Journal Article

Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Bioware; Cell Line; Cells, Cultured; Chemokine CCL2; Chemokine CCL7; Chemokine CXCL1; Chemokines; Female; Humans; Interleukin-8; Leukocytes; Leukocytes, Mononuclear; Macrophages; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Staphylococcal Infections; Staphylococcus aureus; Xen29, Xen14

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-kappaB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Journal Article

Xen14, Xen 14, E. coli Xen14, IVIS, Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides/chemical synthesis/*pharmacology; Bacterial Infections/*metabolism/microbiology/prevention & control; Cell Line; Cells, Cultured; Chemokine CCL2/metabolism; Chemokine CCL7/metabolism; Chemokine CXCL1/metabolism; Chemokines/*metabolism; Female; Humans; Interleukin-8/metabolism; Leukocytes/cytology/*metabolism; Leukocytes, Mononuclear/cytology/drug effects/metabolism; Macrophages/cytology/drug effects/metabolism; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NF-kappa B/metabolism; Phosphatidylinositol 3-Kinases/metabolism; Phosphorylation/drug effects; Staphylococcal Infections/microbiology/prevention & control; Staphylococcus aureus/drug effects; p38 Mitogen-Activated Protein Kinases/metabolism

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-kappaB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Journal Article

Animals, Cell Line, Cell Line, Transformed, Humans, Macrophages, Alveolar/*drug effects/immunology/*microbiology/pathology, Mice, Mice, Inbred C57BL, Morphine/administration & dosage/*therapeutic use, NF-kappa B/*antagonists & inhibitors/physiology, Neutrophil Infiltration/drug effects/immunology, Pneumonia, Pneumococcal/*drug therapy/*immunology/microbiology/mortality, Signal Transduction/*drug effects/immunology, Streptococcus pneumoniae/drug effects/*immunology, Time Factors, Toll-Like Receptor 2/physiology, Toll-Like Receptor 4/physiology, Toll-Like Receptor 9/*antagonists & inhibitors/physiology IVIS, Xenogen, Xen10

Resident alveolar macrophages and respiratory epithelium constitutes the first line of defense against invading lung pneumococci. Results from our study showed that increased mortality and bacterial outgrowth and dissemination seen in morphine-treated mice were further exaggerated following depletion of alveolar macrophages with liposomal clodronate. Using an in vitro alveolar macrophages and lung epithelial cells infection model, we show significant release of MIP-2 from alveolar macrophages, but not from lung epithelial cells, following 4 h of exposure of cells to pneumococci infection. Morphine treatment reduced MIP-2 release in pneumococci stimulated alveolar macrophages. Furthermore, morphine treatment inhibited Streptococcus pneumoniae-induced NF-kappaB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-kappaB activity only in TLR9 stably transfected HEK 293 cells, but not in TLR2 and TLR4 transfected HEK 293 cells, and morphine treatment inhibited S. pneumoniae-induced NF-kappaB activity in these cells. Moreover, morphine treatment also decreased bacterial uptake and killing in alveolar macrophages. Taken together, these results suggest that morphine treatment impairs TLR9-NF-kappaB signaling and diminishes bacterial clearance following S. pneumoniae infection in resident macrophages during the early stages of infection, leading to a compromised innate immune response.

Journal Article

Amine Oxidase (Copper-Containing); Animals; Bacterial Adhesion; Bioware; Cell Adhesion Molecules; Coxsackievirus Infections; Immunity, Mucosal; Immunoglobulin A; Lymphocyte Count; Lymphocytes; Lymphoid Tissue; Mice; Mice, Inbred C57BL; Mice, Knockout; Peyer's Patches; Receptors, Lymphocyte Homing; Staphylococcal Vaccines; Staphylococcus aureus; Xen36

VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer's patches of the gut. IgA concentration in serum was also found to be lower in VAP-1(-/-) animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.

Journal Article

Animals; Bioware; Cattle; Cells, Cultured; Cystic Fibrosis; Flavoproteins; Humans; Hydrogen peroxide; Immunity, Innate; Immunity, Mucosal; Lactoperoxidase; Lung Diseases; Pseudomonas aeruginosa; Rats; Reactive Oxygen Species; Respiratory Mucosa; RNA, Small Interfering; Staphylococcus aureus; Thiocyanates; Trachea; Xen8.1

RATIONALE The respiratory tract is constantly exposed to airborne microorganisms. Nevertheless, normal airways remain sterile without recruiting phagocytes. This innate immune activity has been attributed to mucociliary clearance and antimicrobial polypeptides of airway surface liquid. Defective airway immunity characterizes cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator, a chloride channel. The pathophysiology of defective immunity in CF remains to be elucidated. OBJECTIVE We investigated the ability of non-CF and CF airway epithelia to kill bacteria through the generation of reactive oxygen species (ROS). METHODS ROS production and ROS-mediated bactericidal activity were determined on the apical surfaces of human and rat airway epithelia and on cow tracheal explants. MEASUREMENTS AND MAIN RESULTS Dual oxidase enzyme of airway epithelial cells generated sufficient H(2)O(2) to support production of bactericidal hypothiocyanite (OSCN(-)) in the presence of airway surface liquid components lactoperoxidase and thiocyanate (SCN(-)). This OSCN(-) formation eliminated Staphylococcus aureus and Pseudomonas aeruginosa on airway mucosal surfaces, whereas it was nontoxic to the host. In contrast to normal epithelia, CF epithelia failed to secrete SCN(-), thereby rendering the oxidative antimicrobial system inactive. CONCLUSIONS These data indicate a novel innate defense mechanism of airways that kills bacteria via ROS and suggest a new cellular and molecular basis for defective airway immunity in CF.