Matthew L. Mayer

Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

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-κB, 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.

Potential of immunomodulatory host defense peptides as novel anti-infectives

A fundamentally new strategy for the treatment of infectious disease is the modulation of host immune responses to enhance clearance of infectious agents and reduce tissue damage due to inflammation. Antimicrobial host defense peptides have been investigated for their potential as a new class of antimicrobial drugs. Recently their immunomodulatory activities have begun to be appreciated. Modulation of innate immunity by synthetic variants of host defense peptides, called innate defense regulators (IDRs), is protective without direct antimicrobial action. We discuss the potential and current limitations in exploiting the immunomodulatory activity of IDRs as a novel anti-infective pathway. IDRs show significant promise and current research is uncovering mechanistic information that will aid in the future development of IDRs for clinical use.

INMEX--a web-based tool for integrative meta-analysis of expression data.

The widespread applications of various ‘omics’ technologies in biomedical research together with the emergence of public data repositories have resulted in a plethora of data sets for almost any given physiological state or disease condition. Properly combining or integrating these data sets with similar basic hypotheses can help reduce study bias, increase statistical power and improve overall biological understanding. However, the difficulties in data management and the complexities of analytical approaches have significantly limited data integration to enable meta-analysis. Here, we introduce integrative meta-analysis of expression data (INMEX), a user-friendly web-based tool designed to support meta-analysis of multiple gene-expression data sets, as well as to enable integration of data sets from gene expression and metabolomics experiments. INMEX contains three functional modules. The data preparation module supports flexible data processing, annotation and visualization of individual data sets. The statistical analysis module allows researchers to combine multiple data sets based on P-values, effect sizes, rank orders and other features. The significant genes can be examined in functional analysis module for enriched Gene Ontology terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, or expression profile visualization. INMEX has built-in support for common gene/metabolite identifiers (IDs), as well as 45 popular microarray platforms for human, mouse and rat. Complex operations are performed through a user-friendly web interface in a step-by-step manner. INMEX is freely available at http://www.inmex.ca.

The Pseudomonas aeruginosa autoinducer 3O-C12 homoserine lactone provokes hyperinflammatory responses from cystic fibrosis airway epithelial cells.

The discovery of novel antiinflammatory targets to treat inflammation in the cystic fibrosis (CF) lung stands to benefit patient populations suffering with this disease. The Pseudomonas aeruginosa quorum sensing autoinducer N-3-oxododecanoyl homoserine lactone (3O-C12) is an important bacterial virulence factor that has been reported to induce proinflammatory cytokine production from a variety of cell types. The goal of this study was to examine the ability of 3O-C12 to induce proinflammatory cytokine production in normal and CF bronchial epithelial cells, and better understand the cellular mechanisms by which this cytokine induction occurs. 3O-C12 was found to induce higher levels of IL-6 production in the CF cell lines IB3-1 and CuFi, compared to their corresponding control cell lines C38 and NuLi. Systems biology and network analysis revealed a high predominance of over-represented innate immune pathways bridged together by calcium-dependant transcription factors governing the transcriptional responses of A549 airway cells to stimulation with 3O-C12. Using calcium-flux assays, 3O-C12 was found to induce larger and more sustained increases in intracellular calcium in IB3-1 cells compared to C38, and blocking this calcium flux with BAPTA-AM reduced the production of IL-6 by IB3-1 to the levels produced by C38. These data suggest that 3O-C12 induces proinflammatory cytokine production in airway epithelial cells in a calcium-dependent manner, and that dysregulated calcium storage or signalling in CF cells results in an increased production of proinflammatory cytokines.

Rescue of Dysfunctional Autophagy Attenuates Hyperinflammatory Responses from Cystic Fibrosis Cells

A hallmark feature of cystic fibrosis (CF) is progressive pulmonary obstruction arising from exaggerated host proinflammatory responses to chronic bacterial airway colonization. The mechanisms for these heightened inflammatory responses have been only partially characterized, hampering development of effective anti-inflammatory therapies. The aim of this study was to identify and validate novel dysfunctional processes or pathways driving the hyperinflammatory phenotype of CF cells using systems biology and network analysis to examine transcriptional changes induced by innate defense regulator (IDR)-1018, an anti-inflammatory peptide. IDR-1018 selectively attenuated hyperinflammatory cytokine production from CF airway cells and PBMCs stimulated with multiple bacterial ligands, including flagellin (FliC). Network analysis of CF cell transcriptional responses to FliC and IDR-1018 identified dysfunctional autophagy as the target of the peptide via modulation of upstream adenosine monophosphate–activated protein kinase (AMPK)–Akt signaling. After treatment with FliC, CF cells were found to have elevated levels of the autophagosome marker LC3-II, and GFP-LC3–transfected CF airway cells showed abnormal perinuclear accumulation of GFP+ structures. In both instances, treatment of CF cells with IDR-1018 abolished the accumulation of LC3 induced by FliC. Furthermore, inhibition of autophagosome–lysosome fusion with bafilomycinA1 attenuated the anti-inflammatory and autophagosome-clearing effects of IDR-1018, as did a chemical inhibitor of Akt and an activator of AMPK. These findings were consistent with hypotheses generated in silico, demonstrating the utility of systems biology and network analysis approaches for providing pathway-level insights into CF-associated inflammation. Collectively, these data suggest that dysfunctional autophagosome clearance contributes to heightened inflammatory responses from CF transmembrane receptor mutant cells and highlight autophagy and AMPK–Akt signaling as novel anti-inflammatory targets in CF.

Functional genetic variation in NFKBIA and susceptibility to childhood asthma, bronchiolitis, and bronchopulmonary dysplasia.

Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.

Atypical Activation of the Unfolded Protein Response in Cystic Fibrosis Airway Cells Contributes to p38 MAPK-Mediated Innate Immune Responses

Inflammatory lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF); understanding what produces dysregulated innate immune responses in CF cells will be pivotal in guiding the development of novel anti-inflammatory therapies. To elucidate the molecular mechanisms that mediate exaggerated inflammation in CF following TLR signaling, we profiled global gene expression in immortalized human CF and non-CF airway cells at baseline and after microbial stimulation. Using complementary analysis methods, we observed a signature of increased stress levels in CF cells, specifically characterized by endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and MAPK signaling. Analysis of ER stress responses revealed an atypical induction of the UPR, characterized by the lack of induction of the PERK–eIF2α pathway in three complementary model systems: immortalized CF airway cells, fresh CF blood cells, and CF lung tissue. This atypical pattern of UPR activation was associated with the hyperinflammatory phenotype in CF cells, as deliberate induction of the PERK–eIF2α pathway with salubrinal attenuated the inflammatory response to both flagellin and Pseudomonas aeruginosa. IL-6 production triggered by ER stress and microbial stimulation were both dependent on p38 MAPK activity, suggesting a molecular link between both signaling events. These data indicate that atypical UPR activation fails to resolve the ER stress in CF and sensitizes the innate immune system to respond more vigorously to microbial challenge. Strategies to restore ER homeostasis and normalize the UPR activation profile may represent a novel therapeutic approach to minimize lung-damaging inflammation in CF.

INVEX--a web-based tool for integrative visualization of expression data.

Summary: Gene expression or metabolomics data generated from clinical settings are often associated with multiple metadata (i.e. diagnosis, genotype, gender, etc.). It is of great interest to analyze and to visualize the data in these contexts. Here, we introduce INVEX—a novel web-based tool that integrates the server-side capabilities for data analysis with the browse-based technology for data visualization. INVEX has two key features: (i) flexible differential expression analysis for a wide variety of experimental designs; and (ii) interactive visualization within the context of metadata and biological annotations. INVEX has built-in support for gene/metabolite annotation and a fully functional heatmap builder. Availability and implementation: Freely available at http://www.invex.ca. Contact: bob@hancocklab.ubc.ca

Cathelicidins Link the Endocrine and Immune Systems

Cathelicidin peptides play key roles in host responses to infection. Radek and colleagues (2010) demonstrate that the nicotinic acetylcholine system, activated during stress, suppresses production of mouse cathelicidin, increasing host susceptibility to the pathogen Staphylococcus aureus. This suggests a distinct way by which the endocrine system regulates innate immunity.