Cytokines use different intracellular mechanisms to upregulate the membrane expression of CX3CR1 in human monocytes.

Membrane expression of fractalkine (CX3CL1)-receptor (CX3CR1) is relevant in monocytes (Mo) because CX3CR1-CX3CL1 interactions might participate on both, homeostatic and pathologic conditions. We have previously demonstrated that CX3CR1 levels are decreased during culture and when Mo are differentiated into dendritic cells, but enhanced when differentiated into macrophages. Regarding soluble factors, lipopolysaccharide (LPS) accelerated the loss of CX3CR1, while interleukin (IL)-10 and Interferon-gamma (IFN-γ) prevented it. However, the comprehensive knowledge about the intracellular pathways that underlay the level of CX3CR1 expression in Mo is still incomplete. In the current work, we studied the effect of anti-inflammatory cytokines (IL-4, IL-13, IL-10), alone or together with IFN- γ on CX3CR1 expression. We found that only IL-10 and IFN-γ separately were able to prevent CX3CR1 down-modulation during culture of human Mo. Besides, Mo incubated with IL-10 plus IFN-γ showed the highest CX3CR1 expression by cell, suggesting cooperation between two different mechanism used by both cytokines. By studying intracellular mechanisms triggered by IL-10 and IFN-γ, we demonstrated that they specifically induced PI3K-dependent serine-phosphorylation of signal transducer and activator of transcription (STAT)3 or STAT1, respectively. Moreover, chemical inhibitors of STAT1 or STAT3 abrogated IFN-γ or IL-10 effects on CX3CR1 expression. Strikingly, only IL-10 increased CX3CR1 mRNA level, as consequence of augmenting mRNA stability. CX3CR1 mRNA increase was PI3K-dependent, supporting the causal link between the action of IL-10 at the CX3CR1 transcript and CX3CR1 protein level on Mo. Thus, both cytokines up-regulate CX3CR1 expression on human Mo by different intracellular mechanisms.

Development of a Mouse Model of Shiga Toxin 2 (Stx2) Intoxication for Testing Therapeutic Agents Against Hemolytic Uremic Syndrome (HUS).

BACKGROUND: Hemolytic Uremic Syndrome (HUS) caused by infections with Shiga toxin (Stx)-producing E. coli is a life-threatening complication characterized by acute renal failure, thrombocytopenia and hemolytic anemia. Stx is the main pathogenic factor. Therefore, the mouse model by intravenous administration of a single lethal dose of Stx is often used to explore its pathogenic mechanisms. OBJECTIVE: The aim of this work was to develop an alternative mouse model of Stx type 2 (Stx2) intoxication to evaluate new therapeutic strategies. METHODS AND RESULTS: One lethal dose of Stx2 was divided in four daily doses. We observed a dose-dependent toxicity characterized by neutrophilia, leukocytopenia and renal damage. Most importantly, we demonstrated that the polyclonal anti-Stx2 serum was able to protect mice from fatal evolution even when administered together the third dose of Stx2. CONCLUSION: This model would provide an advantage for evaluation of therapeutic strategies. Furthermore, the results presented herein suggest that appropriate treatment with anti-Stx2 agents following the appearance of initial clinical signs may block the ongoing outcome or may alleviate disease in patients who have just been diagnosed with HUS. However, the delay in the onset of therapy would be unsafe.

Induction of Neutrophil Extracellular Traps in Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS), a vascular disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure, is caused by enterohemorrhagic Shiga toxin (Stx)-producing bacteria, which mainly affect children. Besides Stx, the inflammatory response mediated by neutrophils (PMN) is essential to HUS evolution. PMN can release neutrophil extracellular traps (NET) composed of DNA, histones, and other proteins. Since NET are involved in infectious and inflammatory diseases, the aim of this work was to investigate the contribution of NET to HUS. Plasma from HUS patients contained increased levels of circulating free-DNA and nucleosomes in comparison to plasma from healthy children. Neutrophils from HUS patients exhibited a greater capacity to undergo spontaneous NETosis. NET activated human glomerular endothelial cells, stimulating secretion of the proinflammatory cytokines IL-6 and IL-8. Stx induced PMN activation as judged by its ability to trigger reactive oxygen species production, increase CD11b and CD66b expression, and induce NETosis in PMN from healthy donors. During HUS, NET can contribute to the inflammatory response and thrombosis in the microvasculature and thus to renal failure. Intervention strategies to inhibit inflammatory mechanisms mediated by PMN, such as NETosis, could have a potential therapeutic impact towards amelioration of the severity of HUS.

Leukotriene C4 increases the susceptibility of adult mice to Shiga toxin-producing Escherichia coli infection.

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that causes hemorrhagic colitis. Under some circumstances, Shiga toxin (Stx) produced within the intestinal tract enters the bloodstream, leading to systemic complications that may cause the potentially fatal hemolytic-uremic syndrome (HUS). Despite STEC human infection is characterized by acute inflammation of the colonic mucosa, little is known regarding the role of proinflammatory mediators like cysteine leukotrienes (cysLTs) in this pathology. Thus, the aim of this work was to analyze whether leukotriene C4 (LTC4) influences STEC pathogenesis in mice. We report that exogenous LTC4 pretreatment severely affected the outcome of STEC gastrointestinal infection. LTC4-pretreated (LTC4+) and STEC-infected (STEC+) mice showed an increased intestinal damage by histological studies, and a decreased survival compared to LTC4-non-pretreated (LTC4-) and STEC+ mice. LTC4+/STEC+ mice that died after the infection displayed neutrophilia and high urea levels, indicating that the cause of death was related to Stx2-toxicity. Despite the differences observed in the survival between LTC4+ and LTC4- mice after STEC infection, both groups showed the same survival after Stx2-intravenous inoculation. In addition, LTC4 pretreatment increased the permeability of mucosal intestinal barrier, as assessed by FITC-dextran absorption experiments. Altogether these results suggest that LTC4 detrimental effect on STEC infection is related to the increased passage of pathogenic factors to the bloodstream. Finally, we showed that STEC infection per se increases the endogenous LTC4 levels in the gut, suggesting that this inflammatory mediator plays a role in the pathogenicity of STEC infection in mice, mainly by disrupting the mucosal epithelial barrier.

Association of haemolytic uraemic syndrome with dysregulation of chemokine receptor expression in circulating monocytes.

Haemolytic uraemic syndrome (HUS) is the major complication of Escherichia coli gastrointestinal infections that are Shiga toxin (Stx) producing. Monocytes contribute to HUS evolution by producing cytokines that sensitize endothelial cells to Stx action and migration to the injured kidney. As CC chemokine receptors (CCRs) are involved in monocyte recruitment to injured tissue, we analysed the contribution of these receptors to the pathogenesis of HUS. We analysed CCR1, CCR2 and CCR5 expression in peripheral monocytes from HUS patients during the acute period, with healthy children as controls. We observed an increased expression of CCRs per cell in monocytes from HUS patients, accompanied by an increase in the absolute number of monocytes CCR1+, CCR2+ and CCR5+. It is interesting that prospective analysis confirmed that CCR1 expression positively correlated with HUS severity. The evaluation of chemokine levels in plasma showed that regulated on activation of normal T-cell-expressed and -secreted (RANTES) protein was reduced in plasma from patients with severe HUS, and this decrease correlated with thrombocytopenia. Finally, the expression of the higher CCRs was accompanied by a loss of functionality which could be due to a mechanism for desensitization to compensate for altered receptor expression. The increase in CCR expression correlates with HUS severity, suggesting that the dysregulation of these receptors might contribute to an increased risk of renal damage. Activated monocytes could be recruited by chemokines and then receptors could be dysregulated. The dysregulation of CCRs and their ligands observed during the acute period suggests that a chemokine pathway would participate in HUS development.

Differential expression of the fractalkine chemokine receptor (CX3CR1) in human monocytes during differentiation.

Circulating monocytes (Mos) may continuously repopulate macrophage (MAC) or dendritic cell (DC) populations to maintain homeostasis. MACs and DCs are specialized cells that play different and complementary immunological functions. Accordingly, they present distinct migratory properties. Specifically, whereas MACs largely remain in tissues, DCs are capable of migrating from peripheral tissues to lymphoid organs. The aim of this work was to analyze the expression of the fractalkine receptor (CX3CR1) during the monocytic differentiation process. Freshly isolated Mos express high levels of both CX3CR1 mRNA and protein. During the Mo differentiation process, CX3CR1 is downregulated in both DCs and MACs. However, MACs showed significantly higher CX3CR1 expression levels than did DC. We also observed an antagonistic CX3CR1 regulation by interferon (IFN)-γ and interleukin (IL)-4 during MAC activation through the classical and alternative MAC pathways, respectively. IFN-γ inhibited the loss of CX3CR1, but IL-4 induced it. Additionally, we demonstrated an association between CX3CR1 expression and apoptosis prevention by soluble fractalkine (sCX3CL1) in Mos, DCs and MACs. This is the first report demonstrating sequential and differential CX3CR1 modulation during Mo differentiation. Most importantly, we demonstrated a functional link between CX3CR1 expression and cell survival in the presence of sCX3CL1.

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.

Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice.

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer's patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.

Shiga toxin-producing Escherichia coli O157 : H7 shows an increased pathogenicity in mice after the passage through the gastrointestinal tract of the same host.

Haemolytic uraemic syndrome (HUS) is a rare but life-threatening complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infections, characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. Although the main infection route is the consumption of contaminated food or water, person-to-person transmission has been suggested in several situations. Moreover, epidemiological data indicate that the horizontal transmission of several pathogens, including STEC, among individuals of the same species requires significantly lower doses than those used in animal models infected with laboratory-cultured bacteria. Thus, the aim of this study was to evaluate whether the passage of a clinically isolated STEC strain through the gastrointestinal tract of mice affects its pathogenicity in mice. To test this, weaned mice were orally inoculated by gavage with either an E. coli O157:H7 isolate from an HUS patient, or the same strain recovered from stools after one or two successive passages through the gastrointestinal tract of the mice. We show that stool-recovered strains are able to induce a more generalized and persistent colonization than the parent strain. Furthermore, a 10(4)-fold-reduced inoculum of the stool-recovered strains still causes gut colonization and mouse mortality, which are not observed with the parent strain. These results indicate an increased pathogenicity in stool-recovered strains that may be associated with an increased ability to colonize the mouse intestine.

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.

[Update on the treatment of endemic hemolytic uremic syndrome. Pathogenesis and treatment of the most severe systemic complication of infections by Shiga toxin-producing Escherichia coli]. / Actualización en el tratamiento del síndrome urémico hemolítico endémico. Patogénesis y tratamiento de la complicación sistémica más grave de las infecciones por Escherichia coli productor de toxina Shiga.

The typical form of hemolytic uremic syndrome (HUS) is the major complication of Shiga toxin-producing Escherichia coli (STEC) infections. HUS is a critical health problem in Argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure, giving account for 20% of renal transplants in children and adolescents in our country. In spite of the extensive research in the field, the mainstay of treatment for patients with HUS is supportive therapy, and there are no specific therapies preventing or ameliorating the disease course. In this review, we present the current knowledge about pathogenic mechanisms and discuss traditional and innovative therapeutic approaches, with special focus in national status and contributions made by Argentinean groups.

Actualización en el tratamiento del síndrome urémico hemolítico endémico: Patogénesis y tratamiento de la complicación sistémica más grave de las infecciones por Escherichia coli productor de toxina Shiga / Update on the treatment of endemic hemolytic uremic syndrome: Pathogenesis and treatment of the most severe systemic complication of infections by Shiga toxin-producing Escherichia coli

La forma típica o post-diarreica del síndrome urémico hemolítico (SUH) es la complicación más grave de las infecciones por cepas de Escherichia coli productoras de toxina Shiga (STEC). En la Argentina el SUH es un problema crítico de salud pública, ya que representa la principal causa de falla renal aguda en la infancia, la segunda causa de falla renal crónica, y aporta el 20% de los casos de transplante renal durante la infancia y la adolescencia. A pesar de los avances en el conocimiento de su patogénesis, el único tratamiento actual de los pacientes con SUH es de sostén, y no existen terapias específicas ni preventivas. En la presente revisión expondremos los conocimientos básicos de los mecanismos patogénicos y discutiremos los enfoques terapéuticos tradicionales e innovadores, con especial foco en la situación nacional y los aportes hechos por grupos de la Argentina.

The typical form of hemolytic uremic syndrome (HUS) is the major complication of Shiga toxin-producing Escherichia coli (STEC) infections. HUS is a critical health problem in Argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure, giving account for 20% of renal transplants in children and adolescents in our country. In spite of the extensive research in the field, the mainstay of treatment for patients with HUS is supportive therapy, and there are no specific therapies preventing or ameliorating the disease course. In this review, we present the current knowledge about pathogenic mechanisms and discuss traditional and innovative therapeutic approaches, with special focus in national status and contributions made by Argentinean groups.

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.