ß-sitosterol ameliorates inflammation and Pseudomonas aeruginosa lung infection in a mouse model.

We previously demonstrated that ß-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

New TMA (4,6,4'-Trimethyl angelicin) Analogues as Anti-Inflammatory Agents in the Treatment of Cystic Fibrosis Lung Disease.

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.

Efficacy of selective histone deacetylase 6 inhibition in mouse models of Pseudomonas aeruginosa infection: A new glimpse for reducing inflammation and infection in cystic fibrosis.

The latest studies identified the histone deacetylase (HDAC) class of enzymes as strategic components of the complex molecular machinery underlying inflammation in cystic fibrosis (CF). Compelling new support has been provided for HDAC6 isoform as a key player in the generation of the dysregulated proinflammatory phenotype in CF, as well as in the immune response to the persistent bacterial infection accompanying CF patients. We herein provide in vivo proof-of-concept (PoC) of the efficacy of selective HDAC6 inhibition in contrasting the pro-inflammatory phenotype in a mouse model of chronic P. aeruginosa respiratory infection. Upon careful selection and in-house re-profiling (in vitro and cell-based assessment of acetylated tubulin level through Western blot analysis) of three potent and selective HDAC6 inhibitors as putative candidates for the PoC, we engaged the best performing compound 2 for pre-clinical studies. Compound 2 demonstrated no toxicity and robust anti-inflammatory profile in a mouse model of chronic P. aeruginosa respiratory infection upon repeated aerosol administration. A significant reduction of leukocyte recruitment in the airways, in particular neutrophils, was observed in compound 2-treated mice in comparison with the vehicle; moreover, quantitative immunoassays confirmed a significant reduction of chemokines and cytokines in lung homogenate. This effect was also associated with a modest reduced bacterial load after compound 2-treatment in mice compared to the vehicle. Our study is of particular significance since it demonstrates for the first time the utility of selective drug-like HDAC6 inhibitors in a relevant in vivo model of chronic P. aeruginosa infection, thus supporting their potential application for reverting CF phenotype.

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.

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis.

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1ß, and IL-6, while increasing IL-10 and TGF-ß levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.

The Small RNA ErsA Plays a Role in the Regulatory Network of Pseudomonas aeruginosa Pathogenicity in Airway Infections.

Bacterial small RNAs play a remarkable role in the regulation of functions involved in host-pathogen interaction. ErsA is a small RNA of Pseudomonas aeruginosa that contributes to the regulation of bacterial virulence traits such as biofilm formation and motility. Shown to take part in a regulatory circuit under the control of the envelope stress response sigma factor σ22, ErsA targets posttranscriptionally the key virulence-associated gene algC Moreover, ErsA contributes to biofilm development and motility through the posttranscriptional modulation of the transcription factor AmrZ. Intending to evaluate the regulatory relevance of ErsA in the pathogenesis of respiratory infections, we analyzed the impact of ErsA-mediated regulation on the virulence potential of P. aeruginosa and the stimulation of the inflammatory response during the infection of bronchial epithelial cells and a murine model. Furthermore, we assessed ErsA expression in a collection of P. aeruginosa clinical pulmonary isolates and investigated the link of ErsA with acquired antibiotic resistance by generating an ersA gene deletion mutant in a multidrug-resistant P. aeruginosa strain which has long been adapted in the airways of a cystic fibrosis (CF) patient. Our results show that the ErsA-mediated regulation is relevant for the P. aeruginosa pathogenicity during acute infection and contributes to the stimulation of the host inflammatory response. Besides, ErsA was able to be subjected to selective pressure for P. aeruginosa pathoadaptation and acquirement of resistance to antibiotics commonly used in clinical practice during chronic CF infections. Our findings establish the role of ErsA as an important regulatory element in the host-pathogen interaction.IMPORTANCEPseudomonas aeruginosa is one of the most critical multidrug-resistant opportunistic pathogens in humans, able to cause both lethal acute and chronic lung infections. Thorough knowledge of the regulatory mechanisms involved in the establishment and persistence of the airways infections by P. aeruginosa remains elusive. Emerging candidates as molecular regulators of pathogenesis in P. aeruginosa are small RNAs, which act posttranscriptionally as signal transducers of host cues. Known for being involved in the regulation of biofilm formation and responsive to envelope stress response, we show that the small RNA ErsA can play regulatory roles in acute infection, stimulation of host inflammatory response, and mechanisms of acquirement of antibiotic resistance and adaptation during the chronic lung infections of cystic fibrosis patients. Elucidating the complexity of the networks regulating host-pathogen interactions is crucial to identify novel targets for future therapeutic applications.

Pharmacological modulation of mitochondrial calcium uniporter controls lung inflammation in cystic fibrosis.

Mitochondria physically associate with the endoplasmic reticulum to coordinate interorganelle calcium transfer and regulate fundamental cellular processes, including inflammation. Deregulated endoplasmic reticulum-mitochondria cross-talk can occur in cystic fibrosis, contributing to hyperinflammation and disease progression. We demonstrate that Pseudomonas aeruginosa infection increases endoplasmic reticulum-mitochondria associations in cystic fibrosis bronchial cells by stabilizing VAPB-PTPIP51 (vesicle-associated membrane protein-associated protein B-protein tyrosine phosphatase interacting protein 51) tethers, affecting autophagy. Impaired autophagy induced mitochondrial unfolding protein response and NLRP3 inflammasome activation, contributing to hyperinflammation. The mechanism by which VAPB-PTPIP51 tethers regulate autophagy in cystic fibrosis involves calcium transfer via mitochondrial calcium uniporter. Mitochondrial calcium uniporter inhibition rectified autophagy and alleviated the inflammatory response in vitro and in vivo, resulting in a valid therapeutic strategy for cystic fibrosis pulmonary disease.

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.

Aerosolized Bovine Lactoferrin Counteracts Infection, Inflammation and Iron Dysbalance in A Cystic Fibrosis Mouse Model of Pseudomonas aeruginosa Chronic Lung Infection.

Cystic fibrosis (CF) is a genetic disorder affecting several organs including airways. Bacterial infection, inflammation and iron dysbalance play a major role in the chronicity and severity of the lung pathology. The aim of this study was to investigate the effect of lactoferrin (Lf), a multifunctional iron-chelating glycoprotein of innate immunity, in a CF murine model of Pseudomonas aeruginosa chronic lung infection. To induce chronic lung infection, C57BL/6 mice, either cystic fibrosis transmembrane conductance regulator (CFTR)-deficient (Cftrtm1UNCTgN(FABPCFTR)#Jaw) or wild-type (WT), were intra-tracheally inoculated with multidrug-resistant MDR-RP73 P. aeruginosa embedded in agar beads. Treatments with aerosolized bovine Lf (bLf) or saline were started five minutes after infection and repeated daily for six days. Our results demonstrated that aerosolized bLf was effective in significantly reducing both pulmonary bacterial load and infiltrated leukocytes in infected CF mice. Furthermore, for the first time, we showed that bLf reduced pulmonary iron overload, in both WT and CF mice. In particular, at molecular level, a significant decrease of both the iron exporter ferroportin and iron storage ferritin, as well as luminal iron content was observed. Overall, bLf acts as a potent multi-targeting agent able to break the vicious cycle induced by P. aeruginosa, inflammation and iron dysbalance, thus mitigating the severity of CF-related pathology and sequelae.

Exploring the effect of chirality on the therapeutic potential of N-alkyl-deoxyiminosugars: anti-inflammatory response to Pseudomonas aeruginosa infections for application in CF lung disease.

In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl l-deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I2 reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their d-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding d-enantiomer. The remarkably low dosage of the l-iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its d-counterpart. Biological results herein obtained place ent-1 and congeners among the earliest examples of l-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis.

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.

Aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of Pseudomonas aeruginosa lung infections.

Lactoferrin (Lf), an iron-chelating glycoprotein of innate immunity, produced by exocrine glands and neutrophils in infection/inflammation sites, is one of the most abundant defence molecules in airway secretions. Lf, a pleiotropic molecule, exhibits antibacterial and anti-inflammatory functions. These properties may play a relevant role in airway infections characterized by exaggerated inflammatory response, as in Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) subjects. To verify the Lf role in Pseudomonas aeruginosa lung infection, we evaluated the efficacy of aerosolized bovine Lf (bLf) in mouse models of P. aeruginosa acute and chronic lung infections. C57BL/6NCrl mice were challenged with 106 CFUs of P. aeruginosa PAO1 (acute infection) or MDR-RP73 strain (chronic infection) by intra-tracheal administration. In both acute and chronic infections, aerosolized bLf resulted in nonsignificant reduction of bacterial load but significant decrease of the neutrophil recruitment and pro-inflammatory cytokine levels. Moreover, in chronic infection the bLf-treated mice recovered body weight faster and to a higher extent than the control mice. These findings add new insights into the benefits of bLf as a mediator of general health and its potential therapeutic applications.

The host genetic background defines diverse immune-reactivity and susceptibility to chronic Pseudomonas aeruginosa respiratory infection.

Patients with P. aeruginosa airways infection show markedly variable clinical phenotypes likely influenced by genetic backgrounds. Here, we investigated the cellular events involved in resistance and susceptibility to P. aeruginosa chronic infection using genetically distinct inbred mouse strains. As for patients, different murine genotypes revealed variable susceptibility to infection. When directly compared, resistant C3H/HeOuJ and susceptible A/J strains revealed distinct immune responsiveness to the pathogen. In C3H/HeOuJ resistant mice, IL17-producing cells rapidly and transiently infiltrated the infected lung, and this was paralleled by the acute accumulation of alveolar macrophages, bacterial clearance and resolution of infection. In contrast, A/J susceptible mice revealed a more delayed and prolonged lung infiltration by IL17+ and IFNγ+ cells, persistence of innate inflammatory cells and establishment of chronic infection. We conclude that the host genetic background confers diverse immunoreactivity to P. aeruginosa and IL17-producing cells might contribute to the progress of chronic lung infection.

Genotypic and phenotypic relatedness of Pseudomonas aeruginosa isolates among the major cystic fibrosis patient cohort in Italy.

BACKGROUND: Pseudomonas aeruginosa is the predominant pathogen associated with the decline of pulmonary function in cystic fibrosis (CF) patients. Both environment-to-host acquisition and patient-to-patient transmission have been described for P. aeruginosa infection. Epidemic clones and bacterial phenotypic adaptation to the CF lung have been recognised as independent risk factors for disease progression. So far, there is no established link between genotypic prevalence and phenotypic traits. Here, we look at the major CF patient cohort in Italy to identify shared P. aeruginosa clones and associated common phenotypic traits. RESULTS: A comprehensive analysis of P. aeruginosa genotypes to determine the presence of high-risk shared clones and their association to specific phenotypic traits has been performed in a major Italian CF centre. Pulsed-field gel electrophoresis (PFGE) of P. aeruginosa isolates from 338 CF subjects identified 43 profiles shared by two or more patients and 214 profiles exclusive to individual patients. There was no evidence of a P. aeruginosa outbreak, but four most prevalent pulsotypes were detected. Common phenotypic traits were recorded intra-pulsotypes, but we detected heterogeneity inter-pulsotypes. Two of the four major pulsotypes included P. aeruginosa isolates with hallmarks of adaptation to the CF airways, including loss of motility, low production of siderophore, pyocyanin and proteases, and antibiotic resistance. One of these pulsotypes grouped a high percentage of hypermutable isolates. No clear correlation between epidemiological and clinical data was found. CONCLUSIONS: We conclude that CF patients of this cohort shared common pulsotypes, but their phenotypic heterogeneity indicates an absence of specific traits associated to P. aeruginosa genotypic prevalence.

Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia.

The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections.

Mapping genetic determinants of host susceptibility to Pseudomonas aeruginosa lung infection in mice.

BACKGROUND: P. aeruginosa is one of the top three causes of opportunistic human bacterial infections. The remarkable variability in the clinical outcomes of this infection is thought to be associated with genetic predisposition. However, the genes underlying host susceptibility to P. aeruginosa infection are still largely unknown. RESULTS: As a step towards mapping these genes, we applied a genome wide linkage analysis approach to a mouse model. A large F2 intercross population, obtained by mating P. aeruginosa-resistant C3H/HeOuJ, and susceptible A/J mice, was used for quantitative trait locus (QTL) mapping. The F2 progenies were challenged with a P. aeruginosa clinical strain and monitored for the survival time up to 7 days post-infection, as a disease phenotype associated trait. Selected phenotypic extremes of the F2 distribution were genotyped with high-density single nucleotide polymorphic (SNP) markers, and subsequently QTL analysis was performed. A significant locus was mapped on chromosome 6 and was named P . aeruginosa infection resistance locus 1 (Pairl1). The most promising candidate genes, including Dok1, Tacr1, Cd207, Clec4f, Gp9, Gata2, Foxp1, are related to pathogen sensing, neutrophils and macrophages recruitment and inflammatory processes. CONCLUSIONS: We propose a set of genes involved in the pathogenesis of P. aeruginosa infection that may be explored to complement human studies.

IL-17A impairs host tolerance during airway chronic infection by Pseudomonas aeruginosa.

Resistance and tolerance mechanisms participate to the interplay between host and pathogens. IL-17-mediated response has been shown to be crucial for host resistance to respiratory infections, whereas its role in host tolerance during chronic airway colonization is still unclear. Here, we investigated whether IL-17-mediated response modulates mechanisms of host tolerance during airways chronic infection by P. aeruginosa. First, we found that IL-17A levels were sustained in mice at both early and advanced stages of P. aeruginosa chronic infection and confirmed these observations in human respiratory samples from cystic fibrosis patients infected by P. aeruginosa. Using IL-17a(-/-) or IL-17ra(-/-) mice, we found that the deficiency of IL-17A/IL-17RA axis was associated with: i) increased incidence of chronic infection and bacterial burden, indicating its role in the host resistance to P. aeruginosa; ii) reduced cytokine levels (KC), tissue innate immune cells and markers of tissue damage (pro-MMP-9, elastin degradation, TGF-ß1), proving alteration of host tolerance. Blockade of IL-17A activity by a monoclonal antibody, started when chronic infection is established, did not alter host resistance but increased tolerance. In conclusion, this study identifies IL-17-mediated response as a negative regulator of host tolerance during P. aeruginosa chronic airway infection.

Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections.

Repeated cycles of infections, caused mainly by Pseudomonas aeruginosa, combined with a robust host immune response and tissue injury, determine the course and outcome of cystic fibrosis (CF) lung disease. As the disease progresses, P. aeruginosa adapts to the host modifying dramatically its phenotype; however, it remains unclear whether and how bacterial adaptive variants and their persistence influence the pathogenesis and disease development. Using in vitro and murine models of infection, we showed that P. aeruginosa CF-adaptive variants shaped the innate immune response favoring their persistence. Next, we refined a murine model of chronic pneumonia extending P. aeruginosa infection up to three months. In this model, including CFTR-deficient mice, we unveil that the P. aeruginosa persistence lead to CF hallmarks of airway remodelling and fibrosis, including epithelial hyperplasia and structure degeneration, goblet cell metaplasia, collagen deposition, elastin degradation and several additional markers of tissue damage. This murine model of P. aeruginosa chronic infection, reproducing CF lung pathology, will be instrumental to identify novel molecular targets and test newly tailored molecules inhibiting chronic inflammation and tissue damage processes in pre-clinical studies.