RESUMEN
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.
Asunto(s)
Antiinflamatorios/uso terapéutico , Heparitina Sulfato/química , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/fisiología , Infecciones del Sistema Respiratorio/prevención & control , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Biopelículas/efectos de los fármacos , Quimiocinas/análisis , Quimiocinas/metabolismo , Cromatografía Líquida de Alta Presión , Citocinas/análisis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Heparitina Sulfato/análisis , Heparitina Sulfato/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Infecciones del Sistema Respiratorio/metabolismo , Infecciones del Sistema Respiratorio/microbiologíaRESUMEN
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.
Asunto(s)
Mapeo Cromosómico/métodos , Redes Reguladoras de Genes , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/fisiología , Animales , Modelos Animales de Enfermedad , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Ratones , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
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.
Asunto(s)
Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/microbiología , Animales , Línea Celular , Quimiocinas/metabolismo , Fibrosis Quística/complicaciones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Infecciones Oportunistas , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Infecciones del Sistema Respiratorio/metabolismo , Infecciones del Sistema Respiratorio/patologíaRESUMEN
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.
Asunto(s)
Resistencia a la Enfermedad , Predisposición Genética a la Enfermedad , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/inmunología , Animales , Antecedentes Genéticos , Interleucina-17/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Pulmón/patología , RatonesRESUMEN
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.
Asunto(s)
Fibrosis Quística/microbiología , Interleucina-17/genética , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/patogenicidad , Infecciones del Sistema Respiratorio/microbiología , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Incidencia , Interleucina-17/metabolismo , Ratones , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/inmunología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/inmunología , Regulación hacia ArribaRESUMEN
Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P. aeruginosa infection. These results now provide a basis for mapping genomic regions underlying host susceptibility to P. aeruginosa infection.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Inmunidad Celular/genética , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Carga Bacteriana , Interacciones Huésped-Patógeno/inmunología , Leucocitos/inmunología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones Endogámicos , Neumonía Bacteriana/genética , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/microbiologíaRESUMEN
Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37 °C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37 °C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.