EFFECTS OF BACTERIAL LIPOPOLYSACCHARIDE AND SHIGA TOXIN ON INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELLS.

ABSTRACT: Background : Mesenchymal stem cells (MSCs) can be activated by different bacterial toxins. Lipopolysaccharides and Shiga Toxin (Stx) are the main toxins necessary for hemolytic uremic syndrome development. The main etiological event in this disease is endothelial damage that causes glomerular destruction. Considering the repairing properties of MSC, we aimed to study the response of MSC derived from induced pluripotent stem cells (iPSC-MSC) to LPS and/or Stx and its effect on the restoration of injured endothelial cells. Methods : iPSC-MSC were treated with LPS and or/Stx for 24 h and secretion of cytokines, adhesion, and migration were measured in response to these toxins. In addition, conditioned media from treated iPSC-MSC were collected and used for proteomics analysis and evaluation of endothelial cell healing and tubulogenesis using human microvascular endothelial cells 1 as a source of endothelial cells. Results : The results obtained showed that LPS induced a proinflammatory profile on iPSC-MSC, whereas Stx effects were less evident, even though cells expressed the Gb 3 receptor. Moreover, LPS induced on iPSC-MSC an increment in migration and adhesion to a gelatin substrate. Addition of conditioned media of iPSC-MSC treated with LPS + Stx, decreased the capacity of human microvascular endothelial cells 1 to close a wound, and did not favor tubulogenesis. Proteomic analysis of iPSC-MSC treated with LPS and/or Stx revealed specific protein secretion patterns that support the functional results described. Conclusions : iPSC-MSC activated by LPS acquired a proinflammatory profile that induces migration and adhesion to extracellular matrix proteins but the addition of Stx did not activate any repair program to ameliorate endothelial damage, indicating that the use of iPSC-MSC to regenerate endothelial injury caused by LPS and/or Stx in hemolytic uremic syndrome could not be the best option to consider to regenerate a tissue injury.

Neutrophil Extracellular Traps Induced by Shiga Toxin and Lipopolysaccharide-Treated Platelets Exacerbate Endothelial Cell Damage.

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN-Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN-Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN-Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.

Modulation of neutrophil extracellular traps release by Klebsiella pneumoniae.

One of the main bactericidal mechanisms of polymorphonuclear neutrophils (PMN) is the release of neutrophil extracellular traps (NETs), which capture and destroy pathogens. Klebsiella pneumoniae (Kpn) producer of carbapenemase (KPC) and belonging to the sequence type 258 (ST258), is a hyper epidemic clone that causes a large number of infections worldwide associated with high persistence and mortality. It is necessary to investigate the interaction of Kpn KPC with the immune system to improve prevention and treatment of infections mediated by this bacterium. Based on the hypothesis that Kpn is able to subvert PMN-mediated death, the aim was to assess whether Kpn KPC ST258 could modulate the bactericidal response of PMN, focusing on NETs formation, compared to another opportunistic pathogen, as Escherichia coli (Eco). The results showed that the release of NETs was absent when PMN were challenged with Kpn KPC, while Eco was a strong inducer of NETosis. Moreover, Kpn KPC was able to inhibit NETosis induced by Eco. The inhibition of Kpn KPC-mediated NETs formation still occurred in spite of exogenous addition of hydrogen peroxide (H2 O2 ), did not involve bacterial-released soluble factors or cell wall components, and was dependent on bacterial viability. Moreover, when degranulation was investigated, we found that Kpn KPC affected only the mobilization of primary granules, which harbor the proteins with more potent bactericidal properties and those related to NETosis. In conclusion, Kpn KPC ST258 effectively managed to evade the PMN response by inhibiting the release of NETs, and primary granule mobilization.

Integrin alpha-5 subunit is critical for the early stages of human pluripotent stem cell cardiac differentiation.

The stem cell niche has a strong influence in the differentiation potential of human pluripotent stem cells with integrins playing a major role in communicating cells with the extracellular environment. However, it is not well understood how interactions between integrins and the extracellular matrix are involved in cardiac stem cell differentiation. To evaluate this, we performed a profile of integrins expression in two stages of cardiac differentiation: mesodermal progenitors and cardiomyocytes. We found an active regulation of the expression of different integrins during cardiac differentiation. In particular, integrin α5 subunit showed an increased expression in mesodermal progenitors, and a significant downregulation in cardiomyocytes. To analyze the effect of α5 subunit, we modified its expression by using a CRISPRi technique. After its downregulation, a significant impairment in the process of epithelial-to-mesenchymal transition was seen. Early mesoderm development was significantly affected due to a downregulation of key genes such as T Brachyury and TBX6. Furthermore, we observed that repression of integrin α5 during early stages led to a reduction in cardiomyocyte differentiation and impaired contractility. In summary, our results showed the link between changes in cell identity with the regulation of integrin α5 expression through the alteration of early stages of mesoderm commitment.

Prokaryotic RNA activates endothelial cells promoting neutrophil transmigration.

Endothelial cell (EC)-neutrophil (PMN) interactions are crucial in the resolution of bacterial infections. Prokaryotic RNA (pRNA) has been reported as a pathogen-associated molecular pattern that is released from bacteria upon death and is able to activate PMN. In this work, we studied the effects of pRNA on EC and investigated whether these effects could modulate EC-PMN interaction. For this purpose, we purified total pRNA from Escherichia coli and used it as a stimulus for Human Umbilical Vein Endothelial Cells (HUVEC). We found that the incubation of pRNA with HUVEC caused the increase of surface intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on HUVEC, and the secretion of IL-8 and von Willebrand factor, characteristics consistent with HUVEC activation, without causing toxic effects. Moreover, pRNA-treated HUVEC also induced PMN adhesion and the conditioned medium obtained from treated-HUVEC was chemotactic for PMN and caused their activation, as determined by CD11b upregulation. As reported previously, the degradation products of pRNA induced similar biological effects. The treatment of HUVEC with endocytosis inhibitors revealed that the entry of pRNA partially relied on a clathrin-dependent mechanism, whereas the effects of degradation products could not be inhibited by any of the inhibitors tested. Using a transwell system, we found that pRNA or degraded pRNA were also able to stimulate HUVEC when recognized from the basolateral side. Our results indicate that pRNA activates EC, resulting in the modulation of EC-PMN interaction by inducing PMN chemotaxis, adhesion and activation. In the context of infection, pRNA sensed by EC and PMN could favor bacterial clearance.

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.

Novel Use of All-Trans-Retinoic Acid in A Model of Lipopolysaccharide-Immunosuppression to Decrease the Generation of Myeloid-Derived Suppressor Cells by Reducing the Proliferation of CD34+ Precursor Cells.

All-trans-retinoic acid (ATRA) is a derivative of vitamin A with antiproliferative properties. Endotoxin shock and subsequent immunosuppression (IS) by lipopolysaccharide (LPS) stimulates myelopoiesis with expansion of myeloid-derived suppressor cells (MDSC). Since we have previously shown that ATRA reverses the IS state by decreasing functional MDSC, our aim was to investigate if ATRA was able to modulate MDSC generation by regulating myelopoiesis in murine hematopoietic organs. We found that ATRA administration in vivo and in vitro decreased the number of CD34+ precursor cells that were increased in IS mice. When we studied the cellular mechanisms involved, we did not find any differences in apoptosis of CD34+ precursors or in the differentiation of these cells to their mature counterparts. Surprisingly, ATRA decreased precursor proliferation, in vitro and in vivo, as assessed by a reduction in the size and number of colony forming units generated from CD34+ cells and by a decreased incorporation of H-thymidine. Moreover, ATRA administration to IS mice decreased the number of MDSC in the spleen, with a restoration of T lymphocyte proliferation and a restitution of the histological architecture. Our results indicate, for the first time, a new use of ATRA to abolish LPS-induced myelopoiesis, affecting the proliferation of precursor cells, and in consequence, decreasing MDSC generation, having a direct impact on the improvement of immune competence. Administration of ATRA could overcome the immunosuppressive state generated by sepsis that often leads to opportunistic life-threatening infections. Therefore, ATRA could be considered a complementary treatment to enhance immune responses.

Immature myeloid Gr-1+ CD11b+ cells from lipopolysaccharide-immunosuppressed mice acquire inhibitory activity in the bone marrow and migrate to lymph nodes to exert their suppressive function.

Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Repetitive inoculation of increasing doses of lipopolysaccharide (LPS) into mice mimics the immunosuppression associated with sepsis. Myeloid-derived suppressor cells (MDSCs, Gr-1(+) CD11b(+)) are considered a major component of the immunosuppressive network, interfering with T-cell responses in many pathological conditions. We used LPS-immunosuppressed (IS) mice to address whether MDSCs acquired their suppressive ability in the bone marrow (BM) and whether they could migrate to lymph nodes (LNs) to exert their suppressive function. Our results showed that Gr-1(+) CD11b(+) cells of IS mice already had the potential to inhibit T-cell proliferation in the BM. Moreover, soluble factors present in the BM from IS mice were responsible for inducing this inhibitory ability in control BM cells. In addition, migration of Gr-1(+) CD11b(+) to LNs in vivo was maximal when cells obtained from the BM of IS mice were inoculated into an IS context. In this regard, we found chemoattractant activity in cell-free LN extracts (LNEs) from IS mice and an increased expression of the LN-homing chemokine receptor C-C chemokine receptor type 7 (CCR7) in IS BM Gr-1(+) CD11b(+) cells. These results indicate that Gr-1(+) CD11b(+) cells found in BM from IS mice acquire their suppressive activity in the same niche where they are generated, and migrate to LNs to exert their inhibitory role. A better understanding of MDSC generation and/or regulation of factors able to induce their inhibitory function may provide new and more effective tools for the treatment of sepsis-associated immunosuppression.

all-trans-retinoic acid improves immunocompetence in a murine model of lipopolysaccharide-induced immunosuppression.

Secondary infections due to post-sepsis immunosuppression are a major cause of death in patients with sepsis. Strategies aimed at restoring immune functions offer a new perspective in the treatment of sepsis. In the present study, we used LPS (lipopolysaccharide)-immunosuppressed mice to analyse the effects of ATRA (all-trans retinoic acid) on different immune parameters. The IS (immunocompromised) group had decreased lymphocyte and increased MDSC (myeloid-derived suppressor cell) counts in lymph nodes. They also had an impaired in vitro T-cell proliferation, mediated by MDSCs. ATRA administration restored T-cell proliferation, which was associated with a decreased number of live MDSCs. The IS group treated with ATRA had an increased number of CD4+ and CD8+ T-cells. ATRA partially improved the primary humoral immune response, even when immunosuppression was established first and ATRA was administered subsequently. Our results demonstrate that ATRA restores immunocompetence by modulating the number of leucocytes and the survival of MDSCs, and thus represents an additional potential strategy in the treatment of the immunosuppressive state of sepsis.

Interleukin-10 controls human peripheral PMN activation triggered by lipopolysaccharide.

Large amounts of anti-inflammatory mediators, such as interleukin (IL)-10, are produced and found early in the course of sepsis. We explore the role of IL-10 on neutrophil (PMN) activation/function using an in vitro model. Isolated human PMN were pre-incubated with lipopolysaccharide (LPS) and/or IL-10 for 18h. Subsequently, a second LPS exposure was performed and CD11b and CD66b up-regulation, and the reactive oxygen species (ROS) generation were measured 2h later. We found that IL-10 prevented PMN activation and the secretion of TNF-α and IL-8 induced by the first LPS contact. In the absence of IL-10, a second LPS exposure induced additive effects that were prevented by IL-10. Only ROS generation was highly affected by the blockade of PMN-secreted TNF-α or IL-8. Additionally, IL-10 prevented other possible mechanisms of LPS priming. Therefore, IL-10 modulates PMN activation preventing autocrine activating loops and priming mechanisms, rendering PMN less responsive to a second LPS exposure.

Model of polymicrobial peritonitis that induces the proinflammatory and immunosuppressive phases of sepsis.

Severe sepsis is associated with early release of inflammatory mediators that contribute to the morbidity and mortality observed during the first stages of this syndrome. Although sepsis is a deadly, acute disease, high mortality rates have been observed in patients displaying evidence of sepsis-induced immune deactivation. Although the contribution of experimental models to the knowledge of pathophysiological and therapeutic aspects of human sepsis is undeniable, most of the current studies using animal models have focused on the acute, proinflammatory phase. We developed a murine model that reproduces the early acute phases but also the long-term consequences of human sepsis. We induced polymicrobial acute peritonitis (AP) by establishing a surgical connection between the cecum and the peritoneum, allowing the exit of intestinal bacteria. Using this model, we observed an acute phase with high mortality, leukopenia, increased interleukin-6 levels, bacteremia, and neutrophil activation. A peak of leukocytosis on day 9 or 10 revealed the persistence of the infection within the lung and liver, with inflammatory hepatic damage being shown by histological examination. Long-term (20 days) derangements in both innate and adaptive immune responses were found, as demonstrated by impaired systemic tumor necrosis factor alpha production in response to an inflammatory stimulus; a decreased primary humoral immune response and T cell proliferation, associated with an increased number of myeloid suppressor cells (Gr-1(+) CD11b(+)) in the spleen; and a low clearance capacity. This model provides a good approach to attempt novel therapeutic interventions directed to augmenting host immunity during late sepsis.