M. A. Isturiz

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2‐induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2‐induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2‐induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2‐treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2‐damaging mechanism by potentiating Stx2 toxicity.

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.

Antibody Response to Shiga Toxins in Argentinean Children with Enteropathic Hemolytic Uremic Syndrome at Acute and Long-Term Follow-Up Periods

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.

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

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.

Mifepristone (RU486) restores humoral and T cell-mediated immune response in endotoxin immunosuppressed mice

Sepsis and septic shock can be caused by Gram‐positive and ‐negative bacteria and other microorganisms. In the case of Gram‐negative bacteria, endotoxin, a normal constituent of the bacterial wall, also known as lipopolysaccharide (LPS), has been considered as one of the principal agents causing the undesirable effects in this critical illness. The response to LPS involves a rapid secretion of proinflammatory cytokines such as tumour necrosis factor (TNF)‐α, interleukin (IL)‐1, IL‐6, interferon (IFN)‐γ and the concomitant induction of anti‐inflammatory mediators such as IL‐10, transforming growth factor (TGF)‐β or glucocorticoids, which render the host temporarily refractory to subsequent lethal doses of LPS challenge in a process known as LPS or endotoxin tolerance. Although protective from the development of sepsis or systemic inflammation, endotoxin tolerance has also been pointed out as the main cause of the non‐specific humoral and cellular immunosuppression described in these patients. In this report we demonstrate, using a mouse model, that mifepristone (RU486), a known glucocorticoid receptor antagonist, could play an important role in the restoration of both adaptive humoral and cellular immune response in LPS immunosuppressed mice, suggesting the involvement of endogenous glucocorticoids in this phenomenon. On the other hand, using cyclophosphamide and gemcitabine, we demonstrated that regulatory/suppressor CD4+CD25+forkhead boxP3+ and GR‐1+CD11b+ cells do not play a major role in the establishment or the maintenance of endotoxin tolerance, a central mechanism for inducing an immunosuppression state.

Differential effects of glucocorticoids in the establishment and maintenance of endotoxin tolerance

Gram‐negative infections can result in endotoxic shock, which is the most common cause of death in intensive care units. Most of the undesirable effects in sepsis and septic shock have been ascribed to lipopolysaccharide (LPS), a normal constituent of the bacterial wall. The response to LPS involves rapid secretion of proinflammatory cytokines [tumour necrosis factor‐α, interleukin (IL)‐1, IL‐6, IL‐8, interferon‐γ] and the concomitant induction of anti‐inflammatory mediators such as IL‐10 and transforming growth factor‐β and glucocorticoids (GC), which render the host temporarily refractory to subsequent lethal doses of LPS challenge in a process known as LPS or endotoxin tolerance. Although protective from the development of sepsis or systemic inflammation, endotoxin tolerance has also been pointed out as the principal cause of the non‐specific immunosuppression described in these patients. In this report we demonstrate, using a mouse model, that while the maintenance of tolerance is dependent upon GC, the establishment of tolerance by LPS could be inhibited by dexamethasone (Dex), a synthetic GC. Conversely, we demonstrated that mifepristone (RU486), a known GC receptor antagonist, was capable of inducing a transient and reversible disruption of endotoxin tolerance, also permitting partial restoration of the humoral immune response in LPS tolerant/immunosuppressed mice. These results are encouraging for the management of immunosuppression in sepsis and/or non‐infectious shock, and deserve further investigation in the future.

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.

Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) is caused by Shiga‐toxin‐producing Escherichia coli (STEC). Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS. Previously, we demonstrated that Stx2 injection generates an anti‐inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice. Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model. For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist). We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol‐myristate‐acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect. Conversely, both treatments significantly inhibited in vitro phagocytosis. Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI). Stx2u2003+u2003Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), Stx2u2003+u2003Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2‐treated mice was higher compared with controls, but equal to Stx2u2003+u2003Ru486 treated mice. We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions.

Interleukin‐10 and interferon‐γ modulate surface expression of fractalkine‐receptor (CX3CR1) via PI3K in monocytes

The membrane‐anchored form of the chemokine fractalkine (CX3CL1) has been identified as a novel adhesion molecule that interacts with its specific receptor (CX3CR1) expressed in monocytes, T cells and natural killer cells to induce adhesion. In addition, CX3CL1 can be cleaved from the cell membrane to induce chemotaxis of CX3CR1‐expressing leucocytes. Recently, marked variations in CX3CR1 monocyte expression have been observed during several pathological conditions. Regulation of CX3CR1 in monocytes during basal or inflammatory/anti‐inflammatory conditions is poorly understood. The aim of this study was therefore to examine CX3CR1 expression during monocyte maturation and the effect of soluble mediators on this process. We found that basal expression of CX3CR1 in fresh monocytes was reduced during culture, and that lipopolysacchairde accelerated this effect. In contrast, interleukin‐10 and interferon‐γ treatment abrogated CX3CR1 down‐modulation, through a phosphatidylinositol 3 kinase‐dependent pathway. Most importantly, CX3CR1 membrane expression correlated with monocyte CX3CL1‐dependent function. Taken together, our data demonstrate that CX3CR1 expression in monocytes can be modulated, and suggest that alterations in their environment are able to influence CX3CL1‐dependent functions, such as chemotaxis and adhesion, leading to changes in the kinetics, composition and/or functional status of the leucocyte infiltrate.

Cytokine Production Is Altered in Monocytes from Children with Hemolytic Uremic Syndrome

PurposeThe interaction of Shiga toxin (Stx) and/or lipopolysaccharide (LPS) with monocytes (Mo) may be central to the pathogenesis of hemolytic uremic syndrome (HUS), providing the cytokines necessary to sensitize endothelial cells to Stx action. We have previously demonstrated phenotypical alterations in Mo from HUS patients, including increased number of CD16+ Mo. Our aim was to investigate cytokine production in Mo from HUS patients.MethodsWe evaluated TNF-α and IL-10 intracellular contents and secretion in the different Mo subsets in mild (HUS 1) and moderate/severe (HUS 2u2009+u20093) patients. As controls, we studied healthy (HC) and infected children (IC). We also studied Mo responsive capacity towards LPS, measuring the modulation of Mo surface molecules and cytokine production.ResultsIn basal conditions, the intracellular measurement of TNF-α and IL-10 revealed that the highest number of cytokine-producing Mo was found in HUS 2u2009+u20093 and IC, whereas LPS caused a similar increase in TNF-α and IL-10-producing Mo for all groups. However, when evaluating the release of TNF-α and IL-10, we found a diminished secretion capacity in the entire HUS group and IC compared to HC in basal and LPS conditions. Similarly, a lower Mo response to LPS in HUS 2u2009+u20093 and IC groups was observed when surface markers were studied.ConclusionThese results indicate that Mo from severe cases of HUS, similar to IC but different to mild HUS cases, present functional changes in Mo subpopulations and abnormal responses to LPS.