Mutual Effects of Single and Combined CFTR Modulators and Bacterial Infection in Cystic Fibrosis.

Cystic fibrosis transmembrane conductance regulator (CFTR) modulators improve clinical outcomes with varied efficacies in patients with CF. However, the mutual effects of CFTR modulators and bacterial adaptation, together with antibiotic regimens, can influence clinical outcomes. We evaluated the effects of ivacaftor (IVA), lumacaftor (LUM), tezacaftor, elexacaftor, and a three-modulator combination of elexacaftor, tezacaftor, and ivacaftor (ETI), alone or combined with antibiotics, on sequential CF isolates. IVA and ETI showed direct antimicrobial activities against Staphylococcus aureus but not against Pseudomonas aeruginosa. Additive effects or synergies were observed between the CFTR modulators and antibiotics against both species, independently of adaptation to the CF lung. IVA and LUM were the most effective in potentiating antibiotic activity against S. aureus, while IVA and ETI enhanced mainly polymyxin activity against P. aeruginosa. Next, we evaluated the effect of P. aeruginosa pneumonia on the pharmacokinetics of IVA in mice. IVA and its metabolites in plasma, lung, and epithelial lining fluid were increased by P. aeruginosa infection. Thus, CFTR modulators can have direct antimicrobial properties and/or enhance antibiotic activity against initial and adapted S. aureus and P. aeruginosa isolates. Furthermore, bacterial infection impacts airway exposure to IVA, potentially affecting its efficacy. Our findings suggest optimizing host- and pathogen-directed therapies to improve efficacy for personalized treatment. IMPORTANCE CFTR modulators have been developed to correct and/or enhance CFTR activity in patients with specific cystic fibrosis (CF) genotypes. However, it is of great importance to identify potential off-targets of these novel therapies to understand how they affect lung physiology in CF. Since bacterial infections are one of the hallmarks of CF lung disease, the effects (if any) of CFTR modulators on bacteria could impact their efficacy. This work highlights a mutual interaction between CFTR modulators and opportunistic bacterial infections; in particular, it shows that (i) CFTR modulators have an antibacterial activity per se and influence antibiotic efficacy, and (ii) bacterial airway infections affect levels of CFTR modulators in the airways. These findings may help optimize host- and pathogen-directed drug regimens to improve the efficacy of personalized treatment.

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.

R othia mucilaginosa is an anti-inflammatory bacterium in the respiratory tract of patients with chronic lung disease.

BACKGROUND: Chronic airway inflammation is the main driver of pathogenesis in respiratory diseases such as severe asthma, chronic obstructive pulmonary disease, cystic fibrosis (CF) and bronchiectasis. While the role of common pathogens in airway inflammation is widely recognised, the influence of other microbiota members is still poorly understood. METHODS: We hypothesised that the lung microbiota contains bacteria with immunomodulatory activity which modulate net levels of immune activation by key respiratory pathogens. Therefore, we assessed the immunomodulatory effect of several members of the lung microbiota frequently reported as present in CF lower respiratory tract samples. RESULTS: We show that Rothia mucilaginosa, a common resident of the oral cavity that is also often detectable in the lower airways in chronic disease, has an inhibitory effect on pathogen- or lipopolysaccharide-induced pro-inflammatory responses, in vitro (three-dimensional cell culture model) and in vivo (mouse model). Furthermore, in a cohort of adults with bronchiectasis, the abundance of Rothia species was negatively correlated with pro-inflammatory markers (interleukin (IL)-8 and IL-1ß) and matrix metalloproteinase (MMP)-1, MMP-8 and MMP-9 in sputum. Mechanistic studies revealed that R. mucilaginosa inhibits NF-κB pathway activation by reducing the phosphorylation of IκBα and consequently the expression of NF-κB target genes. CONCLUSIONS: These findings indicate that the presence of R. mucilaginosa in the lower airways potentially mitigates inflammation, which could in turn influence the severity and progression of chronic respiratory disorders.

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

Chronic infection by nontypeable Haemophilus influenzae fuels airway inflammation.

Nontypeable Haemophilus influenzae (NTHi) is commonly isolated from airways of patients suffering from chronic respiratory diseases, such as COPD or cystic fibrosis (CF). However, to what extent NTHi long-term infection contributes to the lung inflammatory burden during chronic airway disease is still controversial. Here, we exploited human respiratory samples from a small cohort of CF patients and found that patients chronically infected with NTHi had significantly higher levels of interleukin (IL)-8 and CXCL1 than those who were not infected. To better define the impact of chronic NTHi infection in fuelling inflammatory response in chronic lung diseases, we developed a new mouse model using both laboratory and clinical strains. Chronic NTHi infection was associated with chronic inflammation of the lung, characterised by recruitment of neutrophils and cytokine release keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), granulocyte colony-stimulating factor (G-CFS), IL-6, IL-17A and IL-17F) at 2 and 14 days post-infection. An increased burden of T-cell-mediated response (CD4+ and γδ cells) and higher levels of pro-matrix metalloproteinase 9 (pro-MMP9), known to be associated with tissue remodelling, were observed at 14 days post-infection. Of note we found that both CD4+IL-17+ cells and levels of IL-17 cytokines were enriched in mice at advanced stages of NTHi chronic infection. Moreover, by immunohistochemistry we found CD3+, B220+ and CXCL-13+ cells localised in bronchus-associated lymphoid tissue-like structures at day 14. Our results demonstrate that chronic NTHi infection exerts a pro-inflammatory activity in the human and murine lung and could therefore contribute to the exaggerated burden of lung inflammation in patients at risk.

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.

A New Model of Chronic Mycobacterium abscessus Lung Infection in Immunocompetent Mice.

Pulmonary infections caused by Mycobacterium abscessus (MA) have increased over recent decades, affecting individuals with underlying pathologies such as chronic obstructive pulmonary disease, bronchiectasis and, especially, cystic fibrosis. The lack of a representative and standardized model of chronic infection in mice has limited steps forward in the field of MA pulmonary infection. To overcome this challenge, we refined the method of agar beads to establish MA chronic infection in immunocompetent mice. We evaluated bacterial count, lung pathology and markers of inflammation and we performed longitudinal studies with magnetic resonance imaging (MRI) up to three months after MA infection. In this model, MA was able to establish a persistent lung infection for up to two months and with minimal systemic spread. Lung histopathological analysis revealed granulomatous inflammation around bronchi characterized by the presence of lymphocytes, aggregates of vacuolated histiocytes and a few neutrophils, mimicking the damage observed in humans. Furthermore, MA lung lesions were successfully monitored for the first time by MRI. The availability of this murine model and the introduction of the successfully longitudinal monitoring of the murine lung lesions with MRI pave the way for further investigations on the impact of MA pathogenesis and the efficacy of novel treatments.

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.

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.

The impact of host genetic background in the Pseudomonas aeruginosa respiratory infections.

Understanding the significance of human genetic diversity in modulating host susceptibility to opportunistic infections is an emerging challenge in the field of respiratory illnesses. While it is recognized that diverse bacterial strains account for differential disease manifestations, emerging data indicate that host genetic diversity is an important determinant factor that influences the severity of opportunistic infections. With particular regard to respiratory illnesses mediated by the gram-negative bacterium Pseudomonas aeruginosa, diverse genetic background is also emerging as a key contributor. Human-genome-wide association studies are a common approach for determining the inter-individual genetic variation associated with variability of the pulmonary infections. Historically, diverse murine inbred mouse strains and ex-vivo cellular models were considered complementary to human studies for establishing the contribution of genetic background to P. aeruginosa respiratory infections. More recently, the development of a new mouse model of infection, mirroring human airway diseases, combined with innovative murine resource populations, modelling human genetic variation, provides additional insights into the mechanisms of genetic susceptibility. In this review, we cover the recent state of the art of human and animal studies and we discuss future potential challenges in the field of P. aeruginosa respiratory infections.

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.

Staphylococcus aureus Impacts Pseudomonas aeruginosa Chronic Respiratory Disease in Murine Models.

Background: Staphylococcus aureus and Pseudomonas aeruginosa are key bacterial pathogens of the respiratory tract in patients with cystic fibrosis (CF). Although P. aeruginosa chronic bronchial infection is associated with a poorer prognosis, the consequences of S. aureus colonization on CF outcomes are controversial. Methods: In this paper, murine models of infection resembling traits of the CF human airways disease have been revisited using an infection schedule that mimics the sequence of events of pulmonary disease in CF patients. First, mice were infected with S. aureus, embedded in agar beads; this was followed by P. aeruginosa infection and analysis of bacterial load, leukocyte infiltration, and lung tissue damage. Results: We reveal that (1) S. aureus promotes severe lesions including abscess formation, (2) S. aureus increases the risk of subsequent chronic P. aeruginosa respiratory infection, and (3) once the chronic infection has been established, P. aeruginosa influences most of the inflammatory responses independent of S. aureus. Conclusions: Our findings established the significance of S. aureus colonization per se and the impact on the subsequent P. aeruginosa infection. This would point towards a thorough assessment for the need of treatment against S. aureus.

The PAPI-1 pathogenicity island-encoded small RNA PesA influences Pseudomonas aeruginosa virulence and modulates pyocin S3 production.

Small non-coding RNAs (sRNAs) are post-transcriptional regulators of gene expression that have been recognized as key contributors to bacterial virulence and pathogenic mechanisms. In this study, we characterized the sRNA PesA of the opportunistic human pathogen Pseudomonas aeruginosa. We show that PesA, which is transcribed within the pathogenicity island PAPI-1 of P. aeruginosa strain PA14, contributes to P. aeruginosa PA14 virulence. In fact, pesA gene deletion resulted in a less pathogenic strain, showing higher survival of cystic fibrosis human bronchial epithelial cells after infection. Moreover, we show that PesA influences positively the expression of pyocin S3 whose genetic locus comprises two structural genes, pyoS3A and pyoS3I, encoding the killing S3A and the immunity S3I proteins, respectively. Interestingly, the deletion of pesA gene results in increased sensitivity to UV irradiation and to the fluoroquinolone antibiotic ciprofloxacin. The degree of UV sensitivity displayed by the PA14 strain lacking PesA is comparable to that of a strain deleted for pyoS3A-I. These results suggest an involvement of pyocin S3 in DNA damage repair and a regulatory role of PesA on this function.

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.

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.

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.

The IL-17A/IL-17RA axis in pulmonary defence and immunopathology.

The interleukin (IL)-17A/IL-17 receptor A (IL-17RA) axis is emerging as a key player in host defence. Several studies have demonstrated that IL-17A-mediated responses play a critical role in both acute and chronic inflammation induced by infectious agents, environmental stimuli and genetic diseases in the airways. In this regard, it is becoming evident that IL-17A/IL-17RA signalling may have a protective and beneficial impact on health, but that it can also result in detrimental outcomes. On one hand, the IL-17A/IL-17RA axis can contribute to the elimination of noxious stimuli and to the resolution of acute inflammatory processes; on the other hand, it can exacerbate immunopathological responses, contributing to the development and progression of chronic respiratory illnesses. In addition, cellular and molecular signatures underlying IL-17A/IL-17RA signalling have been increasingly identified, although further studies are needed to clarify such complex responses. Here, we discuss the latest discoveries on the role of the IL-17A/IL-17RA axis in driving host pulmonary defence and immunopathology.