Shiga Toxin 1 Induces on Lipopolysaccharide-Treated Astrocytes the Release of Tumor Necrosis Factor-alpha that Alter Brain-Like Endothelium Integrity

Verónica I Landoni,María J Lapponi,Marcelo De Campos Nebel,Martín A Isturiz,Cecilia Calatayud,Gabriela C Fernández,Pablo Schierloh,Kenneth A Bradley

doi:10.1371/journal.ppat.1002632

Abstract

The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia and renal dysfunction. The typical form of HUS is generally associated with infections by Gram-negative Shiga toxin (Stx)-producing Escherichia coli (STEC). Endothelial dysfunction induced by Stx is central, but bacterial lipopolysaccharide (LPS) and neutrophils (PMN) contribute to the pathophysiology. Although renal failure is characteristic of this syndrome, neurological complications occur in severe cases and is usually associated with death. Impaired blood-brain barrier (BBB) is associated with damage to cerebral endothelial cells (ECs) that comprise the BBB. Astrocytes (ASTs) are inflammatory cells in the brain and determine the BBB function. ASTs are in close proximity to ECs, hence the study of the effects of Stx1 and LPS on ASTs, and the influence of their response on ECs is essential. We have previously demonstrated that Stx1 and LPS induced activation of rat ASTs and the release of inflammatory factors such as TNF-α, nitric oxide and chemokines. Here, we demonstrate that rat ASTs-derived factors alter permeability of ECs with brain properties (HUVECd); suggesting that functional properties of BBB could also be affected. Additionally, these factors activate HUVECd and render them into a proagregant state promoting PMN and platelets adhesion. Moreover, these effects were dependent on ASTs secreted-TNF-α. Stx1 and LPS-induced ASTs response could influence brain ECs integrity and BBB function once Stx and factors associated to the STEC infection reach the brain parenchyma and therefore contribute to the development of the neuropathology observed in HUS.

Highlights

The epidemic form of hemolytic uremic syndrome (HUS), has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) [1]
In order to accurately model the human blood-brain barrier (BBB), purified endothelial cells (ECs) obtained from human umbilical cord veins (HUVEC) were differentiated into ECs with properties of cerebral endothelium (HUVECd) by incubating them with conditioned media from ASTs (CM-ASTs)
ASTs treated with LPS and Stx1 release factors that induce activation of HUVECd In order to determine whether factors released by ASTs treated with LPS and/or Stx1 induce activation of HUVECd, we evaluated the expression of adhesion molecules such as ICAM-1 and E-selectin, and the release of procoagulant molecules, such as the von Willebrand Factor

Summary

Introduction

The epidemic form of hemolytic uremic syndrome (HUS), has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) [1]. HUS is the most common cause of acute renal failure in children and is related to endothelial damage of kidney glomeruli and arterioles and epithelial cell damage induced by Stx, through the interaction with its globotriaosylceramide (Gb3) receptor [2]. Stx is the main pathogenic factor for HUS development, the inflammatory response is able to potentiate Stx toxicity. Both bacterial lipopolysaccharide (LPS), and polymorphonuclear neutrophils (PMN) play an important role in the full development of HUS [3]. In severe cases of HUS, endothelial cell (ECs) damage is not limited to the kidney but extends to other organs, such as the brain. Central nervous system (CNS) complications are observed in about 30% of infant population with HUS and brain damage is the most common cause of death in this disease [4]

Objectives

Methods

Results

Conclusion