Effects of TLR4 ligands on inflammatory and lymphatic endothelial specific-cell gene expression in CD14+ PBMC

Blood was donated by a healthy volunteer with no known illness or malignancies. Mononuclear cells were separated from the blood by density gradient and CD14+ cells were isolated using anti-CD14 IgG-conjugated magnetic beads and magnetic separation (Miltenyi Biotec). Cells were cultured for 7 days prior to treatment for 96 hours with one of three TLR4 ligands: LPS, HMGB1 or Nab-Paclitaxel. Total RNA was extracted and was analyzed via SYBR Green RT-qPCR. We used an in-house array to analyze the gene expression of a total of 150 targets including 33 cytokines, 34 cytokine receptors, 11 known TLR targets, 64 other inflammation-related proteins, 31 endothelial related transcription factors, 12 myeloid associated genes, 12 genes expressed primarily in lymphatic endothelial cells, and 23 endothelial cell associated proteins. The results show a LPS, HMGB1 and Nab-Paclitaxel upregulated expression of lymphatic specific markers to varying degrees. LPS had the strongest induction of gene expression followed by HMGB1 and Nab-Paclitaxel, in general. CD14+ peripheral blood mononuclear cells (PBMC) were isolate and cultured. Cells were treated for 96 hours with one of three TLR4 ligands: LPS, HMGB1 or Nab-Paclitaxel. Transcript expression was analyzed by RT-qPCR for 150 targets, including inflammatory and lymphatic endothelial specific-cell genes.