Background: Each year 1.7 million people are diagnosed with sepsis in the USA. A putative target to prevent systemic inflammatory response syndrome is via antagonism of toll-like receptors 2 and 4 (TLR2/TLR4) in vascular endothelial cells. Proteoglycan-4 (PRG4) is a mucinous glycoprotein that interacts with CD44 and TLR4 resulting in a blockade of the NLRP3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less IL-6 expression after rhPRG4 treatment in vitro.
Methods: 1) Human lung microvascular endothelial cells (HLMVEC) were seeded into multi-well plates and at 80% confluence were treated with 250 ng/mL LPS-EK (InvivoGen). Three groups were subsequently treated with rhPRG4 (50, 100, 150 µg/mL; Lubris) after 30 mins. Cells were incubated for 24 hrs at 37˚C. IL-6 levels in cellular media were measured via ELISA (Abcam). RNA was collected, reverse transcribed into cDNA and used for qPCR analysis with primers for IL-6 and 18S. 2) HLMVEC were treated with 1:10 diluted plasma from 15 sepsis emergency department patients in culture media. After 30 mins, either 50 or 100 µg/mL rhPRG4 was administered. Cells were incubated at 37˚C for 24 hrs. IL-6 levels in cell media were measured as above. ANOVA was used for statistical analysis with Tukey post-hoc comparison. qPCR results were determined via delta-delta Ct analysis. Patient sample collections were approved by the Institutional Review Board.
Results: 1) HLMVEC treated with LPS had significantly increased IL-6 protein levels (2733 pg/mL) compared to controls (277 pg/mL) (p <0.0001). All concentrations of rhPRG4 significantly reduced IL-6 (566, 348, 242 pg/mL) (p <0.0001). There was no significant change in IL-6 levels in cells treated with rhPRG4 alone. IL-6 gene expression was significantly increased after LPS treatment (p <0.05) compared to controls. This response was reversed by 50 or 100 µg/mL rhPRG4 (p <0.05). There were no significant changes in IL-6 gene expression in cells treated with rhPRG4 alone. 2) 47% and 33% of the cells treated with patient plasma had significantly (p <0.05) reduced protein levels of IL-6 in the cellular media in the presence of either 50 or 100 µg/mL rhPRG4.
Conclusion: rhPRG4 significantly reduces IL-6 protein levels and gene expression after a septic phenotype is induced in human endothelial cells via LPS or septic patient plasma.