Shiga Toxin 1-Induced Inflammatory Response in Lipopolysaccharide-Sensitized Astrocytes Is Mediated by Endogenous Tumor Necrosis Factor Alpha

Hemolytic-uremic syndrome (HUS) is generally caused by Shiga toxin (Stx)-producing Escherichia coli. Endothelial dysfunction mediated by Stx is a central aspect in HUS development. However, inflammatory mediators such as bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) contribute to HUS pathophysiology by potentiating Stx effects. Acute renal failure is the main feature of HUS, but in severe cases, patients can develop neurological complications, which are usually associated with death. Although the mechanisms of neurological damage remain uncertain, alterations of the blood-brain barrier associated with brain endothelial injury is clear. Astrocytes (ASTs) are the most abundant inflammatory cells of the brain that modulate the normal function of brain endothelium and neurons. The aim of this study was to evaluate the effects of Stx type 1 (Stx1) alone or in combination with LPS in ASTs. Although Stx1 induced a weak inflammatory response, pretreatment with LPS sensitized ASTs to Stx1-mediated effects. Moreover, LPS increased the level of expression of the Stx receptor and its internalization. An early inflammatory response, characterized by the release of tumor necrosis factor alpha (TNF-α) and nitric oxide and PMN-chemoattractant activity, was induced by Stx1 in LPS-sensitized ASTs, whereas activation, evidenced by higher levels of glial fibrillary acid protein and cell death, was induced later. Furthermore, increased adhesion and PMN-mediated cytotoxicity were observed after Stx1 treatment in LPS-sensitized ASTs. These effects were dependent on NF-κB activation or AST-derived TNF-α. Our results suggest that TNF-α is a pivotal effector molecule that amplifies Stx1 effects on LPS-sensitized ASTs, contributing to brain inflammation and leading to endothelial and neuronal injury.The epidemic form of hemolytic-uremic syndrome (HUS) has been associated with enterohemorrhagic infections caused by Shiga toxin (Stx)-producing Escherichia coli (STEC) organisms (33). HUS is the most common cause of acute renal failure in children and is related to the endothelial damage of glomeruli and/or arterioles of the kidney and epithelial cell damage induced by Stx through the interaction with its globotriaosylceramide (Gb₃) receptor (35). Although Stx is the main pathogenic factor and is necessary for epidemic HUS development, clinical and experimental evidence suggests that the inflammatory response is able to potentiate Stx toxicity. In fact, both bacterial lipopolysaccharide (LPS) and polymorphonuclear neutrophils (PMN) play a key role in the full development of HUS (15). Moreover, PMN leukocytosis in patients correlates with a poor prognosis (17).Endothelial cell damage is not limited to the kidney but extends to other organs; in severe cases, the brain can be affected. In fact, central nervous system (CNS) complications indicate severe HUS, and brain damage involvement is the most common cause of death (14).However, the pathogenesis of CNS impairment is not yet fully understood. Although it has been demonstrated that human brain endothelial cells (BECs) are relatively resistant to Stx, inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), markedly increase human BEC sensitivity to Stx cytotoxicity (11).BECs are part of the blood-brain barrier (BBB), which protects the brain from potentially harmful substances and leukocytes present in the bloodstream. Thus, the integrity of BBB function is theorized to be a key component in CNS-associated pathologies, and BEC damage is thought to be one of the possible mechanisms involved in the disruption of the BBB in HUS. In fact, LPS from bacterial infections leads to the release of TNF-α, interleukin-1β (IL-1β), and reactive oxygen species (ROS), all of which have the ability to open the BBB.Several in vivo studies demonstrated previously that Stx is able to impair BBB function, increasing its permeability (21). Moreover, Stx itself is able to cross the endothelial barrier and enter into the CNS, since Stx activity in cerebrospinal fluid was previously observed (19, 23), and Stx was previously immunodetected in many brain cells including astrocytes (ASTs) and neurons (44).ASTs, which are inflammatory cells found throughout the CNS, are in close contact with BECs by end-foot processes (24), and their interaction with the cerebral endothelium determines BBB function (2, 4). In addition, ASTs interact with neurons through gap junctions and release neurotrophins that are essential for neuronal survival (6). However, in response to brain injury, ASTs become activated and release inflammatory mediators such as nitric oxide (NO) and TNF-α, altering the permeability of the BBB and affecting neuronal survival and tissue integrity (1, 9). In addition, AST-derived cytokines and chemokines can stimulate the peripheral immune system and attract peripheral inflammatory leukocytes to the site of injury (46).ASTs are therefore in a critical position to influence neuronal viability and BEC integrity once Stx and factors associated with the STEC infection reach the brain parenchyma. We hypothesize that the effects of LPS and Stx on ASTs may be involved in the brain damage observed with severe cases of HUS. Thus, the aim of this study was to evaluate whether Stx type 1 (Stx1) alone or in combination with LPS is capable of inducing an inflammatory response in ASTs.