Expression and Roles of Antimicrobial Peptides in Innate Defense of Airway Mucosa: Potential Implication in Cystic Fibrosis

© Copyright © 2020 Geitani, Moubareck, Xu, Karam Sarkis and Touqui. The treatment of respiratory infections is associated with the dissemination of antibiotic resistance in the community and clinical settings. Development of new antibiotics is notoriously costly and slow; therefore, alternative strategies are needed. Antimicrobial peptides (AMPs), the central effector molecules of the immune system, are being considered as alternatives to conventional antibiotics. Most AMPs are epithelium-derived and play a key role in host defense at mucosal surfaces. They are classified on the basis of their structure and amino acid motifs. These peptides display a range of activities, including not only direct antimicrobial activity, but also immunomodulation and wound repair. In the lung, airway epithelial cells and neutrophils, in particular, contribute to AMP synthesis. The relevance of AMPs for host defense against infection has been demonstrated in animal models and is supported by observations in patient studies, showing altered expression and/or unfavorable circumstances for their action in a variety of lung diseases. Of note, AMPs are active against bacterial strains that are resistant to conventional antibiotics, including multidrug-resistant bacteria. Several strategies have been proposed to use these peptides in the treatment of infections, including direct administration of AMPs. In this review, we focus on studies related to direct bactericidal effects of AMPs and their potential clinical applications with a particular focus on cystic fibrosis

Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention

Multidrug-resistant bacteria are the cause of an increasing number of deadly pulmonary infections. Because there is currently a paucity of novel antibiotics, phage therapy—the use of specific viruses that infect bacteria—is now more frequently being considered as a potential treatment for bacterial infections. Using a mouse lung-infection model caused by a multidrug resistant Pseudomonas aeruginosa mucoid strain isolated from a cystic fibrosis patient, we evaluated bacteriophage treatments. New bacteriophages were isolated from environmental samples and characterized. Bacteria and bacteriophages were applied intranasally to the immunocompetent mice. Survival was monitored and bronchoalveolar fluids were analysed. Quantification of bacteria, bacteriophages, pro-inflammatory and cytotoxicity markers, as well as histology and immunohistochemistry analyses were performed. A curative treatment (one single dose) administrated 2 h after the onset of the infection allowed over 95% survival. A four-day preventive treatment (one single dose) resulted in a 100% survival. All of the parameters measured correlated with the efficacy of both curative and preventive bacteriophage treatments. We also showed that in vitro optimization of a bacteriophage towards a clinical strain improved both its efficacy on in vivo treatments and its host range on a panel of 20 P. aeruginosa cystic fibrosis strains. This work provides an incentive to develop clinical studies on pulmonary bacteriophage therapy to combat multidrug-resistant lung infections

Edema Toxin Impairs Anthracidal Phospholipase A2 Expression by Alveolar Macrophages

Bacillus anthracis, the etiological agent of anthrax, is a spore-forming Gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET). Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA) against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs), sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A–dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deathly pathogen

Peroxiredoxin 6 Fails to Limit Phospholipid Peroxidation in Lung from Cftr-Knockout Mice Subjected to Oxidative Challenge

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult

Mouse models of cystic fibrosis: Phenotypic analysis and research applications

AbstractGenetically modified mice have been studied for more than fifteen years as models of cystic fibrosis (CF). The large amount of experimental data generated illuminates the complex multi-organ pathology of CF and raises new questions relevant to human disease. CF mice have also been used to test experimental therapies prior to clinical trials. This review recapitulates the major phenotypic traits of CF mice and highlights important new findings including aberrant alveolar macrophages, bone and cartilage abnormalities and abnormal bioactive lipid metabolism. Novel data are presented on the intestinal and nasal physiology of F508del-CFTR CF mice backcrossed onto different genetic backgrounds. Caveats, and sources of variability including age, gender and animal husbandry, are discussed. Interspecies differences limit comparison of lung pathology in CF mice to the human disease. The recent development of genetically modified pigs and ferrets heralds the application of more advanced animal models to CF research and drug development

A Crucial Role of Flagellin in the Induction of Airway Mucus Production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells

BACKGROUND:The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-alpha, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production. CONCLUSIONS/SIGNIFICANCE:The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-alpha, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88(-/-) mice