Activation of PPARγ by endogenous prostaglandin J2 mediates the antileukemic effect of selenium in murine leukemia.

Supplementation with nontoxic doses of micronutrient selenium has been shown to alleviate chronic myelogenous leukemia (CML) via the elimination of leukemia stem cells (LSCs) in mice. This treatment provides a new and novel method for eliminating the LSCs that are otherwise not targeted by existing therapies. The antileukemic effect of selenium was dependent on the production of endogenous cyclopentenone prostaglandins (CyPGs), Δ-12 prostaglandin J2 (Δ12-PGJ2), and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). Here, we show that these endogenous CyPGs, produced by mice maintained on selenium-supplemented diets, alleviate the symptoms of CML through their ability to activate the nuclear hormone receptor, peroxisome proliferator activated receptor γ (PPARγ). GW9662, a potent PPARγ antagonist, blocked the antileukemic effect of selenium supplementation by significantly reducing CyPGs. This effect was mediated by an increase in 15-prostaglandin dehydrogenase (15-Pgdh) activity, which oxidizes and inactivates Δ12-PGJ2 and 15d-PGJ2 In contrast, treatment with the PPARγ agonist pioglitazone mimicked selenium supplementation. This treatment led to decreased 15-Pgdh activity and increased CyPG levels, which inhibited CML progression. Selenium-dependent activation of PPARγ mediated by endogenous CyPGs decreased Stat5 expression leading to the downregulation of Cited2, a master regulator of LSC quiescence. These studies suggest a potential role for selenium supplementation as an adjuvant therapy in CML.

Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species.

BACKGROUND: The genus Bordetella consists of nine species that include important respiratory pathogens such as the 'classical' species B. bronchiseptica, B. pertussis and B. parapertussis and six more distantly related and less extensively studied species. Here we analyze sequence diversity and gene content of 128 genome sequences from all nine species with focus on the evolution of virulence-associated factors. RESULTS: Both genome-wide sequence-based and gene content-based phylogenetic trees divide the genus into three species clades. The phylogenies are congruent between species suggesting genus-wide co-evolution of sequence diversity and gene content, but less correlated within species, mainly because of strain-specific presence of many different prophages. We compared the genomes with focus on virulence-associated genes and identified multiple clade-specific, species-specific and strain-specific events of gene acquisition and gene loss, including genes encoding O-antigens, protein secretion systems and bacterial toxins. Gene loss was more frequent than gene gain throughout the evolution, and loss of hundreds of genes was associated with the origin of several species, including the recently evolved human-restricted B. pertussis and B. holmesii, B. parapertussis and the avian pathogen B. avium. CONCLUSIONS: Acquisition and loss of multiple genes drive the evolution and speciation in the genus Bordetella, including large scale gene loss associated with the origin of several species. Recent loss and functional inactivation of genes, including those encoding pertussis vaccine components and bacterial toxins, in individual strains emphasize ongoing evolution.

Horizontally acquired divergent O-antigen contributes to escape from cross-immunity in the classical bordetellae.

BACKGROUND: Horizontal gene transfer (HGT) allows for rapid spread of genetic material between species, increasing genetic and phenotypic diversity. Although HGT contributes to adaptation and is widespread in many bacteria, others show little HGT. This study builds on previous work to analyze the evolutionary mechanisms contributing to variation within the locus encoding a prominent antigen of the classical bordetellae. RESULTS: We observed amongst classical bordetellae discrete regions of the lipopolysaccharide O-antigen locus with higher sequence diversity than the genome average. Regions of this locus had less than 50% sequence similarity, low dN/dS ratios and lower GC content compared to the genome average. Additionally, phylogenetic tree topologies based on genome-wide SNPs were incongruent with those based on genes within these variable regions, suggesting portions of the O-antigen locus may have been horizontally transferred. Furthermore, several predicted recombination breakpoints correspond with the ends of these variable regions. To examine the evolutionary forces that might have selected for this rare example of HGT in bordetellae, we compared in vitro and in vivo phenotypes associated with different O-antigen types. Antibodies against O1- and O2-serotypes were poorly cross-reactive, and did not efficiently kill or mediate clearance of alternative O-type bacteria, while a distinct and poorly immunogenic O-antigen offered no protection against colonization. CONCLUSIONS: This study suggests that O-antigen variation was introduced to the classical bordetellae via HGT through recombination. Additionally, genetic variation may be maintained within the O-antigen locus because it can provide escape from immunity to different O-antigen types, potentially allowing for the circulation of different Bordetella strains within the same host population.

Decreased leukocyte accumulation and delayed Bordetella pertussis clearance in IL-6-/- mice.

IL-6, a pleiotropic cytokine primarily produced by the innate immune system, has been implicated in the development of acquired immune responses, though its roles are largely undefined and may vary in the context of different diseases. Using a murine model of infection, we established that IL-6 influences the adaptive immune responses against the endemic human respiratory pathogen Bordetella pertussis. IL-6 was induced in the lungs of C57BL/6 mice by B. pertussis. IL-6(-/-) mice showed a protracted infectious course and were less efficiently protected by B. pertussis vaccination than wild-type mice. Abs from IL-6(-/-) mice, though lower in titer, efficiently reduced B. pertussis numbers in IL-6-sufficient mice. Pulmonary leukocyte recruitment and splenic or pulmonary T cell cytokine responses to B. pertussis, including Th1 and Th17 cytokine production, were lower in IL-6(-/-) mice than in wild-type mice. Adoptive transfer of immune wild-type CD4(+) cells ameliorated the defect of IL-6(-/-) mice in the control of B. pertussis numbers. Together, these results reveal the dysregulation of multiple aspects of adaptive immune responses in B. pertussis-infected IL-6(-/-) mice and suggest that IL-6 is involved in regulating Ab generation, pulmonary leukocyte accumulation, and T cell cytokine production in response to B. pertussis as well as the generation of effective vaccine-induced immunity against this pathogen.

Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells.

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ12-PGJ2, and 15d-PGJ2, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44-/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.

Denver and Marshall scores successfully predict susceptibility to multiple independent infections in trauma patients.

Trauma patients are at risk of repeated hospital-acquired infections, however predictive scores aiming to identify susceptibility to such infections are lacking. The objective of this study was to investigate whether commonly employed disease-severity scores can successfully predict susceptibility to multiple independent infectious episodes (MIIEs) among trauma patients. A secondary analysis of data derived from the prospective, longitudinal study "Inflammation and the Host Response to Injury" ("Glue Grant") was performed. 1,665 trauma patients, older than 16, were included. Patients who died within seven days from the time of injury were excluded. Five commonly used disease-severity scores [Denver, Marshall, Acute Physiology and Chronic Health Evaluation II (APACHE II), Injury Severity Score (ISS), and New Injury Severity Score (NISS)] were examined as independent predictors of susceptibility to MIIEs. The latter was defined as two or more independent infectious episodes during the index hospital stay. Multivariable logistic regression was used for the statistical analysis. 22.58% of the population was found to be susceptible to MIIEs. Denver and Marshall scores were highly predictive of the MIIE status. For every 1-unit increase in the Denver or the Marshall score, there was a respective 15% (Odds Ratio:1.15; 95% CI: 1.07-1.24; p < 0.001) or 16% (Odds Ratio:1.16; 95% CI: 1.09-1.24; p < 0.001) increase in the odds of MIIE occurrence. APACHE II, ISS, and NISS were not independent predictors of susceptibility to MIIEs. In conclusion, the Denver and Marshall scores can reliably predict which trauma patients are prone to MIIEs, prior to any clinical sign of infection. Early identification of these individuals would potentially allow the implementation of rapid, personalized, preventative measures, thus improving patient outcomes and reducing healthcare costs.

Multi-Biomarker Prediction Models for Multiple Infection Episodes Following Blunt Trauma.

Severe trauma predisposes patients to multiple independent infection episodes (MIIEs), leading to augmented morbidity and mortality. We developed a method to identify increased MIIE risk before clinical signs appear, which is fundamentally different from existing approaches entailing infections' detection after their establishment. Applying machine learning algorithms to genome-wide transcriptome data from 128 adult blunt trauma patients' (42 MIIE cases and 85 non-cases) leukocytes collected ≤48 hr of injury and ≥3 days before any infection, we constructed a 15-transcript and a 26-transcript multi-biomarker panel model with the least absolute shrinkage and selection operator (LASSO) and Elastic Net, respectively, which accurately predicted MIIE (Area Under Receiver Operating Characteristics Curve [AUROC] [95% confidence intervals, CI]: 0.90 [0.84-0.96] and 0.92 [0.86-0.96]) and significantly outperformed clinical models. Gene Ontology and network analyses found various pathways to be relevant. External validation found our model to be generalizable. Our unique precision medicine approach can be applied to a wide range of patient populations and outcomes.

Growth in Egg Yolk Enhances Salmonella Enteritidis Colonization and Virulence in a Mouse Model of Human Colitis.

Salmonella Enteritidis (SE) is one of the most common causes of bacterial food-borne illnesses in the world. Despite the SE's ability to colonize and infect a wide-range of host, the most common source of infection continues to be the consumption of contaminated shell eggs and egg-based products. To date, the role of the source of SE infection has not been studied as it relates to SE pathogenesis and resulting disease. Using a streptomycin-treated mouse model of human colitis, this study examined the virulence of SE grown in egg yolk and Luria Bertani (LB) broth, and mouse feces collected from mice experimentally infected with SEE1 (SEE1 passed through mice). Primary observations revealed that the mice infected with SE grown in egg yolk displayed greater illness and disease markers than those infected with SE passed through mice or grown in LB broth. Furthermore, the SE grown in egg yolk achieved higher rates of colonization in the mouse intestines and extra-intestinal organs of infected mice than the SE from LB broth or mouse feces. Our results here indicate that the source of SE infection may contribute to the overall pathogenesis of SE in a second host. These results also suggest that reservoir-pathogen dynamics may be critical for SE's ability to establish colonization and priming for virulence potential.

Type Six Secretion System of Bordetella bronchiseptica and Adaptive Immune Components Limit Intracellular Survival During Infection.

The Type Six Secretion System (T6SS) is required for Bordetella bronchiseptica cytotoxicity, cytokine modulation, infection, and persistence. However, one-third of recently sequenced Bordetella bronchiseptica strains of the predominantly human-associated Complex IV have lost their T6SS through gene deletion or degradation. Since most human B. bronchiseptica infections occur in immunocompromised patients, we determine here whether loss of Type Six Secretion is beneficial to B. bronchiseptica during infection of immunocompromised mice. Infection of mice lacking adaptive immunity (Rag1-/- mice) with a T6SS-deficient mutant results in a hypervirulent phenotype that is characterized by high numbers of intracellular bacteria in systemic organs. In contrast, wild-type B. bronchiseptica kill their eukaryotic cellular hosts via a T6SS-dependent mechanism that prevents survival in systemic organs. High numbers of intracellular bacteria recovered from immunodeficient mice but only low numbers from wild-type mice demonstrates that B. bronchiseptica survival in an intracellular niche is limited by B and T cell responses. Understanding the nature of intracellular survival during infection, and its effects on the generation and function of the host immune response, are important to contain and control the spread of Bordetella-caused disease.

Chemopreventive Effects of Dietary Eicosapentaenoic Acid Supplementation in Experimental Myeloid Leukemia.

Current therapies for treatment of myeloid leukemia do not eliminate leukemia stem cells (LSC), leading to disease relapse. In this study, we supplemented mice with eicosapentaenoic acid (EPA, C20:5), a polyunsaturated omega-3 fatty acid, at pharmacologic levels, to examine whether the endogenous metabolite, cyclopentenone prostaglandin delta-12 PGJ3 (Δ(12)-PGJ3), was effective in targeting LSCs in experimental leukemia. EPA supplementation for 8 weeks resulted in enhanced endogenous production of Δ(12)-PGJ3 that was blocked by indomethacin, a cyclooxygenase (COX) inhibitor. Using a murine model of chronic myelogenous leukemia (CML) induced by bone marrow transplantation of BCR-ABL-expressing hematopoietic stem cells, mice supplemented with EPA showed a decrease in the LSC population, and reduced splenomegaly and leukocytosis, when compared with mice on an oleic acid diet. Supplementation of CML mice carrying the T315I mutation (in BCR-ABL) with EPA resulted in a similar effect. Indomethacin blocked the EPA effect and increased the severity of BCR-ABL-induced CML and decreased apoptosis. Δ(12)-PGJ3 rescued indomethacin-treated BCR-ABL mice and decreased LSCs. Inhibition of hematopoietic-prostaglandin D synthase (H-PGDS) by HQL-79 in EPA-supplemented CML mice also blocked the effect of EPA. In addition, EPA supplementation was effective in a murine model of acute myeloid leukemia. EPA-supplemented mice exhibited a decrease in leukemia burden and a decrease in the LSC colony-forming unit (LSC-CFU). The decrease in LSCs was confirmed through serial transplantation assays in all disease models. The results support a chemopreventive role for EPA in myeloid leukemia, which is dependent on the ability to efficiently convert EPA to endogenous COX-derived prostanoids, including Δ(12)-PGJ3.

Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

Draft Genome Sequences of 53 Genetically Distinct Isolates of Bordetella bronchiseptica Representing 11 Terrestrial and Aquatic Hosts.

Bordetella bronchiseptica infects a variety of mammalian and avian hosts. Here, we report the genome sequences of 53 genetically distinct isolates acquired from a broad range of terrestrial and aquatic animals. These data will greatly facilitate ongoing efforts to better understand the evolution, host adaptation, and virulence mechanisms of B. bronchiseptica.

Toll-like receptor 4 limits transmission of Bordetella bronchiseptica.

Transmission of pathogens has been notoriously difficult to study under laboratory conditions leaving knowledge gaps regarding how bacterial factors and host immune components affect the spread of infections between hosts. We describe the development of a mouse model of transmission of a natural pathogen, Bordetella bronchiseptica, and its use to assess the impact of host immune functions. Although B. bronchiseptica transmits poorly between wild-type mice and mice lacking other immune components, it transmits efficiently between mice deficient in Toll-Like Receptor 4 (TLR4). TLR4-mutant mice were more susceptible to initial colonization, and poorly controlled pathogen growth and shedding. Heavy neutrophil infiltration distinguished TLR4-deficient responses, and neutrophil depletion did not affect respiratory CFU load, but decreased bacterial shedding. The effect of TLR4 response on transmission may explain the extensive variation in TLR4 agonist potency observed among closely related subspecies of Bordetella. This transmission model will enable mechanistic studies of how pathogens spread from one host to another, the defining feature of infectious disease.

Genome Sequences of Nine Bordetella holmesii Strains Isolated in the United States.

An increasing number of pertussis-like cases are attributed to the emergent pathogen Bordetella holmesii. The genomes of 9 clinical isolates show that they are clonal, lack the virulence factors encoded by B. pertussis, and are more similar to nonpertussis bordetellae. New markers for B. holmesii can be developed using these sequences.

Genome Sequences of 28 Bordetella pertussis U.S. Outbreak Strains Dating from 2010 to 2012.

Despite the availability of highly effective vaccines, Bordetella pertussis incidence has been rapidly rising in highly vaccinated populations. Recent outbreaks have received media attention, feeding concerns about the emergence of dangerous new strains with increased virulence or that escape vaccine-induced immunity. To accelerate the study of this reemerging pathogen, we sequenced the genomes of 28 B. pertussis strains isolated during outbreaks from 2010 through 2012, making both strains and sequence data available to the scientific community.