Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

ABSTRACT Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.

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Human microvascular endothelial cells resist Shiga toxins by IFN-γ treatment in vitro

Microbiology ◽

10.1099/mic.0.26142-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2609-2614 ◽

Cited By ~ 2

Author(s):

Tomoaki Yoshida ◽

Tsuyoshi Sugiyama ◽

Naoki Koide ◽

Isamu Mori ◽

Takashi Yokochi

Keyword(s):

Endothelial Cells ◽

Cell Model ◽

Cell Damage ◽

Shigella Dysenteriae ◽

Microvascular Endothelial Cells ◽

Microvascular Endothelial Cell ◽

Shiga Toxins ◽

Resistant Phenotype ◽

Microvascular Endothelial

Shiga toxins (Stxs) produced by enterohaemorrhagic Escherichia coli or Shigella dysenteriae damage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-α. However, in this study, in vitro IFN-γ pre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-α addition to IFN-γ reverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-γ. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as an N-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-γ in the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.

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Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells

Infection and Immunity ◽

10.1128/iai.71.2.845-849.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 845-849 ◽

Cited By ~ 12

Author(s):

T. G. Obrig ◽

R. M. Seaner ◽

M. Bentz ◽

C. A. Lingwood ◽

B. Boyd ◽

...

Keyword(s):

Endothelial Cells ◽

Shiga Toxin ◽

Microvascular Endothelial Cells ◽

Cytotoxic Action ◽

Ceramide Level ◽

Shiga Toxins ◽

Shiga Toxin 2 ◽

Toxin Receptor ◽

Uremic Syndrome ◽

Microvascular Endothelial

ABSTRACT Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

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Decrease of miR-19b-3p in Brain Microvascular Endothelial Cells Attenuates Meningitic Escherichia coli-Induced Neuroinflammation via TNFAIP3-Mediated NF-κB Inhibition

Pathogens ◽

10.3390/pathogens8040268 ◽

2019 ◽

Vol 8 (4) ◽

pp. 268 ◽

Cited By ~ 2

Author(s):

Amjad ◽

Yang ◽

Li ◽

Fu ◽

Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Negative Regulator ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Inflammatory Damage ◽

Microvascular Endothelial

Meningitic Escherichia coli can traverse the host’s blood–brain barrier (BBB) and induce severe neuroinflammatory damage to the central nervous system (CNS). During this process, the host needs to reasonably balance the battle between bacteria and brain microvascular endothelial cells (BMECs) to minimize inflammatory damage, but this quenching of neuroinflammatory responses at the BBB is unclear. MicroRNAs (miRNAs) are widely recognized as key negative regulators in many pathophysiological processes, including inflammatory responses. Our previous transcriptome sequencing revealed numbers of differential miRNAs in BMECs upon meningitic E. coli infection; we next sought to explore whether and how these miRNAs worked to modulate neuroinflammatory responses at meningitic E. coli entry of the BBB. Here, we demonstrated in vivo and in vitro that meningitic E. coli infection of BMECs significantly downregulated miR-19b-3p, which led to attenuated production of proinflammatory cytokines and chemokines via increasing the expression of TNFAIP3, a negative regulator of NF-κB signaling. Moreover, in vivo injection of miR-19b-3p mimics during meningitic E. coli challenge further aggravated the inflammatory damage to mice brains. These in vivo and in vitro findings indicate a novel quenching mechanism of the host by attenuating miR-19b-3p/TNFAIP3/NF-κB signaling in BMECs in response to meningitic E. coli, thus preventing CNS from further neuroinflammatory damage.

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Th1 and Th2 Cytokine-mediated CXC Chemokine Production in Human Lung Microvascular Endothelial Cells in Vitro

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2005.12.995 ◽

2006 ◽

Vol 117 (2) ◽

pp. S252

Author(s):

A. Matsuda ◽

H. Saito ◽

K. Matsumoto

Keyword(s):

Endothelial Cells ◽

Human Lung ◽

Microvascular Endothelial Cells ◽

Chemokine Production ◽

Th2 Cytokine ◽

Cxc Chemokine ◽

Microvascular Endothelial ◽

Th1 And Th2

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Shiga Toxin 2 Causes Apoptosis in Human Brain Microvascular Endothelial Cells via C/EBP Homologous Protein

Infection and Immunity ◽

10.1128/iai.01581-07 ◽

2008 ◽

Vol 76 (8) ◽

pp. 3679-3689 ◽

Cited By ~ 36

Author(s):

Jun Fujii ◽

Katie Wood ◽

Fumiko Matsuda ◽

Benedito A. Carneiro-Filho ◽

Keilo H. Schlegel ◽

...

Keyword(s):

Endothelial Cells ◽

Human Brain ◽

Shiga Toxin ◽

Caspase 3 ◽

Apoptotic Cell ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

Homologous Protein ◽

Shiga Toxin 2 ◽

Microvascular Endothelial

ABSTRACT Shiga toxin 1 (Stx1) and Stx2 produced by Escherichia coli O157 are known to be cytotoxic to Vero and HeLa cells by inhibiting protein synthesis and by inducing apoptosis. In the present study, we have demonstrated that 10 ng/ml Stx2 induced DNA fragmentation in human brain microvascular endothelial cells (HBMEC), with cleavage activation of caspase-3, -6, -8, and -9. A microarray approach used to search for apoptotic potential signals in response to Stx2 revealed that Stx2 treatment induced a marked upregulation of C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible protein 153 (GADD153). Increased CHOP expression was dependent on enzymatically active Stx1. Knockdown of CHOP mRNA reduced the activation of caspase-3 and prevented apoptotic cell death. These results suggest that Stx2-induced apoptosis is mediated by CHOP in HBMEC and involves activation of both the intrinsic and extrinsic pathways of apoptosis.

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Cell Biological Responses after Shiga Toxin-1 Exposure to Primary Human Glomerular Microvascular Endothelial Cells from Pediatric and Adult Origin

International Journal of Molecular Sciences ◽

10.3390/ijms22115615 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5615

Author(s):

Wouter J. C. Feitz ◽

Petra A. van Setten ◽

Thea J. A. M. van der Velden ◽

Christoph Licht ◽

Lambert P. J. W. van den Heuvel ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Synthesis ◽

Shiga Toxin ◽

Age Groups ◽

Kidney Tissue ◽

Microvascular Endothelial Cells ◽

Dependent Manner ◽

Biological Responses ◽

Microvascular Endothelial

Hemolytic uremic syndrome (HUS) is characterized by a triad of symptoms consisting of hemolytic anemia, thrombocytopenia and acute renal failure. The most common form of HUS is caused by an infection with Shiga toxin (Stx) producing Escherichia coli bacteria (STEC-HUS), and the kidneys are the major organs affected. The development of HUS after an infection with Stx occurs most frequently in children under the age of 5 years. However, the cause for the higher incidence of STEC-HUS in children compared to adults is still not well understood. Human glomerular microvascular endothelial cells (HGMVECs) isolated and cultured from pediatric and adult kidney tissue were investigated with respect to Stx binding and different cellular responses. Shiga toxin-1 (Stx-1) inhibited protein synthesis in both pediatric and adult HGMVECs in a dose-dependent manner at basal conditions. The preincubation of pediatric and adult HGMVECs for 24 hrs with TNFα resulted in increased Stx binding to the cell surface and a 20–40% increase in protein synthesis inhibition in both age groups. A decreased proliferation of cells was found when a bromodeoxyuridine (BrdU) assay was performed. A trend towards a delay in endothelial wound closure was visible when pediatric and adult HGMVECs were incubated with Stx-1. Although minor differences between pediatric HGMVECs and adult HGMVECs were found in the assays applied in this study, no significant differences were observed. In conclusion, we have demonstrated that in vitro primary HGMVECs isolated from pediatric and adult kidneys do not significantly differ in their cell biological responses to Stx-1.

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