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

Brucella abortus–infected platelets modulate the activation of neutrophils

Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Platelets (PLTs) have been widely involved in the modulation of the immune response. We have previously reported the modulation of Brucella abortus–mediated infection of monocytes. As a result, PLTs cooperate with monocytes and increase their inflammatory capacity, promoting the resolution of the infection. Extending these results, in this study we demonstrate that patients with brucellosis present slightly elevated levels of complexes between PLTs and both monocytes and neutrophils. We then assessed whether PLTs were capable of modulating functional aspects of neutrophils. The presence of PLTs throughout neutrophil infection increased the production of interleukin‐8, CD11b surface expression and reactive oxygen species formation, whereas it decreased the expression of CD62L, indicating an activated status of these cells. We next analyzed whether this modulation was mediated by released factors. To discriminate between these options, neutrophils were treated with supernatants collected from B. abortus–infected PLTs. Our results show that CD11b expression was induced by soluble factors of PLTs but direct contact between cell populations was needed to enhance the respiratory burst. Additionally, B. abortus–infected PLTs recruit polymorphonuclear (PMN) cells to the site of infection. Finally, the presence of PLTs did not modify the initial invasion of PMN cells by B. abortus but improved the control of the infection at extended times. Altogether, our results demonstrate that PLTs interact with neutrophils and promote a proinflammatory phenotype which could also contribute to the resolution of the infection.Fil: Trotta, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Milillo, María Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Serafino, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Castillo Montañez, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Birnberg Weiss, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Delpino, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Fernández, Cecilia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Barrionuevo, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

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 (H2O2), 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.Fil: Birnberg Weiss, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Castillo, Luis A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pittaluga, José R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Martire Greco, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gómez, Sonia Alejandra. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Landoni, Verónica Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernández, Gabriela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

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.Fil: Landoni, Verónica Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pittaluga Villareal, Jose Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Carestia, Agostina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Castillo Montañez, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de Campos Nebel, Ildefonso Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Martire Greco, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Birnberg Weiss, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Schierloh, Luis Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernández, Gabriela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin

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.Fil: Castillo Montañez, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Birnberg Weiss, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Rodriguez Rodrigues, Nahuel Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pittaluga, José R.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Martire Greco, Daiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Milillo, María Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Grinstein, Sebastián. Ministerio de Defensa. Ejército Argentino. Hospital Militar Central Cirujano Mayor "Dr. Cosme Argerich"; ArgentinaFil: Camelli, María R.. Ministerio de Defensa. Ejército Argentino. Hospital Militar Central Cirujano Mayor "Dr. Cosme Argerich"; ArgentinaFil: Mena Aybar, Ana J.. Ministerio de Defensa. Ejército Argentino. Hospital Militar Central Cirujano Mayor "Dr. Cosme Argerich"; ArgentinaFil: Landoni, Verónica I. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernández, Gabriela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin