Involvement of aquaporins in Shiga toxin-induced swelling and water transport dysfunction in human renal microvascular endothelial cells.

One of the hallmarks of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is kidney damage. Our previous research demonstrated that Shiga toxin type 2 (Stx2a) decreases cell viability and induces swelling of human glomerular endothelial cells (HGEC). However, Stx2a can disrupt net water transport across HGEC monolayers without affecting cell viability. This work aimed to elucidate the possible mechanisms involved in the water transport disruption caused by Stx2a across HGEC monolayers. We investigated paracellular and transcellular water transfer across HGEC by analyzing the passage of FITC-Dextran and the hydrostatic pressure (Phydr) and measuring the osmotic pressure (Posm), respectively. Stx2a selectively affected the transcellular pathway without impacting the paracellular route. Furthermore, Stx2a cell swelling was prevented by pretreatment with aquaporin inhibitors tetraethylammonium chloride (TEA), Mercury (II) chloride (HgCl2) or TGN-020, suggesting aquaporin involvement in this process. Confocal microscopy revealed that Stx2a increased HGEC total volume, which TEA and TGN-020 counteracted. Additionally, we identified in HGEC not only the expression of aquaporin-1 (AQP1) but also the expression of aquaporin-4 (AQP4). Surprisingly, we observed a decrease in the expression of both AQPs after Stx2a exposure. Our findings suggest that Stx2a may induce water movement into HGEC via AQP1 and AQP4, increasing total cell volume. Subsequently, decreased AQP1 and AQP4 expression could inhibit transcellular water transfer, potentially as a protective mechanism against excessive water entry and cell lysis.

Detection of plasma anti-lipopolysaccharide (LPS)antibodies against enterohemorrhagic Escherichia coli(EHEC) in asymptomatic kindergarten teachers fromBuenos Aires province

Resumen En Argentina, el síndrome urémico hemolítico causado por Escherichia coli enterohemorrágica (EHEC) tiene la más alta incidencia del mundo. Las infecciones por EHEC tienen un comportamiento endemoepidémico y causan del 20 al 30% de los síndromes de diarrea sanguinolenta en niños menores de 5 años. En el período 2016-2020, se notificaron 272 nuevos casos por año al Sistema de Vigilancia de Salud Nacional. Múltiples factores son responsables de la alta incidencia de SUH en Argentina, incluyendo la transmisión persona-persona. Con el objetivo de detectar posibles portadores asintomáticos de EHEC, realizamos un estudio preliminar de la frecuencia de anticuerpos antilipopolisacáridos contra los serotipos de EHEC más prevalentes en Argentina. El estudio se realizó con muestras de plasma obtenidas de 61 maestras y maestros de jardines de infantes de 26 instituciones del distrito de José C. Paz, localizado en el área suburbana de la provincia de Buenos Aires, Argentina. El 51% de las muestras presentaron anticuerpos contra los serotipos de lipopolisacáridos O157, O145, O121 y O103; el 6,4% de las muestras positivas tuvieron el isotipo IgM (n=2), el 61,3% el isotipo IgG (n=19) y el 32,3% los isotipos IgM e IgG (n=10). Dado que los anticuerpos antilipopolisacáridos presentan usualmente una duración corta, la detección de IgM específica podría indicar una infección reciente. Además, el alto porcentaje de muestras positivas hallado podría indicar una exposición frecuente a las cepas de EHEC en la cohorte estudiada. Asimismo, la gran población de adultos portadores asintomáticos de estas cepas patógenas podría contribuir al comportamiento endémico, a través de la transmisión persona-persona. El perfeccionamiento de programas educacionales continuos en jardines de infantes podría constituir una medida importante para reducir los casos de síndrome urémico hemolítico, no solo en Argentina, sino también en el mundo.

Abstract In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Detection of plasma anti-lipopolysaccharide (LPS) antibodies against enterohemorrhagic Escherichia coli (EHEC) in asymptomatic kindergarten teachers from Buenos Aires province.

In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Detection and characterization of circulating microvesicles containing Shiga toxin type 2 in a rat model of Hemolytic Uremic Syndrome.

Shiga toxin producing Escherichia coli (STEC) are foodborne pathogens that release Shiga toxin (Stx), virulence factor responsible for the development of Hemolytic Uremic Syndrome (HUS). Stx causes endothelial cell damage, which leads to platelets deposition and thrombi formation within the microvasculature. It has been described that Stx activates blood cells and induces the shedding of proinflammatory and prothrombotic microvesicles (MVs) containing the toxin. In this sense, it has been postulated that MVs containing Stx2 (MVs-Stx2+) can contribute to the physiopathology of HUS, allowing Stx2 to reach the target organs while evading the immune system. In this work, we propose that circulating MVs-Stx2+ can be a potential biomarker for the diagnosis and prognosis of STEC infections and HUS progression. We developed a rat HUS model by the intraperitoneal injection of a sublethal dose of Stx2 and observed: decrease in body weight, increase of creatinine and urea levels, decrease of creatinine clearance and histological renal damages. After characterization of renal damages, we investigated circulating total MVs and MVs-Stx2+ by flow cytometry at different times after Stx2 injection. Additionally, we evaluated the correlation of biochemical parameters such as creatinine and urea in plasma with MVs-Stx2+. As a result, we found a significant circulation of MVs-Stx2+ at 72 and 96 h after Stx2 injection, nevertheless no correlation with creatinine and urea plasma levels were detected. Our results suggest that MVs-Stx2+ may be an additional biomarker for the characterization and diagnosis of HUS progression. A further analysis is required in order to validate MVs-Stx2+ as biomarker of the disease.

Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells.

BACKGROUND: Shiga toxin-producing Escherichia coli is responsible for post-diarrheal (D+) hemolytic uremic syndrome (HUS), which is a cause of acute renal failure in children. The glycolipid globotriaosylceramide (Gb3) is the main receptor for Shiga toxin (Stx) in kidney target cells. Eliglustat (EG) is a specific and potent inhibitor of glucosylceramide synthase, first step of glycosphingolipid biosynthesis, actually used for the treatment of Gaucher's disease. The aim of the present work was to evaluate the efficiency of EG in preventing the damage caused by Stx2 in human renal epithelial cells. METHODS: Human renal tubular epithelial cell (HRTEC) primary cultures were pre-treated with different dilutions of EG followed by co-incubation with EG and Stx2 at different times, and cell viability, proliferation, apoptosis, tubulogenesis, and Gb3 expression were assessed. RESULTS: In HRTEC, pre-treatments with 50 nmol/L EG for 24 h, or 500 nmol/L EG for 6 h, reduced Gb3 expression and totally prevented the effects of Stx2 on cell viability, proliferation, and apoptosis. EG treatment also allowed the development of tubulogenesis in 3D-HRTEC exposed to Stx2. CONCLUSIONS: EG could be a potential therapeutic drug for the prevention of acute kidney injury caused by Stx2. IMPACT: For the first time, we have demonstrated that Eliglustat prevents Shiga toxin 2 cytotoxic effects on human renal epithelia, by reducing the expression of the toxin receptor globotriaosylceramide. The present work also shows that Eliglustat prevents Shiga toxin 2 effects on tubulogenesis of renal epithelial cells. Eliglustat, actually used for the treatment of patients with Gaucher's disease, could be a therapeutic strategy to prevent the renal damage caused by Shiga toxin.

Combined Action of Shiga Toxin Type 2 and Subtilase Cytotoxin in the Pathogenesis of Hemolytic Uremic Syndrome.

Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.

Glioblastoma cells potentiate the induction of the Th1-like profile in phosphoantigen-stimulated γδ T lymphocytes.

PURPOSE: γδ T lymphocytes are non-conventional T cells that participate in protective immunity and tumor surveillance. In healthy humans, the main subset of circulating γδ T cells express the TCRVγ9Vδ2. This subset responds to non-peptide prenyl-pyrophosphate antigens such as (E)-4-hydroxy-3-methyl-but-enyl pyrophosphate (HMBPP). This unique feature of Vγ9Vδ2 T cells makes them a candidate for anti-tumor immunotherapy. In this study, we investigated the response of HMBPP-activated Vγ9Vδ2 T lymphocytes to glioblastoma multiforme (GBM) cells. METHODS: Human purified γδ T cells were stimulated with HMBPP (1 µM) and incubated with GBM cells (U251, U373 and primary GBM cultures) or their conditioned medium. After overnight incubation, expression of CD69 and perforin was evaluated by flow cytometry and cytokines production by ELISA. As well, we performed a meta-analysis of transcriptomic data obtained from The Cancer Genome Atlas. RESULTS: HMBPP-stimulated γδ T cells cultured with GBM or its conditioned medium increased CD69, intracellular perforin, IFN-γ, and TNF-α production. A meta-analysis of transcriptomic data showed that GBM patients display better overall survival when mRNA TRGV9, the Vγ9 chain-encoding gene, was expressed in high levels. Moreover, its expression was higher in low-grade GBM compared to GBM. Interestingly, there was an association between γδ T cell infiltrates and TNF-α expression in the tumor microenvironment. CONCLUSION: GBM cells enhanced Th1-like profile differentiation in phosphoantigen-stimulated γδ T cells. Our results reinforce data that have demonstrated the implication of Vγ9Vδ2 T cells in the control of GBM, and this knowledge is fundamental to the development of immunotherapeutic protocols to treat GBM based on γδ T cells.

Crosstalk between Human Microvascular Endothelial Cells and Tubular Epithelial Cells Modulates Pro-Inflammatory Responses Induced by Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is a consequence of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection and is the most frequent cause of acute renal failure (ARF) in children. Subtilase cytotoxin (SubAB) has also been associated with HUS pathogenesis. We previously reported that Stx2 and SubAB cause different effects on co-cultures of human renal microvascular endothelial cells (HGEC) and human proximal tubular epithelial cells (HK-2) relative to HGEC and HK-2 monocultures. In this work we have analyzed the secretion of pro-inflammatory cytokines by co-cultures compared to monocultures exposed or not to Stx2, SubAB, and Stx2+SubAB. Under basal conditions, IL-6, IL-8 and TNF-α secretion was different between monocultures and co-cultures. After toxin treatments, high concentrations of Stx2 and SubAB decreased cytokine secretion by HGEC monocultures, but in contrast, low toxin concentrations increased their release. Toxins did not modulate the cytokine secretion by HK-2 monocultures, but increased their release in the HK-2 co-culture compartment. In addition, HK-2 monocultures were stimulated to release IL-8 after incubation with HGEC conditioned media. Finally, Stx2 and SubAB were detected in HGEC and HK-2 cells from the co-cultures. This work describes, for the first time, the inflammatory responses induced by Stx2 and SubAB, in a crosstalk model of renal endothelial and epithelial cells.

Recent Advances in Shiga Toxin-Producing Escherichia coli Research in Latin America.

Pathogenic Escherichia coli are known to be a common cause of diarrheal disease and a frequently occurring bacterial infection in children and adults in Latin America. Despite the effort to combat diarrheal infections, the south of the American continent remains a hot spot for infections and sequelae associated with the acquisition of one category of pathogenic E. coli, the Shiga toxin-producing E. coli (STEC). This review will focus on an overview of the prevalence of different STEC serotypes in human, animals and food products, focusing on recent reports from Latin America outlining the recent research progress achieved in this region to combat disease and endemicity in affected countries and to improve understanding on emerging serotypes and their virulence factors. Furthermore, this review will highlight the progress done in vaccine development and treatment and will also discuss the effort of the Latin American investigators to respond to the thread of STEC infections by establishing a multidisciplinary network of experts that are addressing STEC-associated animal, human and environmental health issues, while trying to reduce human disease. Regardless of the significant scientific contributions to understand and combat STEC infections worldwide, many significant challenges still exist and this review has focus in the Latin American efforts as an example of what can be accomplished when multiple groups have a common goal.

Soluble CD40 Ligand and Oxidative Response Are Reciprocally Stimulated during Shiga Toxin-Associated Hemolytic Uremic Syndrome.

Shiga toxin (Stx), produced by Escherichia coli, is the main pathogenic factor of diarrhea-associated hemolytic uremic syndrome (HUS), which is characterized by the obstruction of renal microvasculature by platelet-fibrin thrombi. It is well known that the oxidative imbalance generated by Stx induces platelet activation, contributing to thrombus formation. Moreover, activated platelets release soluble CD40 ligand (sCD40L), which in turn contributes to oxidative imbalance, triggering the release of reactive oxidative species (ROS) on various cellular types. The aim of this work was to determine if the interaction between the oxidative response and platelet-derived sCD40L, as consequence of Stx-induced endothelium damage, participates in the pathogenic mechanism during HUS. Activated human glomerular endothelial cells (HGEC) by Stx2 induced platelets to adhere to them. Although platelet adhesion did not contribute to endothelial damage, high levels of sCD40L were released to the medium. The release of sCD40L by activated platelets was inhibited by antioxidant treatment. Furthermore, we found increased levels of sCD40L in plasma from HUS patients, which were also able to trigger the respiratory burst in monocytes in a sCD40L-dependent manner. Thus, we concluded that platelet-derived sCD40L and the oxidative response are reciprocally stimulated during Stx2-associated HUS. This process may contribute to the evolution of glomerular occlusion and the microangiopathic lesions.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin.

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Immunization with BLS-Stx2B chimera totally protects dams from early pregnancy loss induced by Shiga toxin type 2 (Stx2) and confers anti-Stx2 immunity to the offspring.

Shiga toxin producing Escherichia coli (STEC) are bacterial pathogens involved in food-borne diseases. Shiga toxin (Stx) is the main virulence factor of STEC and is responsible for systemic complications including Hemolytic Uremic Syndrome (HUS). It has been previously demonstrated that Shiga toxin type 2 (Stx2) induces pregnancy loss in rats in early stage of pregnancy. The main purpose of this study was to determine if an active immunization prevents Stx2 mediated pregnancy loss and confers passive protective immunity to the offspring. For that purpose Sprague Dawley female rats were immunized with the chimera based on the enzyme lumazine synthase from Brucella spp. (BLS) and the B subunit of Shiga toxin 2 (Stx2B) named BLS-Stx2B. After immunization females were mated with males. At day 8 of gestation, dams were challenged intraperitoneally with a sublethal and abortifacient dose of Stx2. The immunization induced high anti-Stx2B-specific antibody titers in sera and most important, prevented pregnancy loss. Pups born and breastfeed by immunized dams had high anti-Stx2B-specific antibody titers in sera. Cross-fostering experiments indicated that passive protective immunity against Stx2 was transmitted through lactation. These results indicate that immunization of adult female rats with BLS-Stx2B prevents Stx2-induced pregnancy loss and confers anti Stx2 protective immunity to the offspring.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20-30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells.

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo.

Effects of Shiga toxin type 2 on maternal and fetal status in rats in the early stage of pregnancy.

Shiga toxin type 2 (Stx2), a toxin secreted by Shiga toxin-producing Escherichia coli (STEC), could be one of the causes of maternal and fetal morbimortality not yet investigated. In this study, we examined the effects of Stx2 in rats in the early stage of pregnancy. Sprague-Dawley pregnant rats were intraperitoneally (i.p.) injected with sublethal doses of Stx2, 0.25 and 0.5 ng Stx2/g of body weight (bwt), at day 8 of gestation (early postimplantation period of gestation). Maternal weight loss and food and water intake were analyzed after Stx2 injection. Another group of rats were euthanized and uteri were collected at different times to evaluate fetal status. Immunolocalization of Stx2 in uterus and maternal kidneys was analyzed by immunohistochemistry. The presence of Stx2 receptor (globotriaosylceramide, Gb3) in the uteroplacental unit was observed by thin layer chromatography (TLC). Sublethal doses of Stx2 in rats caused maternal weight loss and pregnancy loss. Stx2 and Gb3 receptor were localized in decidual tissues. Stx2 was also immunolocalized in renal tissues. Our results demonstrate that Stx2 leads to pregnancy loss and maternal morbidity in rats in the early stage of pregnancy. This study highlights the possibility of human pregnancy loss and maternal morbidity mediated by Stx2.

Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells.

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 -a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

Leukotriene C(4) prevents the complete maturation of murine dendritic cells and modifies interleukin-12/interleukin-23 balance.

Leukotriene C(4) is an important mediator in the development of inflammatory reactions and ischaemia. Previous studies have shown that leukotriene C(4) is able to modulate the function of dendritic cells (DCs) and induce their chemotaxis from skin to lymph node. In this study, we decided to evaluate the modulation exerted by leukotriene C(4) on DCs, depending on their status of activation. We showed for the first time that leukotriene C(4) stimulates endocytosis both in immature and lipopolysaccharide (LPS) -activated DCs. Moreover, it suppressed the interleukin-12p70 (IL-12p70) release, but induces the secretion of IL-23 by DCs activated with LPS and promotes the expansion of T helper type 17 (Th17) lymphocytes. Furthermore, blocking the release of IL-23 reduced the percentages of CD4(+) T cells producing IL-17 in a mixed lymphocyte reaction. Ours results suggest that leukotriene C(4) interferes with the complete maturation of inflammatory DCs in terms of phenotype and antigen uptake, while favouring the release of IL-23, the main cytokine involved in the maintenance of the Th17 profile.

Histamine-treated dendritic cells improve recruitment of type 2 CD8 T cells in the lungs of allergic mice.

Histamine controls the function of dendritic cells (DCs). It appears to be required for the normal development of DCs. It also induces the chemotaxis of immature DCs and promotes the differentiation of CD4(+) T cells into cells with a T helper type 2 (Th2) profile. Moreover, we have recently shown that histamine stimulates both the uptake and the cross-presentation of antigens by DCs, supporting the theory that histamine promotes activation of CD8(+) T cells during the development of allergic pathologies. Here, we investigated whether the course of an allergic response, in a well-defined model of ovalbumin (OVA)-induced allergic airway inflammation, could be modulated by intratracheal injection of OVA-pulsed DCs previously treated with histamine (DCHISs). Compared with control DCs, DCHISs induced: (i) greater recruitment of CD8(+) T cells in the lung, (ii) greater stimulation of the production of interleukin (IL)-5 by lung CD8(+) T cells, and (iii) increased recruitment of CD11c/CD8 double-positive DCs in the lungs of allergic mice. Moreover, mice treated with DCHISs showed increased levels of serum-specific immunoglobulin E (IgE) antibodies directed to OVA, and a higher proportion of eosinophils in bronchoalveolar lavage (BAL) compared with mice treated with OVA-pulsed control DCs. Our results support the notion that histamine, by acting on DCs, increases the severity of allergic processes.

GM-CSF enhances a CpG-independent pathway of neutrophil activation triggered by bacterial DNA.

We have previously demonstrated that bacterial DNA induces neutrophil activation through a CpG- and TLR9-independent but MyD88-dependent-pathway. In this study we determined that GM-CSF enhances the activation of neutrophils by bacterial DNA. Granulocyte-macrophage colony-stimulating factor increased IL-8 and IL-1beta secretion, and CD11b-upregulation induced by single-stranded bacterial DNA. It also enhanced neutrophil IL-8 production induced by double-stranded bacterial DNA, methylated single-stranded DNA, plasmid DNA, and phosphorothioated-CpG and non-CpG-oligodeoxynucleotides. Together these observations indicated that GM-CSF enhances neutrophil responses triggered by bacterial DNA in a CpG-independent fashion. We also found that GM-CSF enhanced the activation of the MAPKs p38 and ERK1/2 induced by bacterial DNA. Moreover, the pharmacological inhibition of these pathways significantly diminished GM-CSF ability to increase neutrophil activation by bacterial DNA. Finally, we observed that GM-CSF was unable to increase the activation of MyD88(-/-) neutrophils by bacterial DNA. Our findings suggest that GM-CSF modulates the CpG-independent, MyD88-dependent neutrophil response to bacterial DNA, by increasing the activation of the MAPKs p38 and ERK1/2.

Interplay of pathogens, cytokines and other stress signals in the regulation of dendritic cell function.

Dendritic cells (DCs) are the only antigen-presenting cell capable of activating naïve T lymphocytes, and hence they play a crucial role in the induction of adaptive immunity. Immature DCs sample and process antigens, and efficiently sense a large variety of signals from the surrounding environment. Upon activation, they become capable to activate naïve T cells and to direct the differentiation and polarization of effector T lymphocytes. It is becoming increasingly clear that different signals are able to determine distinct programs of DC differentiation and different forms of immunity and tolerance. In the past few years many advances have been made in addressing the action exerted by pathogen-associated molecular patterns (PAMPs), cytokines, chemokines, and other less characterized stress molecules on the activity of DCs. In this review we focus on the multiplicity of innate signals able to modulate the functional profile of DCs.