Assessing the Potential of N-Butyl-l-deoxynojirimycin (l-NBDNJ) in Models of Cystic Fibrosis as a Promising Antibacterial Agent.

Over the past few years, l-iminosugars have revealed attractive pharmacological properties for managing rare diseases including Cystic Fibrosis (CF). The iminosugar N-butyl-l-deoxynojirimycin (l-NBDNJ, ent-1), prepared by a carbohydrate-based route, was herein evaluated for its anti-inflammatory and anti-infective potential in models of CF lung disease infection. A significant decrease in the bacterial load in the airways was observed in the murine model of Pseudomonas aeruginosa chronic infection in the presence of l-NBDNJ, also accompanied by a modest reduction of inflammatory cells. Mechanistic insights into the observed activity revealed that l-NBDNJ interferes with the expression of proteins regulating cytoskeleton assembly and organization of the host cell, downregulates the main virulence factors of P. aeruginosa involved in the host response, and affects pathogen adhesion to human cells. These findings along with the observation of the absence of an in vitro bacteriostatic/bactericidal action of l-NBDNJ suggest the potential use of this glycomimetic as an antivirulence agent in the management of CF lung disease.

Pamoic acid is an inhibitor of HMGB1·CXCL12 elicited chemotaxis and reduces inflammation in murine models of Pseudomonas aeruginosa pneumonia.

BACKGROUND: High-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers. METHODS: PAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid. RESULTS: Our data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation. CONCLUSIONS: This work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.

Pseudomonas aeruginosa Elastase Contributes to the Establishment of Chronic Lung Colonization and Modulates the Immune Response in a Murine Model.

Chronic infection by Pseudomonas aeruginosa in cystic fibrosis (CF) patients is a major contributor to progressive lung damage and is poorly treated by available antibiotic therapy. An alternative approach to the development of additional antibiotic treatments is to identify complementary therapies which target bacterial virulence factors necessary for the establishment and/or maintenance of the chronic infection. The P. aeruginosa elastase (LasB) has been suggested as an attractive anti-virulence target due to its extracellular location, its harmful degradative effects on host tissues and the immune system, and the potential to inhibit its activity using small molecule inhibitors. However, while the relevance of LasB in acute P. aeruginosa infection has been demonstrated, it is still unclear whether this elastase might also play a role in the early phase of chronic lung colonization. By analyzing clinical P. aeruginosa clonal isolates from a CF patient, we found that the isolate RP45, collected in the early phase of persistence, produces large amounts of active LasB, while its clonal variant RP73, collected after years of colonization, does not produce it. When a mouse model of persistent pneumonia was used, deletion of the lasB gene in RP45 resulted in a significant reduction in mean bacterial numbers and incidence of chronic lung colonization at Day 7 post-challenge compared to those mice infected with wild-type (wt) RP45. Furthermore, deletion of lasB in strain RP45 also resulted in an increase in immunomodulators associated with innate and adaptive immune responses in infected animals. In contrast, deletion of the lasB gene in RP73 did not affect the establishment of chronic infection. Overall, these results indicate that LasB contributes to the adaptation of P. aeruginosa to a persistent lifestyle. In addition, these findings support pharmacological inhibition of LasB as a potentially useful therapeutic intervention for P. aeruginosa-infected CF patients prior to the establishment of a chronic infection.

Antibiotic efficacy varies based on the infection model and treatment regimen for Pseudomonas aeruginosa.

Antibiotic discovery and preclinical testing are needed to combat the Pseudomonas aeruginosa health threat. Most frequently, antibiotic efficacy is tested in models of acute respiratory infection, with chronic pneumonia remaining largely unexplored. This approach generates serious concerns about the evaluation of treatment for chronically infected patients, and highlights the need for animal models that mimic the course of human disease.In this study, the efficacy of the marketed antibacterial drugs tobramycin (TOB) and colistin (COL) was tested in murine models of acute and chronic P. aeruginosa pulmonary infection. Different administration routes (intranasal, aerosol or subcutaneous) and treatment schedules (soon or 7 days post-infection) were tested.In the acute infection model, aerosol and subcutaneous administration of TOB reduced the bacterial burden and inflammatory response, while intranasal treatment showed modest efficacy. COL reduced the bacterial burden less effectively but dampened inflammation. Mice treated soon after chronic infection for 7 days with daily aerosol or subcutaneous administration of TOB showed higher and more rapid body weight recovery and reduced bacterial burden and inflammation than vehicle-treated mice. COL-treated mice showed no improvement in body weight or change in inflammation. Modest bacterial burden reduction was recorded only with aerosol COL administration. When treatment for chronic infection was commenced 7 days after infection, both TOB and COL failed to reduce P. aeruginosa burden and inflammation, or aid in recovery of body weight.Our findings suggest that the animal model and treatment regimen should be carefully chosen based on the type of infection to assess antibiotic efficacy.

Synthesized Heparan Sulfate Competitors Attenuate Pseudomonas aeruginosa Lung Infection.

Several chronic respiratory diseases are characterized by recurrent and/or persistent infections, chronic inflammatory responses and tissue remodeling, including increased levels of glycosaminoglycans which are known structural components of the airways. Among glycosaminoglycans, heparan sulfate (HS) has been suggested to contribute to excessive inflammatory responses. Here, we aim at (i) investigating whether long-term infection by Pseudomonas aeruginosa, one of the most worrisome threat in chronic respiratory diseases, may impact HS levels, and (ii) exploring HS competitors as potential anti-inflammatory drugs during P. aeruginosa pneumonia. P. aeruginosa clinical strains and ad-hoc synthesized HS competitors were used in vitro and in murine models of lung infection. During long-term chronic P. aeruginosa colonization, infected mice showed higher heparin/HS levels, evaluated by high performance liquid chromatography-mass spectrometry after selective enzymatic digestion, compared to uninfected mice. Among HS competitors, an N-acetyl heparin and a glycol-split heparin dampened leukocyte recruitment and cytokine/chemokine production induced by acute and chronic P. aeruginosa pneumonia in mice. Furthermore, treatment with HS competitors reduced bacterial burden during chronic murine lung infection. In vitro, P. aeruginosa biofilm formation decreased upon treatment with HS competitors. Overall, these findings support further evaluation of HS competitors as a novel therapy to counteract inflammation and infection during P. aeruginosa pneumonia.