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.     

Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei

Background

Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling.

Methods/Principal Findings

An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and ‘low-tech’ methodology that is applicable in both developed and developing countries.

Conclusions/Significance

The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.     

T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients

Background

Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.

Methods

We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43).

Results

We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl.Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA_1c 10.2%, interquartile range 9.2–13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.

Conclusions

The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.     

Expression and Function of Macrophage Migration Inhibitory Factor (MIF) in Melioidosis

Background

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast-Asia. We aimed to characterize the expression and function of MIF in melioidosis.

Methodology and Principal Findings

MIF expression was determined in leukocytes and plasma from 34 melioidosis patients and 32 controls, and in mice infected with B. pseudomallei. MIF function was investigated in experimental murine melioidosis using anti-MIF antibodies and recombinant MIF. Patients demonstrated markedly increased MIF mRNA leukocyte and MIF plasma concentrations. Elevated MIF concentrations were associated with mortality. Mice inoculated intranasally with B. pseudomallei displayed a robust increase in pulmonary and systemic MIF expression. Anti-MIF treated mice showed lower bacterial loads in their lungs upon infection with a low inoculum. Conversely, mice treated with recombinant MIF displayed a modestly impaired clearance of B. pseudomallei. MIF exerted no direct effects on bacterial outgrowth or phagocytosis of B. pseudomallei.

Conclusions

MIF concentrations are markedly elevated during clinical melioidosis and correlate with patients'' outcomes. In experimental melioidosis MIF impaired antibacterial defense.     

MyD88 dependent signaling contributes to protective host defense against Burkholderia pseudomallei

Background

Toll-like receptors (TLRs) have a central role in the recognition of pathogens and the initiation of the innate immune response. Myeloid differentiation primary-response gene 88 (MyD88) and TIR-domain-containing adaptor protein inducing IFNβ (TRIF) are regarded as the key signaling adaptor proteins for TLRs. Melioidosis, which is endemic in SE-Asia, is a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei. We here aimed to characterize the role of MyD88 and TRIF in host defense against melioidosis.

Methodology and Principal Findings

First, we found that MyD88, but not TRIF, deficient whole blood leukocytes released less TNFα upon stimulation with B. pseudomallei compared to wild-type (WT) cells. Thereafter we inoculated MyD88 knock-out (KO), TRIF mutant and WT mice intranasally with B. pseudomallei and found that MyD88 KO, but not TRIF mutant mice demonstrated a strongly accelerated lethality, which was accompanied by significantly increased bacterial loads in lungs, liver and blood, and grossly enhanced liver damage compared to WT mice. The decreased bacterial clearance capacity of MyD88 KO mice was accompanied by a markedly reduced early pulmonary neutrophil recruitment and a diminished activation of neutrophils after infection with B. pseudomallei. MyD88 KO leukocytes displayed an unaltered capacity to phagocytose and kill B. pseudomallei in vitro.

Conclusions

MyD88 dependent signaling, but not TRIF dependent signaling, contributes to a protective host response against B. pseudomallei at least in part by causing early neutrophil recruitment towards the primary site of infection.     

Induction of Mouse Melioidosis with Meningitis by CD11b+ Phagocytic Cells Harboring Intracellular B. pseudomallei as a Trojan Horse

Background

Approximately 3–5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited.

Methods and Findings

We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b⁺ populations. The CD11b⁺Ly6C^high inflamed monocytes, CD11b⁺Ly6C^low resident monocytes, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages and CD11b⁺CD19⁺ B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b⁺ cells induced bacterial colonization in the brain, whereas CD11b⁻ cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b⁺ cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b⁺ cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b⁺ CD62L-negative cells did not.

Conclusions/Significance

We suggest that B. pseudomallei-infected CD11b⁺ selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.     

Ethanol Inhibits Activation of NLRP3 and AIM2 Inflammasomes in Human Macrophages–A Novel Anti-Inflammatory Action of Alcohol

Objective

In the pathogenesis of coronary atherosclerosis, local macrophage-driven inflammation and secretion of proinflammatory cytokines, interleukin-1β (IL-1β) in particular, are recognized as key factors. Moderate alcohol consumption is associated with a reduced risk of coronary artery disease mortality. Here we examined in cultured human macrophages whether ethanol modulates the intracellular processes involved in the secretion of IL-1β.

Results

Ethanol decreased dose-dependently the production of mature IL-1β induced by activators of the NLRP3 inflammasome, i.e. ATP, cholesterol crystals, serum amyloid A and nigericin. Ethanol had no significant effect on the expression of NLRP3 or IL1B mRNA in LPS-primed macrophages. Moreover, secretion of IL-1β was decreased in parallel with reduction of caspase-1 activation, demonstrating that ethanol inhibits inflammasome activation instead of synthesis of pro-IL-1β. Acetaldehyde, a highly reactive metabolite of ethanol, had no effect on the ATP-induced IL-1β secretion. Ethanol also attenuated the secretion of IL-1β triggered by synthetic double-stranded DNA, an activator of the AIM2 inflammasome. Ethanol conferred the inhibitory functions by attenuating the disruption of lysosomal integrity and ensuing leakage of the lysosomal protease cathepsin B and by reducing oligomerization of ASC.

Conclusion

Ethanol-induced inhibition of the NLRP3 inflammasome activation in macrophages may represent a biological pathway underlying the protective effect of moderate alcohol consumption on coronary heart disease.     

Persistent Inflammation and Endothelial Activation in HIV-1 Infected Patients after 12 Years of Antiretroviral Therapy

Objective

The study investigated markers of inflammation and endothelial activation in HIV infected patients after 12 years of successful combination antiretroviral treatment (cART).

Methods

Inflammation and endothelial activation were assessed by measuring levels of immunoglobulins, β2-microglobulin, interleukin (IL) 8, tumor necrosis factor α (TNFα), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), sE-Selectin, and sP-Selectin.

Results

HIV infected patients had higher levels of β2-microglobulin, IL-8, TNFα, and sICAM-1 than uninfected controls, and HIV infected patients lacked correlation between platelet counts and sP-Selectin levels found in uninfected controls.

Conclusion

Discrete signs of systemic and vascular inflammation persist even after very long term cART.     

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils

Background

PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results

We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd^−/− mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions

These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.     

Burkholderia pseudomallei Is Genetically Diverse in Agricultural Land in Northeast Thailand

Background

The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined.

Methods

Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10×10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).

Principal Findings

B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations.

Conclusions

Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.     

Osteopontin Impairs Host Defense during Established Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Background

Melioidosis, caused by infection with Burkholderia (B.) pseudomallei, is a severe illness that is endemic in Southeast Asia. Osteopontin (OPN) is a phosphorylated glycoprotein that is involved in several immune responses including induction of T-helper 1 cytokines and recruitment of inflammatory cells.

Methodology and Principal Findings

OPN levels were determined in plasma from 33 melioidosis patients and 31 healthy controls, and in wild-type (WT) mice intranasally infected with B. pseudomallei. OPN function was studied in experimental murine melioidosis using WT and OPN knockout (KO) mice. Plasma OPN levels were elevated in patients with severe melioidosis, even more so in patients who went on to die. In patients who recovered plasma OPN concentrations had decreased after treatment. In experimental melioidosis in mice plasma and pulmonary OPN levels were also increased. Whereas WT and OPN KO mice were indistinguishable during the first 24 hours after infection, after 72 hours OPN KO mice demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, especially due to less necrosis, and decreased neutrophil infiltration. Moreover, OPN KO mice displayed a delayed mortality as compared to WT mice. OPN deficiency did not influence the induction of proinflammatory cytokines.

Conclusions

These data suggest that sustained production of OPN impairs host defense during established septic melioidosis.     

Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells attenuates Escherichia coli-induced acute lung injury in mice

Background

Human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) attenuate hyperoxic neonatal lung injury primarily through anti-inflammatory effects. We hypothesized that intratracheal transplantation of human UCB-derived MSCs could attenuate Escherichia coli (E. coli)-induced acute lung injury (ALI) in mice by suppressing the inflammatory response.

Methods

Eight-week-old male ICR mice were randomized to control or ALI groups. ALI was induced by intratracheal E. coli instillation. Three-hours after E. coli instillation, MSCs, fibroblasts or phosphate-buffered saline were intratracheally administered randomly and survival was analyzed for 7 days post-injury. Lung histology including injury scores, myeloperoxidase (MPO) activity, and protein levels of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 as well as the wet-dry lung ratio and bacterial counts from blood and bronchoalveolar lavage (BAL) were evaluated at 1, 3, and 7 days post-injury. Levels of inflammatory cytokines in the lung were also profiled using protein macroarrays at day 3 post-injury which showed peak inflammation.

Results

MSC transplantation increased survival and attenuated lung injuries in ALI mice, as evidenced by decreased injury scores on day 3 post-injury and reduced lung inflammation including increased MPO activity and protein levels of IL-1α, IL-1β, IL-6, TNF-α, and MIP-2 on day 3 and 7 post-injury. Inflammatory cytokine profiles in the lungs at day 3 post-injury were attenuated by MSC transplantation. MSCs also reduced the elevated lung water content at day 3 post-injury and bacterial counts in blood and BAL on day 7 post-injury.

Conclusions

Intratracheal transplantation of UCB-derived MSCs attenuates E. coli-induced ALI primarily by down-modulating the inflammatory process and enhancing bacterial clearance.     

Anti Diabetic effect of CL 316,243 (A β3-Adrenergic Agonist) by Down Regulation of Tumour Necrosis Factor (TNF-α) Expression

Objective

Obesity is a risk factor for the development of insulin resistance and is one of the most important contributors to the pathogenesis of type2 diabetes, which acts mainly through the secretion of adipokines such as TNF-α that may influence insulin sensitivity. TNF-α affects many aspects of adipocyte function, such as adipocyte development and lipid metabolism.

Material and Methods

We demonstrated that there is a correlation between the expressions of TNF-α in retroperitoneal WAT and insulin-resistance in 8 genetically obese fa/fa rats. Treatment of animals with CL 316,243, a β3-adrenergic agonist, showed an improvement of insulin-resistance that was linked with the suppression of TNF-α mRNA expression in WAT.

Results

These results confirm the association between TNF-α expression and the insulin-resistant condition in rats. Our finding indicates that the hyperglycaemia and hyperinsulinemia induced by insulin-resistance correlated positively with the expression of TNF-α mRNA in an abdominal WAT depot.

Conclusion

We conclude that CL 316,243 possesses both anti-diabetic effects and anti-obesity effects in rodents.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea

Background

The island of New Guinea is located midway between the world''s two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales.

Methodology/Principal Findings

We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability.

Conclusions/Significance

Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.