Blood-Brain Barrier in a Haemophilus influenzae Type a In Vitro Infection: Role of Adenosine Receptors A2A and A2B

Mol Neurobiol. 2018 Jun;55(6):5321-5336. doi: 10.1007/s12035-017-0769-y. Epub 2017 Sep 18.

Abstract

The blood-brain barrier (BBB) is mainly made up of tightly connected microvascular endothelial cells (BMECs), surrounded by pericytes (BMPCs) which regulate BBB tightness by providing soluble factors that control endothelial proliferation. Haemophilus influenzae type a (Hia) is able to reach the BBB, crossing it, thus causing meningitis. In this study, by using an in vitro model of BBB, performed with human BMECs and human BMPCs in co-culture, we demonstrated that, after Hia infection, the number of hBMPCs decreased whereas the number of hBMECs increased in comparison with non-infected cells. SEM and TEM images showed that Hia was able to enter hBMECs and reduce TEER and VE-cadherin expression. When the cells were infected in presence of SCH58261 and PSB603 but not DPCPX, an increase in TEER values was observed thus demonstrating that A2A and A2B adenosine receptors play a key role in BBB dysfunction. These results were confirmed by the use of adenosine receptor agonists CGS21680, CCPA, and NECA. In infected co-cultures cAMP and VEGF increased and TEER reduction was counter-balanced by VEGF-R1 or VEGF-R2 antibodies. Moreover, the phosphorylated CREB and Rho-A significantly increased in infected hBMECs and hBMPCs and the presence of SCH58261 and PSB603 significantly abrogated the phosphorylation. In conclusion, this study demonstrated that the infection stimulated A2A and A2B adenosine receptors in hBMECs and hBMPCs thus inducing the pericytes to release large amounts of VEGF. The latter could be responsible for both, pericyte detachment and endothelial cell proliferation, thus provoking BBB impairment.

Keywords: Adenosine receptors; Bacterial infection; Blood–brain barrier; Co-cultures; Human brain microvascular endothelial cells; Human brain microvascular pericytes; VEGF.

MeSH terms

  • Blood-Brain Barrier / metabolism*
  • Blood-Brain Barrier / pathology
  • Blood-Brain Barrier / ultrastructure
  • Cadherins / metabolism
  • Cell Count
  • Coculture Techniques
  • Cyclic AMP / biosynthesis
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Electric Impedance
  • Endothelial Cells / metabolism
  • Endothelial Cells / ultrastructure
  • Haemophilus Infections / metabolism*
  • Haemophilus Infections / virology*
  • Haemophilus influenzae / physiology*
  • Haemophilus influenzae / ultrastructure
  • Humans
  • Microvessels / pathology
  • Pericytes / metabolism
  • Phosphorylation
  • Receptor, Adenosine A2A / metabolism*
  • Receptor, Adenosine A2B / metabolism*
  • Vascular Endothelial Growth Factor A / metabolism
  • rho GTP-Binding Proteins / metabolism

Substances

  • Cadherins
  • Cyclic AMP Response Element-Binding Protein
  • Receptor, Adenosine A2A
  • Receptor, Adenosine A2B
  • Vascular Endothelial Growth Factor A
  • Cyclic AMP
  • rho GTP-Binding Proteins