Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.     

The oxidative stress induced in vivo by Shiga toxin‐2 contributes to the pathogenicity of haemolytic uraemic syndrome

Typical haemolytic uraemic syndrome (HUS) is caused by Shiga toxin (Stx)‐producing Escherichia coli infections and is characterized by thrombotic microangiopathy that leads to haemolytic anaemia, thrombocytopenia and acute renal failure. Renal or neurological sequelae are consequences of irreversible tissue damage during the acute phase. Stx toxicity and the acute inflammatory response raised by the host determine the development of HUS. At present there is no specific therapy to control Stx damage. The pathogenic role of reactive oxygen species (ROS) on endothelial injury has been largely documented. In this study, we investigated the in‐vivo effects of Stx on the oxidative balance and its contribution to the development of HUS in mice. In addition, we analysed the effect of anti‐oxidant agents as therapeutic tools to counteract Stx toxicity. We demonstrated that Stx induced an oxidative imbalance, evidenced by renal glutathione depletion and increased lipid membrane peroxidation. The increased ROS production by neutrophils may be one of the major sources of oxidative stress during Stx intoxication. All these parameters were ameliorated by anti‐oxidants reducing platelet activation, renal damage and increasing survival. To conclude, Stx generates a pro‐oxidative state that contributes to kidney failure, and exogenous anti‐oxidants could be beneficial to counteract this pathogenic pathway.     

Chemokine receptor CCR1 disruption limits renal damage in a murine model of hemolytic uremic syndrome

Shiga toxin (Stx)-producing Escherichia coli is the main etiological agent that causes hemolytic uremic syndrome (HUS), a microangiopathic disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Although direct cytotoxic effects on endothelial cells by Stx are the primary pathogenic event, there is evidence that indicates the inflammatory response mediated by polymorphonuclear neutrophils and monocytes as the key event during HUS development. Because the chemokine receptor CCR1 participates in the pathogenesis of several renal diseases by orchestrating myeloid cell kidney infiltration, we specifically addressed the contribution of CCR1 in a murine model of HUS. We showed that Stx type 2-treated CCR1(-/-) mice have an increased survival rate associated with less functional and histological renal damage compared with control mice. Stx type 2-triggered neutrophilia and monocytosis and polymorphonuclear neutrophil and monocyte renal infiltration were significantly reduced and delayed in CCR1(-/-) mice compared with control mice. In addition, the increase of the inflammatory cytokines (tumor necrosis factor-α and IL-6) in plasma was delayed in CCR1(-/-) mice compared with control mice. These data demonstrate that CCR1 participates in cell recruitment to the kidney and amplification of the inflammatory response that contributes to HUS development. Blockade of CCR1 could be important to the design of future therapies to restrain the inflammatory response involved in the development of HUS.     

The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.     

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1β, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1β) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1β, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.     

Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48 hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.Contamination of food and water sources with Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) is a global cause of sporadic outbreaks of painful diarrhea and hemorrhagic colitis^1–3 with an estimated 176,000 cases in the United States annually and approximately one death for every 1000 infections.^4,5 Symptoms arise within 3 to 4 days after infection and most resolve, but 5% to approximately 10% of patients progress to develop hemolytic uremic syndrome (HUS).⁶ Postdiarrheal HUS is characterized by thrombocytopenia, nonimmune hemolytic anemia, and thrombotic microangiopathy, often progressing to acute renal injury with severe cases requiring renal dialysis.⁷ The most vulnerable to infection are the young and elderly,⁸ and EHEC infections are a leading cause of acute renal failure in otherwise healthy children in the United States.EHEC bacteria attach to the intestinal epithelium with characteristic attaching and effacing lesions, which allows type III secretion of bacterial effector proteins and the Shiga toxin type-1 and type-2 toxins (Stx1, Stx2) and several variants into the host.⁹ Bacteremia is rare, and these toxins are primary contributors to the development of HUS and organ damage.¹⁰ The strain often associated with greatest severity is the O157:H7 serotype,¹¹ although there are dozens of pathogenic strains. New strains are emerging with greater virulence as experienced in Germany during summer 2011 when a rare enteroaggregative E. coli O104:H4 strain that causes otherwise self-limiting diarrhea acquired both a stx2 gene and aggressive virulence.^12–14 This is a matter of considerable concern because antibiotics increase HUS risk,¹⁵ and no toxin-specific therapies are available.The relative contribution of the two toxins to organ injury is difficult to distinguish in patients because EHEC strains can secrete one or both toxins in differing ratios, and the EHEC strain may not be identified or reported. Organ injury is assumed to be roughly equivalent between the toxins, although postdiarrheal renal injury is more commonly associated with EHEC strains that secrete Stx2.¹⁰ There is suggestion that inhibition of only Stx2 is necessary for therapeutic relief,¹⁶ but no data are available that directly compares the toxins in an animal model that presents with full-spectrum HUS.In ongoing studies to develop clinically relevant EHEC and HUS animal models, we are characterizing the pathophysiology elicited by Stx1 or Stx2 in juvenile baboons (Papio). We previously showed that, when the toxins are administered intravenously, they elicit thrombocytopenia, hemolytic anemia, thrombotic microangiopathy, and acute kidney injury, consistent with HUS.¹⁷ Using this model, we demonstrated rescue of the animals from an otherwise lethal Stx2 challenge and preservation of kidney function, with a custom-designed anti-Stx2 synthetic peptide.¹⁸ When comparing effects of the two toxins, we observed substantial distinguishing features, including different proinflammatory responses and different timing with delayed organ injury after Stx2 challenge. We present here detailed pathology examinations of kidney tissue from the animals challenged with Stx1 or Stx2 and cytokine analyses that extend our prior characterizations of kidney injury. In the baboon model, as in humans, the glomeruli are a particular target of the toxins, but injury is not exclusive to that structure. Stx1 and Stx2 had distinct effects on the glomeruli, with endothelial injury predominating with Stx1 and mesangial injury a predominant feature with Stx2. Both toxins elicited a marked chemotactic environment in the kidneys and other organs that may contribute to the pathophysiology.     

Oral Intoxication of Mice with Shiga Toxin Type 2a (Stx2a) and Protection by Anti-Stx2a Monoclonal Antibody 11E10

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB₅ toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD₅₀) of 2.9 μg, but no morbidity occurred after oral intoxication with up to 157 μg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.     

Apoptosis of Renal Cortical Cells in the Hemolytic-Uremic Syndrome: In Vivo and In Vitro Studies

This study examined apoptotic cell death associated with Shiga-like toxin (Stx)-producing Escherichia coli. Renal cortices from three children with postenteropathic hemolytic-uremic syndrome (HUS) and from mice infected with E. coli O157:H7 and pediatric renal tubular epithelial cells stimulated with Stx and E. coli O157:H7 extracts were examined for apoptotic changes. Apoptotic cells were detected by terminal dUTP nick end labeling of tubuli and glomeruli from HUS patients and from mice inoculated with Stx-2-positive and Stx-negative strains. Apoptosis was more extensive and severe ultramorphological nuclear and cytoplasmic changes were seen in the Stx-2-positive group. Stx caused DNA fragmentation and ultramorphological changes indicating apoptosis in cultured pediatric tubular cells. DNA fragmentation increased when cells were prestimulated with tumor necrosis factor alpha. Polymyxin extracts from Stx-2-positive and Stx-negative strains induced DNA fragmentation, but only extracts from Stx-2-positive strains caused ultramorphological changes and extensive DNA fragmentation. The results indicate that HUS is accompanied by increased apoptosis of kidney cells and that bacterial factors, possibly together with host cytokines in vivo, may activate apoptotic tissue injury.     

Mouse model of hemolytic-uremic syndrome caused by endotoxin-free Shiga toxin 2 (Stx2) and protection from lethal outcome by anti-Stx2 antibody

Hemolytic-uremic syndrome (HUS) results from infection by Shiga toxin (Stx)-producing Escherichia coli and is the most common cause of acute renal failure in children. We have developed a mouse model of HUS by administering endotoxin-free Stx2 in multiple doses over 7 to 8 days. At sacrifice, moribund animals demonstrated signs of HUS: increased blood urea nitrogen and serum creatinine levels, proteinuria, deposition of fibrin(ogen), glomerular endothelial damage, hemolysis, leukocytopenia, and neutrophilia. Increased expression of proinflammatory chemokines and cytokines in the sera of Stx2-treated mice indicated a systemic inflammatory response. Currently, specific therapeutics for HUS are lacking, and therapy for patients is primarily supportive. Mice that received 11E10, a monoclonal anti-Stx2 antibody, 4 days after starting injections of Stx2 recovered fully, displaying normal renal function and normal levels of neutrophils and lymphocytes. In addition, these mice showed decreased fibrin(ogen) deposition and expression of proinflammatory mediators compared to those of Stx2-treated mice in the absence of antibody. These results indicate that, when performed during progression of HUS, passive immunization of mice with anti-Stx2 antibody prevented the lethal effects of Stx2.     

Protection of Mice against Shiga Toxin 2 (Stx2)-Associated Damage by Maternal Immunization with a Brucella Lumazine Synthase-Stx2 B Subunit Chimera

Hemolytic-uremic syndrome (HUS) is defined as the triad of anemia, thrombocytopenia, and acute kidney injury. Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC), which causes a prodromal hemorrhagic enteritis, remains the most common etiology of the typical or epidemic form of HUS. Because no licensed vaccine or effective therapy is presently available for human use, we recently developed a novel immunogen based on the B subunit of Shiga toxin 2 (Stx2B) and the enzyme lumazine synthase from Brucella spp. (BLS) (BLS-Stx2B). The aim of this study was to analyze maternal immunization with BLS-Stx2B as a possible approach for transferring anti-Stx2 protection to the offspring. BALB/c female mice were immunized with BLS-Stx2B before mating. Both dams and pups presented comparable titers of anti-Stx2B antibodies in sera and fecal extracts. Moreover, pups were totally protected against a lethal dose of systemic Stx2 injection up to 2 to 3 months postpartum. In addition, pups were resistant to an oral challenge with an Stx2-producing EHEC strain at weaning and did not develop any symptomatology associated with Stx2 toxicity. Fostering experiments demonstrated that anti-Stx2B neutralizing IgG antibodies were transmitted through breast-feeding. Pups that survived the EHEC infection due to maternally transferred immunity prolonged an active and specific immune response that protected them against a subsequent challenge with intravenous Stx2. Our study shows that maternal immunization with BLS-Stx2B was very effective at promoting the transfer of specific antibodies, and suggests that preexposure of adult females to this immunogen could protect their offspring during the early phase of life.     

Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model.

The haemolytic uraemic syndrome (HUS) is a clinical syndrome consisting of haemolytic anaemia, thrombocytopenia, and acute renal insufficiency. HUS is the most frequent cause of acute renal failure in childhood. It has been previously suggested that the presence of Shiga toxin (Stx) is necessary but not sufficient for HUS development, and cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-1beta appear to be necessary to develop the syndrome. Since the mononuclear phagocytic system (MPS) is the major source of these cytokines, macrophages might be one of the relevant targets for Stx action in the pathophysiology of HUS. In this study our objective was to examine the role of the hepatic and splenic macrophages in a mouse model of HUS induced by injection of Shiga toxin type-2 (Stx2) or Stx2 plus lipopolysaccharide (LPS). For this purpose, depletion of mice macrophages by liposome-encapsulated clodronate (lip-clod), followed by injection of STx2 or Stx2 plus LPS, was assayed. In this study we show that depletion of hepatic and splenic macrophages by clodronate treatment induces a survival of 50% in animals treated with Stx2 alone or in presence of LPS. This maximal effect was observed when lip-clod was injected 48-72 h before Stx2 injection. Biochemical and histological parameters show characteristics of the lesion produced by Stx2, discarding non-specific damage due to LPS or lip-clod. In addition, we determined that the toxic action of Stx2 is similar in BALB/c and N:NIH nude mice, indicating the T cell compartment is not involved in the Stx2 toxicity. Briefly, we demonstrate that macrophages play a central role in the pathophysiology of HUS, and that the systemic production of cytokines by liver and/or spleen is for Stx2 to manifest its full cytotoxic effect. In addition, the toxicity of Stx2 alone, or in presence of LPS, is independent of the T cell compartment.     

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.     

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB₅ toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.     

Shiga toxin subtypes display dramatic differences in potency

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.     

Role of Tumor Necrosis Factor Alpha in Disease Using a Mouse Model of Shiga Toxin-Mediated Renal Damage

Mice have been extensively employed as an animal model of renal damage caused by Shiga toxins. In this study, we examined the role of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in the development of toxin-mediated renal disease in mice. Mice pretreated with TNF-α and challenged with Shiga toxin type 1 (Stx1) showed increased survival compared to that of mice treated with Stx1 alone. Conversely, mice treated with Stx1 before TNF-α administration succumbed more quickly than mice given Stx1 alone. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with evidence of glomerular damage and the loss of renal function. No differences in renal histopathology were noted between animals treated with Stx1 alone and the TNF-α pretreatment group, although we noted a sparing of renal function when TNF-α was administered before toxin. Compared to that of treatment with Stx1 alone, treatment with TNF-α after toxin altered the renal cytokine profile so that the expression of proinflammatory cytokines TNF-α and interleukin-1β (IL-1β) increased, and the expression of the anti-inflammatory cytokine IL-10 decreased. Increased lethality in mice treated with Stx1 followed by TNF-α was associated with higher numbers of dUTP-biotin nick end labeling-positive renal tubule cells, suggesting that increased lethality involved enhanced apoptosis. These data suggest that the early administration of TNF-α is a candidate interventional strategy blocking disease progression, while TNF-α production after intoxication exacerbates disease.Shiga toxins are a family of genetically and functionally related cytotoxic proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and certain serotypes of Escherichia coli. Antigenic similarity to Shiga toxin expressed by S. dysenteriae serotype 1 is used to define Shiga toxin type 1 (Stx1) and type 2 (Stx2) expressed by Shiga toxin-producing E. coli (STEC) (44). Shiga toxins consist of a single A subunit in noncovalent association with a pentamer of B subunits. B subunits mediate binding to the neutral glycolipid receptor globotriaosylceramide (Gb₃), while the A subunit possesses an N-glycosidase activity (38). Following toxin internalization and routing to the endoplasmic reticulum (ER), a fragment of the toxin A subunit generated by furin or a furin-like protease is translocated across the ER membrane and mediates the cleavage of a single adenine residue (A4256 in the rat) from the 28S rRNA component of ribosomes (39). Stx-induced depurination leads to the disruption of elongation factor-dependent aminoacyl-tRNA binding to nascent polypeptides (30). Thus, Shiga toxins are potent protein synthesis inhibitors, with 50% cytotoxic doses measured in pg/ml amounts for many cell types in vitro. Shiga toxins also activate the ribotoxic and ER stress pathways, which are important in the activation of proinflammatory cytokine/chemokine production and apoptosis (6, 22, 41).The ingestion of small quantities of Stx-producing bacteria may lead to the development of bloody diarrhea with progression to acute renal failure, designated diarrhea-associated hemolytic uremic syndrome (D+HUS) (33). Epidemiologic studies have shown that the ingestion of STEC strains expressing Stx2 alone or Stx1 and Stx2 are more likely to progress to life-threatening extraintestinal complications (3, 17, 31). D+HUS is a leading cause of pediatric acute renal failure. D+HUS is characterized by rapid-onset oligouria or anuria, azotemia, microangiopathic hemolytic anemia with schistocytosis, and thrombocytopenia (33, 47). The histopathological examination of D+HUS renal tissues showed that glomerular microvascular endothelial cells were frequently swollen and detached from the basement membrane, and glomerular capillary lumina may be occluded with fibrin-rich microthrombi (21, 36).Numerous animal models have been employed to characterize the role of Stx1 and Stx2 in pathogenesis. Studies utilizing nonhuman primates showed that Shiga toxins are essential virulence determinants in the development of microangiopathic lesions. Fontaine et al. (9) showed that macaque monkeys fed toxigenic strains of S. dysenteriae developed colonic microvascular lesions, while baboons given purified intravenous Stx1 developed acute renal failure (48). The bolus intravenous administration of Stx1 or Stx2 into baboons revealed that the animals were more sensitive to Stx2, although the mean times to death were prolonged in Stx2-treated animals compared to that with Stx1 treatment. Both toxins mediated hematologic changes such as thrombocytopenia and schistocytosis, and both toxins produced renal pathology, but with different presentations. Renal damage caused by Stx1 was characterized by moderate congestion at the cortico-medullary junction, while Stx2-treated animals showed severe medullary congestion with cortical ischemia (42). Mice fed Stx2-producing E. coli or given a single bolus injection of purified Shiga toxins died without the development of glomerular thrombotic microangiopathy (50, 54). However, the administration of multiple low doses of Stx2 allowed the animals to survive initial toxin challenge and develop glomerular lesions characteristic of HUS in humans (40). In addition to the toxins, host response factors may contribute to D+HUS pathogenesis. Prodromal hemorrhagic colitis may alter normal colonic barrier function, and patients with D+HUS may be endotoxemic or show evidence of elevated antibody titers against lipopolysaccharides (LPS) expressed by Stx-producing E. coli (2, 10, 26). LPS elicit the expression of a broad array of pro- and anti-inflammatory cytokines and chemokines (45). In accordance with this, D+HUS patients frequently have increased serum or urinary proinflammatory cytokine and chemokine levels (15, 23). Studies using small-animal models support the hypothesis that additional bacterial and host response factors facilitate the development of renal disease. Keepers et al. (19) demonstrated that the coadministration of Stx2 and LPS to C57BL/6 mice did not produce major changes in lethality but resulted in pathophysiological changes more consistent with disease in humans: intraglomerular platelet and fibrin deposition, decreased renal function, neutrophilia, and lymphocytopenia. Barrett et al. (1) showed that the timing of toxin and LPS challenges were critical in disease outcome. LPS enhanced the lethal effects of purified Stx2 when administered to rabbits or mice after toxin challenge, whereas LPS protected the animals from Stx2 toxicity when administered before the toxin. Palermo et al. (32) showed that the LPS-induced modulation of Stx2 lethality was cytokine time and dose dependent. Mice given low doses of TNF-α or IL-1β 1 h before Stx2 treatment showed increased lethality when treated with Stx2, while mice given higher doses of IL-1β (sufficient to elicit corticosteroid production) were protected from Stx2 lethality.The proinflammatory cytokines TNF-α and IL-1β sensitize vascular endothelial cells to the cytotoxic action of Shiga toxins in vitro (24, 34, 53) through a mechanism involving the increased expression of genes involved in the biosynthesis of Gb₃ (43). Murine and human macrophages or macrophage-like cell lines express proinflammatory cytokines and chemokines when treated with purified Shiga toxins (12, 35, 51). Keepers et al. (18) showed a marked monocytic cell infiltrate into the kidneys of mice given Stx2 and LPS. Collectively, these data suggest that the innate immune response to Shiga toxins, in the presence or absence of LPS, alters the outcome of renal disease. In the present study, we have examined the role of a single proinflammatory cytokine, TNF-α, in pathogenesis using the mouse model of Stx-induced renal damage. Our data suggest that the timing of TNF-α production affects the outcome of disease in that the presence of elevated TNF-α levels prior to toxin challenge protects animals from disease, while high TNF-α levels occurring after toxin administration result in accelerated lethality.     

Distinct Physiologic and Inflammatory Responses Elicited in Baboons after Challenge with Shiga Toxin Type 1 or 2 from Enterohemorrhagic Escherichia coli

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-α]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-α. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.Infection with Shiga toxin-producing Escherichia coli (STEC) results in intestinal cramps and bloody diarrhea, followed 5 to 12 days later in some patients by the development of hemolytic-uremic syndrome (HUS) (16, 18). HUS is characterized clinically by the triad of thrombocytopenia, hemolytic microangiopathy, and renal injury and is the leading cause of acute renal failure in otherwise healthy children in the United States. An antibiotic regimen is not recommended, and treatment options are limited to critical care support (47). Patients with diarrhea-associated HUS can have long-term renal impairment of varying severity, and approximately one-fourth of patients have neurologic sequelae, including seizures, coma/stupor, cortical blindness, ataxia, and paraplegia (10, 14).The natural infection route is gastrointestinal, via contaminated food or water. The bacteria colonize the intestinal lumen, with most strains forming characteristic attaching-and-effacing lesions, and the organisms may synthesize and release one or more toxins that are primary virulence factors contributing to the clinical manifestations of HUS (19). The toxins are AB₅ holotoxins, referred to as Shiga toxins due to their functional and structural similarities to Shiga toxin expressed by Shigella dysenteriae serotype 1 (4). Shiga toxin type 1 (Stx1) is essentially identical to the Shigella toxin (4), differing by one amino acid, but shares only 58% amino acid identity with Shiga toxin type 2 (Stx2). Stx1 and Stx2 have distinct spatial conformations (8) and dissociation rates from receptor-lipid surfaces (24). STEC strains may secrete one or both toxins and several toxin variants, and clinical studies have demonstrated that HUS is most often associated with the expression of Stx2 (3), particularly following infection with E. coli O157:H7 strains (12, 20). All Shiga toxins share a cellular intoxication mechanism in which B subunits oligomerize into pentamers for interaction with a cell surface globotriaosylceramide Gb₃ (CD77) receptor. Following binding, holotoxins are internalized via clathrin-dependent or clathrin-independent mechanisms and undergo retrograde transport through the trans-Golgi network and Golgi apparatus to reach the endoplasmic reticulum (33, 46). During transport, the A subunit undergoes limited proteolysis, and once in the endoplasmic reticulum, a fragment of the A subunit translocates into the cytoplasm, where its N-glycosidase activity inactivates the 28S rRNA component of eukaryotic ribosomes to inhibit protein synthesis and cause cell death (25, 43).While Stx1 and Stx2 share many characteristics, they are not identical and there is evidence that toxin-specific activities may be clinically relevant. Both toxins are internalized after binding to Gb₃, but the mechanisms of their intracellular trafficking through polarized intestinal epithelial cells to reach the intestinal endothelium are very different (15). Also, endothelial sensitivities to Stx1 and Stx2 differ depending on the vascular bed, with intestinal endothelium being more sensitive to the Shiga toxins than saphenous vein endothelium (12), and glomerular endothelial cells are about 1,000 times more sensitive to Stx2 than human umbilical vein endothelial cells (17). The mechanisms for these differences are not completely understood but may be related to receptor density, toxin effects on endoplasmic reticulum stress responses and apoptosis (22, 41), or local availability of sensitizing cytokines (5, 7, 11).Most animal models show greater sensitivity to Stx2, including murine, rabbit, and gnotobiotic piglet models, although renal and neurologic micropathologies differ from those in humans and between animal species (6, 9, 45). Earlier studies with the baboon (Papio) model showed that a bolus infusion of purified Stx1 induced intestinal injury, kidney glomerular injury, microangiopathic anemia, thrombocytopenia, and neurologic abnormalities similar to those in humans, suggesting that the baboon represents a promising preclinical animal model (44). A systemic inflammatory response was minimal after Stx1 challenge, but urinary tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels were consistent with local kidney inflammatory responses. Baboons were also more sensitive to Stx2 (38), but a direct comparison of the pathophysiologies elicited by the two toxins was difficult because of differing experimental designs. We sought to expand these earlier studies of baboons to identify similarities and differences elicited by Stx1 and Stx2 under reproducible experimental conditions. Given the clinical relevance of Stx2 production during STEC infection in patients, we were particularly interested in responses after Stx2 challenge, for which few data are available from the baboon model. We present the metabolic, physiologic, and inflammatory responses in baboons after intravenous challenge with Stx1 or Stx2. The observed differences in pathophysiology elicited by the two toxins may contribute to a better understanding of the differences in clinical manifestations produced by the toxins.     

Protective Efficacy and Pharmacokinetics of Human/Mouse Chimeric Anti-Stx1 and Anti-Stx2 Antibodies in Mice

In the United States, Shiga toxin (Stx)-producing Escherichia coli (STEC) is the most frequent infectious cause of hemorrhagic colitis. Hemolytic uremic syndrome (HUS) is a serious sequela that may develop after STEC infection that can lead to renal failure and death in up to 10% of cases. STEC can produce one or more types of Stx, Stx1 and/or Stx2, and Stx1 and Stx2 are responsible for HUS-mediated kidney damage. We previously generated two monoclonal antibodies (MAbs) that neutralize the toxicity of Stx1 or Stx2. In this study, we evaluated the protective efficacy of human/mouse chimeric versions of those monoclonal antibodies, named cαStx1 and cαStx2. Mice given an otherwise lethal dose of Stx1 were protected from death when injected with cαStx1 either 1 h before or 1 h after toxin injection. Additionally, streptomycin-treated mice fed the mouse-lethal STEC strain B2F1 that produces the Stx2 variant Stx2d were protected when given a dose of 0.1 mg of cαStx2/kg of body weight administered up to 72 h post-oral bacterial challenge. Since many STEC strains produce both Stx1 and Stx2 and since either toxin may lead to the HUS, we also assessed the protective efficacy of the combined MAbs. We found that both antibodies were required to protect mice from the presence of both Stx1 and Stx2. Pharmacokinetic studies indicated that cαStx1 and cαStx2 had serum half-lives (t_1/2) of about 50 and 145 h, respectively. We propose that cαStx1 and cαStx2, both of which have been tested for safety in humans, could be used therapeutically for prevention or treatment early in the development of HUS.     

Endogenous glucocorticoids attenuate Shiga toxin-2-induced toxicity in a mouse model of haemolytic uraemic syndrome

The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models. The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram-negative bacteria that produce Shiga toxin (Stx). Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS. The aim of this study was to investigate the influence of endogenous GC on Stx-toxicity in a mouse model. Stx2 was injected into GC-deprived mice and survival rate, renal damage and serum urea levels were evaluated. Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post-Stx2 treatment. Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage. Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion. Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils. These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection.     

Interaction of Shiga toxin 2 with complement regulators of the factor H protein family

Shiga toxin 2 (Stx2) is believed to be a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) contributing to hemolytic uremic syndrome (HUS). The complement system has recently been found to be involved in the pathogenesis of EHEC-associated HUS. Stx2 was shown to activate complement via the alternative pathway, to bind factor H (FH) at short consensus repeats (SCRs) 6–8 and 18–20 and to delay and reduce FH cofactor activity on the cell surface.