Liesbeth M. Kager

Urokinase Receptor Is Necessary for Bacterial Defense against Pneumonia-Derived Septic Melioidosis by Facilitating Phagocytosis

Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR], CD87), a GPI-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in Southeast Asia. In this study, we investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with wild-type mice, corresponding with increased pulmonary and hepatic inflammation. uPAR knockout mice demonstrated significantly reduced neutrophil migration toward the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR-deficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils toward the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei.

Plasminogen activator inhibitor type I contributes to protective immunity during experimental Gram-negative sepsis (melioidosis)

Summary.  Background: Melioidosis is a frequent cause of sepsis in Southeast Asia caused by the Gram‐negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of plasminogen activator inhibitor type 1 (PAI‐1), an important regulator of inflammation and fibrinolysis. Objectives: In this study, we aimed to investigate the role of PAI‐1 during melioidosis. Methods: Wild‐type (WT) and PAI‐1‐deficient (PAI‐1–/1−/−) mice were intranasally infected with B. pseudomallei. Mice were killed after 24, 48 or 72 h. Lungs, liver and blood were harvested for measurement of bacterial loads, cytokines, clinical chemistry, histopathology, and coagulation parameters. Additionally, survival studies were performed. Results: PAI‐1−/− mice demonstrated enhanced susceptibility to B. pseudomallei infection, as shown by a strongly increased mortality rate (100% vs. 58% among WT mice, P < 0.001), associated with enhanced bacterial loads in lungs, liver, and blood. Additionally, PAI‐1−/− mice showed elevated levels of proinflammatory cytokines in lungs and plasma, accompanied by enhanced local and systemic coagulation activation (thrombin–antithrombin complexes and D‐dimer), increased hepatocellular injury (plasma aspartate aminotransferase and alanine aminotransferase), and renal failure (plasma creatinine and urea). Conclusions: PAI‐1 has a protective role during severe Gram‐negative sepsis caused by B. pseudomallei by limiting bacterial growth, inflammation, and coagulation, and probably, as a consequence thereof, distant organ injury.

Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis

Background Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis. Methods Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×102 cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM). Results Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion. Conclusions Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.

Endogenous protein C has a protective role during Gram-negative pneumosepsis (melioidosis)

Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality.

Endogenous α2-antiplasmin is protective during severe gram-negative sepsis (melioidosis).

RATIONALE α2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit plasmin. Although the fibrinolytic system is strongly affected by infection, the functional role of A2AP in the host response to sepsis is unknown. OBJECTIVES To study the role of A2AP in melioidosis, a common form of community-acquired sepsis in Southeast Asia and Northern Australia caused by the gram-negative bacterium Burkholderia pseudomallei. METHODS In a single-center observational study A2AP was measured in patients with culture-proven septic melioidosis. Wild-type and A2AP-deficient (A2AP(-/-)) mice were intranasally infected with B. pseudomallei to induce severe pneumosepsis (melioidosis). Parameters of inflammation and coagulation were measured, and survival studies were performed. MEASUREMENTS AND MAIN RESULTS Patients with melioidosis showed elevated A2AP plasma levels. Likewise, A2AP levels in plasma and lung homogenates were elevated in mice infected with B. pseudomallei. A2AP-deficient (A2AP(-/-)) mice had a strongly disturbed host response during experimental melioidosis as reflected by enhanced bacterial growth at the primary site of infection accompanied by increased dissemination to distant organs. In addition, A2AP(-/-) mice showed more severe lung pathology and injury together with an increased accumulation of neutrophils and higher cytokine levels in lung tissue. A2AP deficiency further was associated with exaggerated systemic inflammation and coagulation, increased distant organ injury, and enhanced lethality. CONCLUSIONS This study is the first to identify A2AP as a protective mediator during gram-negative (pneumo)sepsis by limiting bacterial growth, inflammation, tissue injury, and coagulation.

Pulmonary tuberculosis induces a systemic hypercoagulable state

OBJECTIVES Human tuberculosis (TB) remains an important cause of death globally. Bangladesh is one of the most affected countries. We aimed to investigate the impact of pulmonary TB on pro- and anticoagulant mechanisms. METHODS This prospective study was conducted in Chittagong, Bangladesh. We performed an in-depth analysis of coagulation activation and inhibition in plasma obtained from 64 patients with primary lung TB and 11 patients with recurrent lung TB and compared these with 37 healthy controls. Additionally, in nine patients coagulation activation was studied in bronchoalveolar lavage fluid (BALF) harvested from the site of infection and compared with BALF from a contralateral unaffected lung subsegment. RESULTS Relative to uninfected controls, primary and recurrent TB were associated with a systemic net procoagulant state, as indicated by enhanced activation of coagulation (elevated plasma levels of thrombin-antithrombin complexes, D-dimer and fibrinogen) together with impaired anticoagulant mechanisms (reduced plasma levels of antithrombin, protein C activity, free protein S, and protein C inhibitor). Activation of coagulation did not correlate with plasma concentrations of established TB biomarkers. Coagulation activation could not be detected at the primary site of infection in a subset of TB patients. CONCLUSIONS Pulmonary TB is associated with a systemic hypercoagulable state.

Endogenous tissue-type plasminogen activator impairs host defense during severe experimental Gram-negative sepsis (melioidosis)*.

Objective:Melioidosis is a frequent cause of severe sepsis in Southeast Asia caused by the gram-negative bacterium Burkholderia pseudomallei. Patients with melioidosis have elevated circulating levels of tissue-type plasminogen activator, an important regulator of fibrinolysis. In this study, we aimed to investigate the role of tissue-type plasminogen activator during melioidosis. Design:Animal study. Setting:University research laboratory. Subjects:Wild-type and tissue-type plasminogen activator–deficient C57BL/6 mice. Interventions:Mice were intranasally infected with viable Burkholderia pseudomallei and killed after 24, 48, or 72 hrs for harvesting of lungs, liver, and blood. Additionally, survival studies were performed. Measurements and Main Results:Experimentally induced melioidosis was associated with elevated levels of tissue-type plasminogen activator in lungs of infected wild-type mice. During infection with Burkholderia pseudomallei, tissue-type plasminogen activator–deficient mice were protected when compared to wild-type mice as demonstrated by a strongly decreased mortality (62% vs. 100% amongst wild-type mice, p < .0001), together with decreased pulmonary bacterial loads, less severe histopathological scores, and decreased fibrinolysis. These results were accompanied with an early increase in cytokine levels in tissue-type plasminogen activator–deficient mice. Conclusions:During severe gram-negative sepsis caused by Burkholderia pseudomallei, endogenous tissue-type plasminogen activator has harmful effects with respect to survival and pulmonary bacterial growth. These effects are related to tissue-type plasminogen activator–associated plasmin-induced fibrinolysis and/or a tissue-type plasminogen activator–associated decrease in proinflammatory cytokine production.

Differential Toll-Like Receptor-Signalling of Burkholderia pseudomallei Lipopolysaccharide in Murine and Human Models.

The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia. Methodology/Principal Findings Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma. Conclusion/Significance Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.

Overexpression of activated protein C is detrimental during severe experimental gram-negative sepsis (melioidosis)

Objective:The interplay between inflammation and blood coagulation is an essential part of host defense during severe pneumosepsis. Melioidosis, instigated by the Gram-negative bacterium Burkholderia pseudomallei, is a frequent cause of pneumosepsis in Southeast Asia. Patients with severe pneumosepsis, including melioidosis, have decreased circulating levels of protein C. Activated protein C has anticoagulant and anti-inflammatory properties. In this study, we aimed to investigate the effect of sustained elevated activated protein C levels on the host response during melioidosis. Design:Animal study. Setting:University research laboratory. Subjects:Wild type and activated protein C overexpressing C57BL/6 mice. Interventions:Mice were intranasally infected with viable B. pseudomallei and killed after 24, 48, or 72 hours for harvesting of lungs, liver, spleen, and blood. Additionally, survival studies were performed. Measurements and Main Results:Plasma activated protein C concentrations in activated protein C overexpressing mice (median 18.1 ng/mL) were in the same range as previously measured in patients treated with recombinant human activated protein C. Activated protein C overexpressing mice demonstrated enhanced susceptibility to B. pseudomallei infection compared with wild type mice as evidenced by a strongly increased mortality accompanied by enhanced bacterial loads in the lungs, blood, and distant organs 48 hours after infection. Additionally, at this time point, activated protein C overexpressing mice showed elevated levels of proinflammatory cytokines in lungs and plasma, together with increased pulmonary histopathology scores and neutrophil influx. At 72 hours postinfection, decreased levels of thrombin-antithrombin complexes, reflecting inhibition of coagulation, were measured in lungs of activated protein C overexpressing mice. Conclusion:Constitutively enhanced expression of activated protein C impairs host defense during severe Gram-negative sepsis caused by B. pseudomallei.