributing to aberrant inflammation. TLR4 blockade resulted in approximately 3-fold greater suppression of LPS-induced CXCL8 release in preterm neonates (38%) than in adults (14%). CD14 blockade (ϳ80%) in neonates induced approximately 3-fold greater inhibition of CXCL8 release, compared with anti-TLR4 (ϳ30%). Anti-TLR4 partly (50 -60%) inhibited IL-10 and TNF-␣, whereas anti-CD14 completely suppressed their release. Our findings reveal that neonates depend more on TLR4 for CXCL8 release. Furthermore, neonatal LPS-induced CXCL8 release, apart from TLR4/CD14-mediated signaling, is regulated by LPS interactions with other TLRs and/or immune receptors. IL-10 and TNF-␣ release depends on LPS binding not only to CD14/TLR4 but also to CD14 associated with another TLR. Our findings reveal the contribution of TLR4 and CD14 in neonatal cytokine and chemokine release and could aid in design of antagonists to prevent harmful inflammation. (Pediatr Res 66: 179-184, 2009) N eonates are highly susceptible to Gram-negative bacteria that induce high morbidity and mortality. The principal pathogenic agent involved in neonatal sepsis induced by Gramnegative bacteria is endotoxin lipopolysaccharide (LPS), an essential component of their surface. Defense against pathogens is offered by immune cells, such as granulocytes, monocytes, and dendritic cells (DCs), which express pattern-recognition receptors (PRRs) that recognize specific structures present on microorganisms, termed pathogen-associated molecular patterns (PAMPs) (1). Binding of PAMPs to PRRs triggers antimicrobial responses to combat the infection (1,2).LPS is one of the best characterized PAMPs that binds to the CD14/toll-like receptor (TLR)4/MD2 complex of PRRs and activates intracellular signaling (3). CD14 binds to LPS but lacks an intracellular component and is, thus, incapable of signaling. MD2 is a molecule necessary for LPS recognition by TLR4, which also cannot mediate signaling. TLR4, upon LPS binding, leads to intracellular activation of mitogenactivated protein kinase and nuclear factor-B that mediate the transcription of proinflammatory cytokine and chemokine genes (4). TLR4 signaling also activates DCs that subsequently present pathogenic peptides to T lymphocytes and, thus, stimulate T-cell-mediated immunity (5).The specific roles of TLR4 and CD14 in LPS-induced inflammatory responses by neonatal leukocytes remain not clearly defined. Studies in neona...
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