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

Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. B. pseudomallei is the major cause of community-acquired septicemia in northeast Thailand with a mortality rate in severe cases of around 40% Little is known, however, about the mechanisms of the host defense to B. pseudomallei infection. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has emerged as an important mediator of the host defense in severe bacterial infections. In this article, we studied the expression and function of MIF both in patients with melioidosis and in mice during experimental melioidosis. We found that MIF concentrations were elevated in patients with melioidosis. Furthermore, high MIF concentrations are associated with poor outcome in patients with melioidosis. Also, in mice with experimentally induced melioidosis, we observed an upregulation of MIF concentrations. Furthermore, mice with melioidosis that were treated with a MIF blocking treatment showed lower bacterial counts in their lungs during infection. In conclusion, MIF seems to impair host defense mechanisms during melioidosis

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

Melioidosis is a severe tropical disease caused by infection with the bacterium Burkholderia (B.) pseudomallei. In northeast Thailand infection with this bacterium is the major cause of community-acquired septicemia with a mortality rate up to 40%. Extending the knowledge on the mechanisms of host defense against B. pseudomallei infection would be helpful to improve treatment of this severe illness. Osteopontin (OPN) is a cytokine that is involved in several immune responses that occur during bacterial infection. In this study, we investigated levels of OPN in patients with melioidosis, and studied the function of OPN during experimental melioidosis in mice. We found that OPN concentrations were elevated in patients with severe melioidosis, and that high OPN concentrations are associated with poor outcome in patients with melioidosis. In experimental melioidosis in mice plasma and lung OPN levels were also increased. Moreover, mice with melioidosis that were deficient for OPN demonstrated reduced bacterial numbers in their lungs, diminished pulmonary tissue injury, and decreased neutrophil infiltration into the lungs during established melioidosis. Moreover, these mice displayed a delayed mortality as compared to control mice. In conclusion, sustained production of OPN impairs host defense during melioidosis

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 IFNb (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 TNFa upon stimulation with B. pseudomallei compared to wild-type (WT) cells. Thereafter we inoculated MyD88 knockout (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 respons

PART I: MINIMUM QUALITY THRESHOLD IN PRECLINICAL SEPSIS STUDIES (MQTiPSS) FOR STUDY DESIGN AND HUMANE MODELING ENDPOINTS

The Austrian Science Fund T707-B13 (SD, MFO)The William Harvey Research Foundation (CT)NWO VIDI (no.: 91716475)MC-ITN ‘‘European Sepsis Academy’’ (WJW)National Institutes of Health R01 GM067202 and GM115973 (BZ)The National Institute of General Medical Sciences GM072808, GM104323, GM109779, and GM113228 (CMC)

Incidence, risk factors and clinical epidemiology of melioidosis: a complex socio-ecological emerging infectious disease in the Alor Setar region of Kedah, Malaysia

<p>Abstract</p> <p>Background</p> <p>Melioidosis, a severe and fatal infectious disease caused by <it>Burkholderia pseudomallei</it>, is believed to an emerging global threat. However, data on the natural history, risk factors, and geographic epidemiology of the disease are still limited.</p> <p>Methods</p> <p>We undertook a retrospective analysis of 145 confirmed cases extracted from a hospital-based Melioidosis Registry set up from 2005 in Hospital Sultanah Bahiyah, Alor Setar, Kedah state, Malaysia, in order to provide a first description of the contemporary incidence, risk factors, and clinical epidemiology of the disease in this putatively high risk region of the country.</p> <p>Results</p> <p>The incidence of melioidosis in Alor Setar is remarkably high at 16.35 per 100,000 population per year. The mean age of patients was 50.40 years, with infection varying nonlinearly with age. Males (75.2%; <it>P </it>< 0.0001) predominated and the majority of cases were Malays (88.9%). The overall, crude mortality rate among the study patients was 33.8%. The proportions of cases and deaths were significantly greater among patients involved in farming, forestry and fishing and the unemployed (χ²= 30.57, <it>P </it>< 0.0001). A majority of cases (62.75%) were culture positive, with mortality in these patients being 45.05%. A large proportion (83.0%) of culture positives was also bacteremic. Pneumonia accounted for 42.06% of primary diagnoses followed in importance by soft tissue abscess. In patients with pneumonia and who were culture positive, the mortality rate was as high as 65.00%. Diabetes mellitus constituted the major underlying risk factor for developing and dying from melioidosis, occurring in 57% of all diagnosed cases. The age distribution of diabetes paralleled that of melioidosis cases. There were linear associations between cases and deaths with monthly rainfall.</p> <p>Conclusions</p> <p>Melioidosis represents a complex socio-ecological public health problem in Kedah, being strongly related with age, occupation, rainfall and predisposing chronic diseases, such as diabetes mellitus. Among cases, bacteremic patients were associated with significantly high mortality despite provision of the recommended antibacterial therapy. The burden of this disease is likely to grow in this region unless better informed interventions targeted at high-risk groups and associated diseases are urgently implemented.</p

Bayesian modeling of recombination events in bacterial populations

Background: We consider the discovery of recombinant segments jointly with their origins within multilocus DNA sequences from bacteria representing heterogeneous populations of fairly closely related species. The currently available methods for recombination detection capable of probabilistic characterization of uncertainty have a limited applicability in practice as the number of strains in a data set increases. Results: We introduce a Bayesian spatial structural model representing the continuum of origins over sites within the observed sequences, including a probabilistic characterization of uncertainty related to the origin of any particular site. To enable a statistically accurate and practically feasible approach to the analysis of large-scale data sets representing a single genus, we have developed a novel software tool (BRAT, Bayesian Recombination Tracker) implementing the model and the corresponding learning algorithm, which is capable of identifying the posterior optimal structure and to estimate the marginal posterior probabilities of putative origins over the sites. Conclusion: A multitude of challenging simulation scenarios and an analysis of real data from seven housekeeping genes of 120 strains of genus Burkholderia are used to illustrate the possibilities offered by our approach. The software is freely available for download at URL http://web.abo.fi/fak/ mnf//mate/jc/software/brat.html

Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016.

OBJECTIVE: To provide an update to "Surviving Sepsis Campaign Guidelines for Management of Sepsis and Septic Shock: 2012." DESIGN: A consensus committee of 55 international experts representing 25 international organizations was convened. Nominal groups were assembled at key international meetings (for those committee members attending the conference). A formal conflict-of-interest (COI) policy was developed at the onset of the process and enforced throughout. A stand-alone meeting was held for all panel members in December 2015. Teleconferences and electronic-based discussion among subgroups and among the entire committee served as an integral part of the development. METHODS: The panel consisted of five sections: hemodynamics, infection, adjunctive therapies, metabolic, and ventilation. Population, intervention, comparison, and outcomes (PICO) questions were reviewed and updated as needed, and evidence profiles were generated. Each subgroup generated a list of questions, searched for best available evidence, and then followed the principles of the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system to assess the quality of evidence from high to very low, and to formulate recommendations as strong or weak, or best practice statement when applicable. RESULTS: The Surviving Sepsis Guideline panel provided 93 statements on early management and resuscitation of patients with sepsis or septic shock. Overall, 32 were strong recommendations, 39 were weak recommendations, and 18 were best-practice statements. No recommendation was provided for four questions. CONCLUSIONS: Substantial agreement exists among a large cohort of international experts regarding many strong recommendations for the best care of patients with sepsis. Although a significant number of aspects of care have relatively weak support, evidence-based recommendations regarding the acute management of sepsis and septic shock are the foundation of improved outcomes for these critically ill patients with high mortality

Inflammasome-dependent Pyroptosis and IL-18 Protect against Burkholderia pseudomallei Lung Infection while IL-1β Is Deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β

Immunospecific Responses to Bacterial Elongation Factor Tu during Burkholderia Infection and Immunization

Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection

The Burkholderia pseudomallei Type III Secretion System and BopA Are Required for Evasion of LC3-Associated Phagocytosis

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes