Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis

Sara Brilha,Catherine W M Ong,Nacho Romero,Babette Weksler,Jon S Friedland,Pierre-Olivier Couraud

doi:10.1038/s41598-017-16250-3

Abstract

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB.

Highlights

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation
The co-culture blood-brain barrier (BBB) model was stimulated with conditioned medium from Mtb-infected monocytes (CoMtb) or control medium (CoMCont) to mimic in vivo cellular networks between astrocytes/endothelial cells and Mycobacterium tuberculosis (Mtb)-infected monocyte-derived cells, which are important in immune responses against Mtb
Since Mtb invasion was associated with decreased expression of the tight junction protein (TJP) claudin-5 and occludin, and the Sonic hedgehog (Shh) pathway is involved in TJP expression and maintaining tight junctions (TJ) integrity[6] (Fig. 6a), we evaluated whether Mtb stimulation decreased production of Shh

Summary

Introduction

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. The blood-brain barrier (BBB) protects the brain, by regulating the transport of substances into the CNS to maintain homeostasis of the microenvironment It is composed by capillary endothelial cells, surrounded by a basement membrane (comprised of type IV collagen, laminin and fibronectin), pericytes, and the astrocytic perivascular end-feet[3]. In the adult CNS, the hedgehog (Hh) pathway is involved in inhibition of endothelial secretion of chemokines and expression of adhesion proteins required for leukocyte extravasation to the brain[6]

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Conclusion