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.

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.