The functional and inflammatory response of brain endothelial cells to Toll-Like Receptor agonists

Rebecca H Johnson,Dan T Kho,E Scott Graham,Simon J O’ Carroll,Catherine E Angel

doi:10.1038/s41598-018-28518-3

Rebecca H Johnson, Dan T Kho + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-28518-3

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Abstract

Toll-Like receptors (TLRs) represent an important early warning mechanism for the immune system to detect infection or tissue damage. The focus of this research was to determine the neuroinflammatory responses to commercial TLR ligands and their effects on brain endothelial barrier strength. Using biosensor technology we screened TLR ligands to all human TLRs and found that the brain endothelial hCMVECs cell line only responded to Poly(I:C) (TLR3-ligand), LPS (TLR4-ligand) and Imiquimod (TLR7 ligand). Both Poly(I:C) and LPS induced pronounced pro-inflammatory cytokine secretion as expected, whereas Imiquimod did not induce secretion of any pro-inflammatory cytokines. Using ECIS technology to measure endothelial barrier function, LPS and Poly(I:C) both acutely reduced barrier-strength, whereas Imiquimod caused immediate and sustained strengthening of the barrier. Further cytokine and ECIS studies showed that Imiquimod could abrogate some of the pro-inflammatory responses to Poly(I:C) and LPS. Most surprisingly, PCR revealed that the hCMVECs lacked TLR7 but expressed both TLR3 and TLR4 and did not respond to other structurally different TLR7 ligands. These data demonstrate that brain endothelial cells can be regulated by TLR 3 and TLR4 ligands in a pro-inflammatory manner and have receptors to Imiquimod, distinct to the classical TLR7, that function in an anti-inflammatory manner.

Highlights

Endothelial cells of the blood-brain barrier (BBB) are an integral component in the regulation of immune cell entry into the central nervous system (CNS)
5 μg/mL of Imiquimod exerted an immediate and sustained significant increase in Cell Index’ (CI) compared to control. This data indicates that human cerebral microvascular endothelial cells (hCMVECs) are responsive to this small Imidazoquinoline (Fig. 1), but in a manner that differs to the response to Poly(I:C) and LPS
Using the RTCA xCELLigence Biosensor technology we were able to assess the temporal effects of Toll-Like receptors (TLRs) ligands on the brain (cerebrovascular) endothelial cells (BECs), and determine the global response of these cells[17]

Summary

Introduction

Endothelial cells of the blood-brain barrier (BBB) are an integral component in the regulation of immune cell entry into the central nervous system (CNS). The primary function of these receptors is the recognition of specific and distinct conserved endogenous and exogenous molecular patterns (Danger Associated Molecular Patterns (DAMP) and Pathogen Associated Molecular Patterns (PAMP) respectively)[4,5] These receptors mediate their effects through the TIR-domain-containing adaptor-inducing interferon-β (TRIF) or myeloid differentiation primary response gene 88 (MyD88) pathways[6,7]. The data presented here are the very first demonstration of Imiquimod mediating a barrier enhancing and immunosuppressive effect on human BECs via a non-TLR7 receptor These data reveal the power of biosensor technologies to uncover the cellular responsiveness to ligands, irrespective of knowledge pertaining to the presence or absence of the ligand’s target receptors. More importantly with regards to neuroinflammation, these data indicate that brain endothelial cells have anti-inflammatory mechanisms to protect themselves against the pro-inflammatory milieu that may occur during peripheral or neuroinflammatory events

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