Abstract

BackgroundVascular 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.MethodsHuman 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.ResultsHMVECs 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.ConclusionsThese 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.

Highlights

  • Vascular dysfunction is commonly seen during severe viral infections

  • Knockdown of Endothelial nitric oxide synthase (eNOS) potentiates cytokine and chemokine release after toll-like receptor 3 (TLR3) activation Data has demonstrated that knockdown of eNOS potentiates TLR4-mediated inflammation [8]

  • This is in comparison to the TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling pathway utilized by endosomal toll-like receptors (TLRs), such as TLR3

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Summary

Introduction

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. Koch et al Cell Communication and Signaling (2019) 17:33 expressed enzyme, endothelial nitric oxide synthase (eNOS) [6] Both the over and under production of NO during inflammatory challenge has been linked to alterations in endothelial barrier dysfunction, though its role remains controversial, especially in the setting of sepsis and severe, acute infections [7]. While there certainly is a role for NO in that pathology, it has been shown that eNOS can alter inflammatory signaling in response to infectious challenge in an NO-independent manner [8] In this setting, the modulatory effects of eNOS on inflammation appear to be related through regulation of the p38 mitogen activated protein kinase (MAPK) pathway in TLR4 activation. Though homology exists among all TLRs, within endothelial cells, TLR4 activation is dependent more on cell surface activation while TLR3, the TLR responsible for activation by double stranded RNA (dsRNA) viruses, requires endocytosis prior to inflammatory stimulation

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