Pam3Cys-treated HUVEC before and after TLR2 knockdown

The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) Toll-like receptor 2 (TLR2). In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of IL6, IL8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that bacterial lipopeptides activate human EC exclusively through TLR2, that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, and that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis. qPCR gene expression profiling. HUVEC monolayers were left untreated or treated with 10 μg/mL of Pam3Cys for 3 hr prior to lysis. RQ values were calculated using the 2-ΔΔCt method. Three biological replicates and one technical replicate were analyzed in both the untreated and Pam3Cys-treated TLR2 transfected groups. Four biological replicates and one technical replicate were analyzed in the Pam3Cys-treated group (the data for three untransfected samples and one control siRNA-transfected sample were combined).