A model of the Blood Brain Barrier to investigate immune trafficking in neurological disorders
Lauranzano E1, Molteni R2, Filipello F1, Grassi F3, Pardi R2, Matteoli M1,4
1Humanitas Clinical and Research Center, Rozzano, Italy; 2San Raffaele Scientific Institute, Milan, Italy;3 Institute for Research in Biomedicine, Bellinzona, Switzerland; 4 CNR Institute of Neuroscience, Milan, Italy
Progressive blood-brain barrier (BBB) breakdown and loss of solute barrier are associated with a diverse range of neuroinflammatory and neurodegenerative conditions. Extracellular ATP and adenosine are soluble mediators and important signaling molecules acting locally and in a reversible manner at the BBB both in physiological and pathological conditions. Their actions are mediated by purinergic receptors, and terminated by metabolizing enzymes, named ectonucleotidases (NTPDases). NTPDases are expressed at the BBB by endothelial cells (EC), pericytes and astrocytes (As). To date, the role of ATP and adenosine in modulating neuroinflammatory processes is not fully understood yet.
We developed an in vitro BBB model consisting of a contact co-culture of brain EC growing as a monolayer on top of a matrix-coated permeable membrane, and As cultured on the opposite side with As endfeet taking contact with EC. We monitored transendothelial electrical resistance (TEER) as the main indicator of the functional formation of the barrier. Barrier integrity was confirmed by the formation of tight junctions between adjacent EC, by immunofluorescence for claudin-5 and ZO-1. Permeability assays using the impermeable dye LY or the hydrophilic dye NaF, and Dextran confirmed low solute passive transport and protein extravasation, respectively. Inserts with fully differentiated BBB were placed in a microfluidic platform to assess transmigration of immune cells. Real-time 4D tracking of cells under flow conditions revealed the initial rolling and adhesion of leukocytes to EC, followed by subsequent diapedesis across the BBB and interstitial migration. NTPDases expression correlated with T cells interaction with the BBB and were involved in the control of T cells transmigration.
This microfluidic platform provides a new and versatile tool to investigate in vitro the stepwise process of circulating immune cell extravasation across the BBB and to test the role of ATP/adenosine axis as a druggable target to modulate immune infiltration and interfere with the defective neuroimmune interaction at the BBB.