Toll-like receptor 3-mediated inflammation by p38 is enhanced by endothelial nitric oxide synthase knockdown.

Koch SR, Choi H, Mace EH, Stark RJ
Cell Commun Signal. 2019 17 (1): 33

PMID: 30987646 · PMCID: PMC6466662 · DOI:10.1186/s12964-019-0345-3

BACKGROUND - Vascular dysfunction is commonly seen during severe viral infections. Endothelial nitric oxide synthase (eNOS), has been postulated to play an important role in regulating vascular homeostasis as well as propagation of the inflammatory reaction. We hypothesized that the loss of eNOS would negatively impact toll-like receptor 3 (TLR3) signaling and worsen vascular function to viral challenge.

METHODS - Human microvascular endothelial cells (HMVECs) were exposed to either control or eNOS siRNA and then treated with Poly I:C, a TLR3 agonist and mimicker of dsRNA viruses. Cells were assessed for protein-protein associations, cytokine and chemokine analysis as well as transendothelial electrical resistance (TEER) as a surrogate of permeability.

RESULTS - HMVECs that had reduced eNOS expression had a significantly elevated increase in IL-6, IL-8 and IP-10 production after Poly I:C. In addition, the knockdown of eNOS enhanced the change in TEER after Poly I:C stimulation. Western blot analysis showed enhanced phosphorylation of p38 in sieNOS treated cells with Poly I:C compared to siControl cells. Proximity ligation assays further demonstrated direct eNOS-p38 protein-protein interactions. The addition of the p38 inhibitor, SB203580, in eNOS knockdown cells reduced both cytokine production after Poly I:C, and as well as mitigated the reduction in TEER, suggesting a direct link between eNOS and p38 in TLR3 signaling.

CONCLUSIONS - These results suggest that reduction of eNOS increases TLR3-mediated inflammation in human endothelial cells in a p38-dependent manner. This finding has important implications for understanding the pathogenesis of severe viral infections and the associated vascular dysfunction.

MeSH Terms (14)

Capillary Permeability Cells, Cultured Chemokine CXCL10 Endothelium, Vascular Gene Knockdown Techniques Humans Inflammation Interleukin-6 Interleukin-8 Nitric Oxide Synthase Type III p38 Mitogen-Activated Protein Kinases Poly I-C RNA, Small Interfering Toll-Like Receptor 3

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