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.

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice.

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.

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)-ß 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-alpha, interleukin (IL)-1, IL-6, IL-8, interferon-gamma] and the concomitant induction of anti-inflammatory mediators such as IL-10 and transforming growth factor-beta 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.

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.

Activación de la respuesta inmune innata y específica en los pacientes con sindrome urémico hemolítico: papel dual a traves de la paticipacion en los mecanismos pagogenicos y protectores / Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)-patients

The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.

Activación de la respuesta inmune innata y específica en los pacientes con sindrome urémico hemolítico: papel dual a traves de la paticipacion en los mecanismos pagogenicos y protectores / Activation of innate and specific immune responses in hemolytic uremic syndrome (HUS)-patients

The central role of the immune system is the preservation of the health against several pathogenic microbes and injury agents. However, on special conditions defensive mechanisms triggered towards the foreign agent can damage the host. Clinical and experimental evidence indicate that inflammatory reaction triggered by the main components of Shiga toxin (Stx)-producing Escherichia coil (STEC), participate in the evolution to the complete form of HUS. When children are diagnosed of HUS, they present evidence that have suffered a very strong and early inflammatory response. These features include: the presence of a marked neutrophilia, the polymorfonuclear leucocytes (PMN) are "deactivated or exhausted" and the monocytes are differentiated towards an inflammatory phenotype (CD14-reduced and CD16-enhanced membrane expression). In addition, HUS-patients show a marked reduction in the absolute and relative number of leucocytes carrying the receptor (CX3CR1) for the chemokine "Fractalkine" (FKN, CX3CL1), which are the classic monocytes and Natural Killer cells (NK). All these cells express a high cytotoxic potencial. The chemokine FKN is expressed in endothelial and epithelial renal cells, and is involved in the pathogenic mechanism of different nephropathies. Noteworthy, we found a significant correlation between the severity of the renal damage (as days of anuria) and the alterations described above. Finally, the protective role of specific immune response, mainly through the antibody production with Stx-neutralizing capacity, is discussed.(AU)

Inhibition of allospecific response in pancreatic islet transplantation: the glycan approach.

UNLABELLED: Host resistance has precluded clinical islet transplantation from becoming a consistent therapy for type I diabetic patients, mainly due to both specific and nonspecific processes. O-glycosylated proteins have a primary role in immunologic synapses. Therefore, we investigated the effects of a putative immunomodulatory effect of the cleavage of these molecules on islet allotransplantation. METHODS: Murine islets were treated with O-sialoglycoprotein endopeptidase. Three endpoints were studied: (1) proliferation in allogeneic mixed islet mononuclear cell reactions using treated and control irradiated islets as stimulator cells of mononuclear cells; (2) expression of IA-d on monocytes using 48-hour transplants of treated versus control mouse islets into subcutaneous capsules; (3) posttransplant graft function in an in vivo model of islet allotransplantation. Treated and control islets were transplanted in diabetic mice treated daily with cyclosporine. Glycemia was monitored to determine diabetes reversion. RESULTS: The allogeneic proliferative response was maximal when allogeneic mononuclear cells were mixed with control islets; it was significantly decreased with treated islets. Mean proliferative inhibition rate of treated vs. control was 62%. IA-d expression on monocytes was maximal in control islets. Reversion was significantly different for treated versus control islets with its duration varied from 3 to 7 days. CONCLUSION: These results suggest that treatment of islets with O-sialoglycoprotein endopeptidase may modulate allogeneic immunologic reactions.

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). Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2. Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process. The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 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.

Macrophage derived signalling regulates negatively the megakaryocyte compartment.

Megakaryocytopoiesis is the process by which stem cells go through a process of commitment, proliferation and differentiation leading to the production of platelets. In the mouse, this process is accomplished within the bone marrow (BM) and spleen microenvironment and is carried out by regulatory molecules and accessory cells including macrophages, fibroblasts and endothelial-like cells. Previously, we have reported that macrophage depletion following administration of liposomal clodronate (LIP-CLOD) provokes enhancement of both, megakaryocytopoiesis and thrombocytopoiesis. In this report, we investigated the changes in the compartment of megakaryocyte progenitor cells (MK-CFU), their correlation with plasmatic thrombopoietin (TPO) and TPO transcription levels after macrophage depletion. LIP-CLOD-treated mice showed an increase of the MK-CFU in BM and spleen. Concerning TPO plasma levels, kinetic studies revealed a 1.5- and 1.3-fold increase in the TPO concentration at 12 and 24 hr of treatment. We also show evidence of regulation of TPO transcription in the liver and spleen. Although empty liposomes also enhanced TPO gene regulation in these organs, transcriptional TPO up regulation correlated with an increase of protein synthesis only in those animals where macrophages were effectively removed. Taken together, these results suggest that BM and spleen macrophages derived signalling regulates negatively the megakaryocyte compartment.

Immune complex-FcgammaR interaction modulates monocyte/macrophage molecules involved in inflammation and immune response.

The interaction between receptors for the Fc portion of IgG (FcgammaRs) from monocytes/macrophages and immune complexes (IC) triggers regulatory and effector functions. Recently, we have demonstrated that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. Taking into account that the regulation of MHC class II molecules is a crucial event in the immune response, in this report we extend our previous studies analysing the effect of STAT-1 phosphorylation in the down-regulatory process, the fate of the intracellular pool of MHC class II molecules and the effect of complement on MHC class II down-regulation induced by IC. We also studied the effect of IC on the expression of MHC class II (I-A(d)) in macrophages using a mouse model of chronic inflammation. We demonstrate that IC induce a depletion not only on surface expressed but also on intracellular MHC class II content and that IC-induced down-regulation of MHC class II is not mediated by the inhibition of STAT-1 phosphorylation. On the other hand, the effect of IC is not specific for the down-regulation of MHC class II, for it could be restricted to other molecules involved in inflammatory processes. Our experiments also show that the activation of the complement system could be a crucial step on the regulation of the effect of IC on MHC class II expression. In agreement with our in vitro experiments using human monocytes, IC treatment reduces the expression of MHC class II in a mouse model of chronic inflammation.

Monocytes and neutrophils from tuberculosis patients are insensitive to anti-inflammatory effects triggered by the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP).

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis where formyl peptides, which are cleavage products of bacterial and mitochondrial proteins, are present. In this study, we demonstrated that interferon gamma (IFN)-gamma and interleukin (IL)-10 induced the overexpression of the receptor for the Fc portion of IgG I (FcgammaRI) in monocytes from tuberculosis (TB) patients, showing that these cells respond to IFN-gamma and IL-10 signals. We also demonstrated that lower doses of IL-10 render monocytes from TB patients less responsive to higher doses of the cytokine. Although the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) is a well-known proinflammatory agonist, we have demonstrated previously that preincubation of monocytes with FMLP inhibited the up-regulation of FcgammaRI induced by IFN-gamma or IL-10. This effect was not observed in monocytes from TB patients. FMLP also induced the down-regulation of the expression of FcgammaRI in monocytes that had been activated already with IFN-gamma. However, this effect of FMLP was not observed in monocytes from TB patients and supernatants from monocytes obtained from these patients were incapable of inducing the down-regulation of FcgammaRI. In contrast to normal donors, supernatants from FMLP-treated neutrophils from TB patients did not modify the basal level of expression of FcgammaRI in monocytes from normal donors. In conclusion, in this study we demonstrated the existence of two novel mechanisms that may contribute to the pathological effects generated by M. tuberculosis: the enhancement of FcgammaRI in response to IFN-gamma and IL-10, and the unresponsiveness to the anti-inflammatory effects induced by formyl peptides.

The formyl peptide N-formyl-methionyl-leucyl-phenylalanine downregulates the expression of FcgammaRs in interferon-gamma-activated monocytes/macrophages in vitro and in vivo.

N-Formyl peptides are cleavage products of bacterial and mitochondrial proteins that have pro-inflammatory activities and play an important role in antibacterial host defence. FcgammaRI is a receptor for the Fc portion of immunoglobulin G expressed in monocytes that mediates cytotoxicity and is upregulated by interferon-gamma (IFN-gamma) and interleukin-10 (IL-10). In this report, we demonstrate that N-formyl-methionyl-leucyl-phenylalanine (FMLP) downregulates the expression of FcgammaRI in IFN-gamma-treated monocytes, but not in IL-10-treated monocytes. We determine that supernatants obtained from monocytes treated with IFN-gamma and then exposed to FMLP induce the downregulation of FcgammaRI in naïve monocytes. This effect is abrogated by the protease inhibitors phenylmethylsulphonyl fluoride and phosphoramidon, which inhibit serine and metalloproteases, respectively. Supernatants from FMLP-treated neutrophils also induce the downregulation of FcgammaRI, when added to naïve monocytes. Similar observations were obtained in vivo in a mouse model of chronic inflammation. In vivo, FMLP also downregulates the expression of FcgammaRs in IFN-gamma-activated macrophages. Our results support the existence of a new mechanism through which FMLP could modulate the activity of monocytes/macrophages during bacterial infections.

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.

Interleukin-1beta induces in vivo tolerance to lipopolysaccharide in mice.

Endotoxin or lipopolysaccharide (LPS) tolerance may be partially due to the secretion of potent anti-inflammatory cytokines following severe Gram-negative infections, or by low doses of LPS. In this work, we describe the effects of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF-), two early cytokines secreted after LPS exposure, in the induction of LPS tolerance. Our results demonstrate that mice treated with three daily doses of 100 ng of IL-1 were tolerant to LPS-induced shock. However, TNF- was unable to induce an LPS refractory state. Given the fact that 100 ng of IL-1 increase the plasma levels of glucocorticoids, we evaluated whether a daily injection of dexamethasone (DEX) alone was able to reproduce the LPS-like tolerant state. However, no signs of LPS refractoriness were detected, except when DEX was administered concomitantly with a dose of IL-1 that does not induce corticosterone secretion (12 ng/mouse). This dose was found to induce in vitro up-regulation of the glucocorticoid receptors (GcR) of peritoneal macrophages following 24 h of treatment. In addition, we demonstrate that IL-1 is capable of inducing the down-regulation of Toll-like receptor 4 (TLR4), a crucial molecule in the signal transduction of LPS. Taken together, our results indicate that IL-1 can generate tolerance to LPS in vivo, and suggest that the regulation of mechanisms of the down-regulation of TLR4, as well as those involved in the expression of GcR and/or in the secretion of glucocorticoids, would be crucial for these effects.

Immune complexes (IC) down-regulate the basal and interferon-gamma-induced expression of MHC class II on human monocytes.

The interaction of Fc receptors for IgG (FcgammaRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcgammaR-IC interactions inhibit the IFN-gamma-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-gamma are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-gamma. We demonstrate that IC exert a drastic inhibition of basal and IFN-gamma-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcgammaRI and FcgammaRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class II expression, antigen presentation was markedly inhibited in the presence of IC.

Activation of peripheral blood neutrophils from patients with active advanced tuberculosis.

Activation of peripheral blood neutrophils (PMN) was investigated in order to determine whether they might contribute to the inflammatory process during active advanced tuberculosis. Receptors for the Fc portion of IgG (FcgammaR) (FcgammaRI, FcgammaRII, and FcgammaRIIIB), CD66 (degranulation marker), and receptors for tumor necrosis factor-alpha (TNF-R55 and TNF-R75) were analyzed on PMN obtained from normal controls and tuberculosis patients (TB-PMN). Functional parameters such as cytotoxicity, superoxide anion generation triggered by N-formyl-methionyl-leucyl-phenyl-alanine (FMLP), and TNF-alpha and IL-1beta production were evaluated. A high expression of TNF-R55, CD66, and FcgammaRIIIB and the appearance of FcgammaRI were detected in TB-PMN. In addition, cytotoxicity, superoxide anion release, and TNF-alpha and IL-1beta production were enhanced in TB-PMN. Thus, in tuberculosis, the activation of PMN outside the focus of infection strongly suggests the possibility of a systemic inflammation that could modulate the inflammatory response.