Examining the Role of Specific Virulence Mechanisms During Pseudomonas Aeruginosa Infection in a Zebrafish Model of Cystic Fibrosis

Cystic fibrosis (CF) is the most common lethal hereditary disease. CF is caused by recessive mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is associated with multi-­-organ defects resulting from improper ion transport across epithelial membranes. Chronic lung infection by the environmentally ubiquitous opportunistic human pathogen Pseudomonas aeruginosa and the subsequent hyperinflammation that occurs as the host immune system combats the bacterium cause substantial morbidity and mortality in CF. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were also found to display a iv significant iron deficiency (ID) compared to control embryos, a symptom commonly diagnosed in CF patients. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, a phenomenon that was not observed with any of the other bacterial species examined. The bacterial burden in Cftr morphants infected with a P. aeruginosa LasR mutant, a quorum sensing (QS)-­-deficient strain, was comparable to that in control fish indicating that the regulation of virulence factors through QS is required for enhancement of infection in the absence of Cftr. Cftr morphants were then challenged with P. aeruginosa mutants defective in the expression of QS regulated virulence factors. A mutant defective in the production of Exotoxin A (ETA) resulted in similar bacterial clearance in both the Cftr morphant and control embryos. The reduction in directed neutrophil migration to a P. aeruginosa infection was also restored when zebrafish embryos were challenged with the ETA mutant. Taken together, these data point towards a possible explanation for the specificity between P. aeruginosa and CFTR. The zebrafish system provides a multitude of advantages for studying the unique pathophysiology resulting from defective expression of CFTR, investigating the pathogenesis of P. aeruginosa and elucidating the role that the innate system plays in the host response to acute bacterial infections commonly associated with cystic fibrosis

Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells

Dendritic cell nediated inhibition of lentiviral infection

Lentiviral entry to quiescent lymphocytes represents a 'time bomb' waiting for cellular activation to spread infection. In order to undergo immune activation T cells interact with dendritic cells presenting peptide:MHC complexes 'sampling' them to look for agonist peptides and receiving survival signals from self peptides. This makes the dendritic cell:T cell interaction an ideal checkpoint to contain lentiviral infection of quiescent lypmhocytes

Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling.

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells

Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish Is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator ▿ †

Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ΔLasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System▿ †

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacIq carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen

Induction and Therapeutic Targeting of Human NPM1c+ Myeloid Leukemia in the Presence of Autologous Immune System in Mice

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123 + leukemic stem or initiating cells (LSC), resembles NPM1c + AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123 + LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c + AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.National Cancer Institute (Grant P30-CA14051