interleukin-8 and Pseudomonas-Infections

Long-term administration of azithromycin (AZM) in children with cystic fibrosis (CF) has improved outcomes. However, the doses and schedule of administration are not very well studied in children with CF.. A randomized controlled trial was conducted to compare the effect of two doses of azithromycin (5mg/kg/day and 15mg/kg/day) on FEV(1) and pulmonary exacerbations in children with cystic fibrosis. Enrolled children were randomly allocated to receive daily azithromycin (5mg/kg/day or 15mg/kg/day) for 6months. Clinical assessment and FEV(1) measurement were performed monthly.. 56 children (28 in high dose group and 28 in low dose group) were enrolled. 47 (24 and 23 children in low and high dose groups) completed 12months of follow up. There was no difference in clinical scores, FEV(1), pulmonary exacerbation rates between two groups at baseline, 6months and at 12months. Per protocol analysis revealed that pulmonary exacerbation increased after discontinuing AZM and there was significantly more increase after 12months of enrolment in children getting high dose azithromycin. There was no improvement in FEV(1) in either group at the end of treatment period. Children tolerated daily low as well as high dose AZM well for 6months. There was no significant side effect of azithromycin.. In this randomized controlled trial, we did not find differences in the effect of 2 doses (5mg/kg/day or 15mg/kg/day) of AZM on change in percentage predicted FEV(1), clinical scores, Pseudomonas colonization rates, pulmonary exacerbations and need for antibiotics. There was increase in exacerbations after stopping azithromycin in both the groups. Our results also suggest that the decrease in the incidence of LRTI persists only till 6months after discontinuing azithromycin.

Topics: Anti-Bacterial Agents; Azithromycin; Body Weight; Candidiasis; Child; Child, Preschool; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Spirometry; Sputum; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus pneumoniae; Treatment Outcome

Treatment strategies for cystic fibrosis (CF) lung disease include antibiotics, mucolytics, and anti-inflammatory therapies. Increasing evidence suggests that macrolide antibiotics might be beneficial in patients with CF.. To determine if an association between azithromycin use and pulmonary function exists in patients with CF.. A multicenter, randomized, double-blind, placebo-controlled trial conducted from December 15, 2000, to May 2, 2002, at 23 CF care centers in the United States.. Of the 251 screened participants with a diagnosis of CF, 185 (74%) were randomized. Eligibility criteria included age 6 years or older, infection with Pseudomonas aeruginosa for 1 or more years, and a forced expiratory volume in 1 second (FEV1) of 30% or more. Participants were stratified by FEV1 (> or =60% predicted vs <60% predicted), weight of less than 40 kg vs 40 kg or more, and CF center.. The active group (n = 87) received 250 mg (weight <40 kg) or 500 mg (weight > or =40 kg) of oral azithromycin 3 days a week for 168 days; placebo group (n = 98) received identically packaged tablets.. Change in FEV1 from day 0 to completion of therapy at day 168 and determination of safety. Secondary outcomes included pulmonary exacerbations and weight gain.. The azithromycin group had a mean 0.097-L (SD, 0.26) increase in FEV1 at day 168 compared with 0.003 L (SD, 0.23) in the placebo group (mean difference, 0.094 L; 95% confidence interval [CI], 0.023-0.165; P =.009). Nausea occurred in 17% more participants in the azithromycin group (P =.01), diarrhea in 15% more (P =.009), and wheezing in 13% more (P =.007). Participants in the azithromycin group had less risk of experiencing an exacerbation than participants in the placebo group (hazard ratio, 0.65; 95% CI, 0.44-0.95; P =.03) and weighed at the end of the study an average 0.7 kg more than participants receiving placebo (95% CI, 0.1-1.4 kg; P =.02).. Azithromycin treatment was associated with improvement in clinically relevant end points and should be considered for patients with CF who are 6 years or older and chronically infected with P aeruginosa.

Topics: Adolescent; Anti-Bacterial Agents; Azithromycin; Child; Chronic Disease; Cystic Fibrosis; Double-Blind Method; Female; Forced Expiratory Flow Rates; Hospitalization; Humans; Interleukin-8; Male; Pancreatic Elastase; Proportional Hazards Models; Pseudomonas Infections; Quality of Life; Treatment Outcome

Cystic fibrosis (CF) patients continue to have reservoirs of Pseudomonas aeruginosa infection in their sinuses and trachea after transplantation, and studies indicate that nontransplanted CF patients have high bronchoalveolar lavage (BAL) levels of proinflammatory factors, interleukin-8 (IL-8), and elastase and decreased airway lavage levels of IL-10. The aims of our study were to measure the IL-8 and IL-10 levels and elastase activity in the BAL of lung transplant patients, with correlation to microbiologic and pathologic findings, and to identify any differences in the findings between CF and non-CF patients. Fifty serial BAL samples were collected from 38 lung transplant recipients over 8 months. The BAL supernatant fluid was cultured for bacterial, viral, and fungal organisms. Histologic tissue analysis was performed as indicated. The fluid IL-10 and IL-8 levels were measured in duplicate using ELISA techniques. Elastase activity was measured using a colorimetric assay system. The mean IL-8, IL-10, and elastase levels for the group studied were 1894 pg/ml, 394 pg/ml, and 4.2 U/ml, respectively. The CF patients had significantly higher levels of IL-8, with a mean value of 4093 pg/ml (p < 0.02), and lower IL-10, mean 217 pg/ml (n = 9). Elastase activity correlated strongly with IL-8 level (p < 0.04). Pseudomonas growth was associated with higher elastase and IL-8 concentrations (p < 0.02). There was no association between allograft rejection and the markers studied. CF transplanted patients have higher airway lavage concentrations of IL-8 and elastase correlated to the presence of Pseudomonas in the lower airway. They also have lower BAL levels of anti-inflammatory cytokine IL-10.

Topics: Adult; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-10; Interleukin-8; Leukocyte Elastase; Lung Transplantation; Maxillary Sinus; Middle Aged; Pseudomonas Infections; Trachea

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

Topics: Adult; Cystic Fibrosis; Epithelium; Humans; Interleukin-8; Pseudomonas Infections; Quality of Life

Cytokines play a crucial role in the immune system's regulation by mediating protective responses to infections. anti-inflammatory and pro-inflammatory cytokines are in equilibrium. Therefore, any alteration in cytokine production or cytokine receptor expression might result in pathological illnesses and health issues. Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane regulator (CFTR) gene. Lung infection in these patients is related to chronic bacterial airway infection and inflammation, which is triggered by some inflammatory cytokines. Our goal was to compare the cytokine patterns in CF patient's serum and PBMCs caused by microbial pathogens that colonized their airways to controls.. ELISA and Real-time PCR were used to determine the levels of IL-10, IFN-γ, IL-4, TGF-β, IL-8, and IL-17 in serum and PBMC cells. Blood parameters in both patients and healthy people were studied.. An increase in IL-10, IFN-γ, IL-4 (p-v = 0.03, 0.024 and 0.003) levels and a decrease in IL-17 (p-v = 0.004) was found in Pseudomonas aeruginosa positive patients. There were no different in TGF-β and IL-8 (p-value = 0.778 and 0.903) in this patients. IL-10, IFN-γ, and IL-4 (p-value = 0.023, 0.001 and 0.002) levels were high in Staphylococcus aureus positive patients and TGF-β, IL-17, and IL-8 (p-value = 0.085, 0.167 and 0.362) were not significantly different in the patient and control groups. IFN-γ and IL-4 levels were higher in patients without infection who had normal microbiota (p-v = 0.002 and 0.024). In patients with P. aeruginosa, WBC and platelets increased, and MCH and MCV decreased. Patients with normal microbiota had less MCV.. According to our research, patients with P. aeruginosa, S. aureus, and normal microbiota are exposed to cytokine alterations and changes in blood factors. The link between the CF patient's airway microbiota and the kind of generated cytokines might lead to the modulation of inflammatory cytokines alone or in combination with antibiotics, reducing disease-causing effects while avoiding drug resistance.

Topics: Anti-Bacterial Agents; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Humans; Interleukin-10; Interleukin-17; Interleukin-4; Interleukin-8; Leukocytes, Mononuclear; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Cytokine; Staphylococcus aureus; Transforming Growth Factor beta

To explore the clinical benefits of azithromycin in the treatment of Pseudomonas aeruginosa induced bronchiectasis and to evaluate its effect on MUC5AC. From April 2018 to June 2020, 160 patients with bronchiectasis due to Pseudomonas aeruginosa infection were selected. The patients were divided into a control groupand an azithromycin group. Statistics of patients' general clinical data, lung function indexes, sputum volume, oxidative stress level, Bhalla score before and after treatment; Western blot analysis of MUC5AC expression; RT-PCR analysis of TNF-α, IL-8, IL- 1β mRNA expression. The mRNA expression of TNF-α, IL-8 and IL-1β in the azithromycin group was lower than that in the control group (P<0.05). After treatment, the protein expression of MUC5AC in the azithromycin group was lower than that in the control group (P<0.05). The improvement rate in the azithromycin group was significantly higher than that in the control group (P<0.05). The azithromycin group had a lower lung infection rate than the control group (P<0.05). The azithromycin group had a lower dyspnea rate than the control group (P<0.05). Azithromycin treatment has certain clinical benefits for patients with bronchiectasis induced by Pseudomonas aeruginosa and inhibits the MUC5AC expression.

Topics: Adult; Anti-Bacterial Agents; Azithromycin; Bronchiectasis; Dyspnea; Female; Humans; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Mucin 5AC; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Messenger; Tumor Necrosis Factor-alpha

Isoflurane and sevoflurane are volatile anesthetics (VA) widely used in clinical practice to provide general anesthesia. We and others have previously shown that VAs have immunomodulatory effects and may have a significant impact on the progression of disease states. Flagellin is a component of Gram negative bacteria and plays a significant role in the pathophysiology of bacterial pneumonia through its binding to Toll-like Receptor 5 (TLR5). Our results showed that VAs, not an intravenous anesthetic, significantly attenuated the activation of TLR5 and the release of the neutrophil chemoattractant IL-8 from lung epithelial cells. Furthermore, flagellin-induced lung injury was significantly attenuated by VAs by inhibiting neutrophil migration to the bronchoalveolar space. The lungs of cystic fibrosis (CF) patients are highly colonized by Pseudomonas aeruginosa, which causes inflammation. The retrospective study of oxygenation in patients with CF who had received VA versus intravenous anesthesia suggested that VAs might have the protective effect for gas exchange. To understand the interaction between VAs and TLR5, a docking simulation was performed, which indicated that isoflurane and sevoflurane docked into the binding interphase between TLR5 and flagellin.

Topics: Anesthetics, Inhalation; Animals; Cell Line, Tumor; Cystic Fibrosis; Epithelial Cells; Female; Flagellin; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Isoflurane; Lung; Male; Mice; Molecular Docking Simulation; Neutrophils; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Retrospective Studies; Sevoflurane; Toll-Like Receptor 5

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD-CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD-CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm-forming bacterial strains. Forty-one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C-reactive protein (CRP) and NLR (neutrophil-lymphocyte ratio) were examined as representative blood-based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil-derived defence proteins. Isolated sputum bacteria were identified and their biofilm-forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm-forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)-8 and low concentrations of IL-6 and IL-10. The low concentration of sputum IL-6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL-6, IL-8 and IL-10.

Topics: Adolescent; Adult; Biofilms; Biomarkers; C-Reactive Protein; Child; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Inflammation Mediators; Interleukin-10; Interleukin-6; Interleukin-8; Leukocyte Elastase; Lymphocyte Count; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Sputum; Young Adult

High concentrations of particulate matter less than 2.5 μm in diameter (PM2.5) in poultry houses is an important cause of respiratory disease in animals and humans.. In this study, we focused on the synergistic induction of inflammatory injury in the respiratory system and the related molecular mechanisms induced by PM2.5 and. High-throughput 16S rDNA sequence analysis was used for characterizing the bacterial diversity and relative abundance of the PM2.5 samples, and the effects of PM2.5 and. Sequencing results indicated that the PM2.5 in poultry houses contained a high abundance of potentially pathogenic genera, such as. The results confirmed that poultry house PM2.5 in combination with

Topics: Animals; Body Weight; Interleukin-6; Interleukin-8; Mice; Mice, Inbred C57BL; NF-kappa B p50 Subunit; Particulate Matter; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Specific Pathogen-Free Organisms; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.

Topics: A549 Cells; Aryldialkylphosphatase; beta-Defensins; Cystic Fibrosis; Endoplasmic Reticulum Stress; Epithelial Cells; Host-Pathogen Interactions; Humans; Immunity, Innate; Interleukin-8; Mitochondria; Pioglitazone; PPAR gamma; Pseudomonas aeruginosa; Pseudomonas Infections; Transcription Factor CHOP; Unfolded Protein Response

The human pathogen

Topics: beta-Defensins; Bifidobacterium longum; Cell Line, Tumor; Humans; Inflammation Mediators; Interleukin-8; Intestinal Mucosa; Lacticaseibacillus rhamnosus; Nod1 Signaling Adaptor Protein; Pore Forming Cytotoxic Proteins; Probiotics; Proto-Oncogene Proteins c-akt; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Small Interfering; Signal Transduction

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.

Topics: Bronchi; Cystic Fibrosis; Epithelial Cells; Humans; Interleukin-8; NF-kappa B; Prevotella; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Toll-Like Receptors

Children with CF are insulin deficient from infancy but very little is known about the impact of glucose abnormalities in early life. We aimed to identify and describe interstitial glucose levels in CF children <6 years and to evaluate the association with pulmonary infection and inflammation.. We assessed 18 children (5 females) with median age of 3.2 years (range 0·9-5.5) with Continuous Glucose Monitoring for 3 days. Bronchoalveolar lavage (BAL) fluid was cultured for known pathogenic microbial agents and assessed for total white blood cells, percentage of neutrophils and IL-8 level.. Children with CF frequently demonstrate elevated SG levels before age 6 years, which are associated with increased pulmonary inflammation and Pseudomonas aeruginosa infection. Transient SG elevations into the diabetic range (≥11.1 mmol/L) were identified in children from 1 year of age.

Topics: Australia; Blood Glucose; Bronchoalveolar Lavage Fluid; Child, Preschool; Correlation of Data; Cystic Fibrosis; Female; Humans; Hyperglycemia; Infant; Interleukin-8; Leukocyte Count; Male; Monitoring, Physiologic; Neutrophils; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections

Aroylated phenylenediamines (APDs) are novel inducers of innate immunity enhancing cathelicidin gene expression in human bronchial epithelial cell lines. Here we present two newly developed APDs and aimed at defining the response and signaling pathways for these compounds with reference to innate immunity and antimicrobial peptide (AMP) expression. Induction was initially defined with respect to dose and time and compared with the APD Entinostat (MS-275). The induction applies to several innate immunity effectors, indicating that APDs trigger a broad spectrum of antimicrobial responses. The bactericidal effect was shown in an infection model against Pseudomonas aeruginosa by estimating bacteria entering cells. Treatment with a selected APD counteracted Pseudomonas mediated disruption of epithelial integrity. This double action by inducing AMPs and enhancing epithelial integrity for one APD compound is unique and taken as a positive indication for host directed therapy (HDT). The APD effects are mediated through Signal transducer and activator of transcription 3 (STAT3) activation. Utilization of induced innate immunity to fight infections can reduce antibiotic usage, might be effective against multidrug resistant bacteria and is in line with improved stewardship in healthcare.

Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Benzamides; Bronchi; Cathelicidins; Cell Line; Cell Survival; Epithelial Cells; Gene Expression; Humans; Immunity, Innate; Interleukin-8; Phenylenediamines; Pseudomonas aeruginosa; Pseudomonas Infections; Pyridines; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Topics: Aminophenols; Aminopyridines; Benzodioxoles; Bronchi; Cells, Cultured; Chloride Channel Agonists; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-8; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolones

The aim of the present study was to explore the effect of overexpressed suppressor of cytokine signaling‑3 (SOCS3) on T-helper (Th)17 cell responses and neutrophilic airway inflammation in mice with chronic Pseudomonas aeruginosa (PA) infections. SOCS3 expression was enhanced via the administration of tail vein injections of therapeutic lentivirus in mice with chronic PA lung infections. SOCS3 expression in the blood and lung tissue was assessed using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Total and differential cell numbers and myeloperoxidase levels in the bronchoalveolar lavage (BAL) fluid were assessed, as well as the number of bacterial colonies in the lungs. Histological analysis of lung tissue was performed using hematoxylin and eosin staining and phosphorylated‑signal transducer and activator of transcription‑3 (p‑STAT3) expression was measured by western blot analysis and immunohistochemistry. The expression of STAT3 mRNA and retinoid‑related orphan receptor (ROR)γt were measured by RT‑qPCR. The percentage of interleukin (IL)‑17+ cells among cluster of differentiation (CD)4+ cells was calculated using flow cytometry and levels of IL‑17A and IL‑6 were assessed by ELISA. The expression of SOCS3 was significantly increased in CD4+ T cells following lentivirus injection and the inflammation of neutrophilic airways was notably ameliorated. Enhanced SOCS3 expression was associated with a significant decrease in the expression of p‑STAT3 and RORγt in CD4+ T cells. Additionally, the percentage of IL‑17+ cells among CD4+ T cells and the IL‑17 contents in the BAL fluid were significantly decreased. Lentivirus‑mediated overexpression of SOCS3 was revealed to ameliorate neutrophilic airway inflammation by inhibiting pulmonary Th17 responses in mice with chronic PA lung infections.

Topics: Animals; Chronic Disease; Female; Genetic Therapy; Interleukin-17; Interleukin-8; Lentivirus; Lung; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Suppressor of Cytokine Signaling 3 Protein; Th17 Cells; Up-Regulation

In children with cystic fibrosis (CF) the associations between oropharyngeal swabs (OPSs) for detection of

Topics: Australia; Biomarkers; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child, Preschool; Cystic Fibrosis; Disease Progression; Female; Humans; Infant; Interleukin-8; Leukocyte Elastase; Male; Oropharynx; Predictive Value of Tests; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; ROC Curve; Severity of Illness Index; Tomography, X-Ray Computed

IL-8-dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases, including chronic obstructive pulmonary disease, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible, pathological changes associated with elevated levels of IL-8 in the lung. To better understand the duality of IL-8-dependent host immunity to bacterial infection and lung pathology, we expressed human IL-8 transgenically in murine bronchial epithelium, and investigated the impact of overexpression on lung bacterial clearance, host immunity, and lung pathology and function. Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation and activation, and chemotaxis. There was enhanced protection against challenge with Pseudomonas aeruginosa, and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL-2, and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen OprF, indicating a regulatory T-cell phenotype. However, this enhanced bacterial immunity came at a high price of progressive lung remodeling, with increased inflammation, mucus hypersecretion, and fibrosis. There was increased expression of Ccl3 and reduced expression of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all of which resulted in impaired lung function with reduced compliance, increased resistance, and bronchial hyperreactivity as measured by whole-body plethysmography. These results show that IL-8 overexpression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodeling, and damaged tight junctions, leading to impaired lung function.

Topics: Animals; Chronic Disease; Humans; Immunity, Innate; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Fibrosis

Cystic fibrosis (CF) lung infection is a complex condition where opportunistic pathogens and defective immune system cooperate in developing a constant cycle of infection and inflammation. The major pathogen, Pseudomonas aeruginosa, secretes a multitude of virulence factors involved in host immune response and lung tissue damage. In this study, we examined the possible anti-inflammatory effects of molecules inhibiting P. aeruginosa virulence factors.. Pyocyanin, pyoverdine and proteases were measured in bacterial culture supernatant from different P. aeruginosa strains. Inhibition of virulence factors by sub-inhibitory concentrations of clarithromycin and by protease inhibitors was evaluated. Lung inflammatory response was monitored by in vivo bioluminescence imaging in wild-type and CFTR-knockout mice expressing a luciferase gene under the control of a bovine IL-8 promoter.. The amount of proteases, pyocyanin and pyoverdine secreted by P. aeruginosa strains was reduced after growth in the presence of a sub-inhibitory dose of clarithromycin. Intratracheal challenge with culture supernatant containing bacteria-released products induced a strong IL-8-mediated response in mouse lungs while lack of virulence factors corresponded to a reduction in bioluminescence emission. Particularly, sole inactivation of proteases by inhibitors Ilomastat and Marimastat also resulted in decreased lung inflammation.. Our data support the assumption that virulence factors are involved in P. aeruginosa pro-inflammatory action in CF lungs; particularly, proteases seem to play an important role. Inhibition of virulence factors production and activity resulted in decreased lung inflammation; thus, clarithromycin and protease inhibitors potentially represent additional therapeutic therapies for P. aeruginosa-infected patients.

Topics: Animals; Bacterial Proteins; Clarithromycin; Cystic Fibrosis; Disease Models, Animal; Female; Humans; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Pseudomonas aeruginosa; Pseudomonas Infections; Virulence Factors

Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulated their respective levels, suggesting its regulation of lung inflammation. Surprisingly, P. aeruginosa flagellin, and not its lipopolysaccharide moiety, was the primary inducer of inflammatory cytokines in lung cells, which were similarly down-regulated by TP359. Blocking of TLR5, the putative flagellin receptor, completely abrogated the capacity of infected lung cells to secrete cytokines, underscoring that TP359 regulates inflammation via the TLR5-dependent signaling pathway. Downstream pathway-specific inhibition studies further revealed that the MAPK pathway, essentially p38 and JNK are necessary for induction of P. aeruginosa elicited inflammatory cytokines and their down-regulation by TP359. Collectively, our data provides evidence to support exploring the relevancy of TP359 as an anti-microbial and anti-inflammatory agent against P. aeruginosa for clinical applications.

Topics: A549 Cells; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Blotting, Western; Dose-Response Relationship, Drug; Humans; Interleukin-6; Interleukin-8; MAP Kinase Signaling System; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Toll-Like Receptor 5; Tumor Necrosis Factor-alpha

Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P. aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.

Topics: 4-Butyrolactone; Antioxidant Response Elements; Biofilms; Cell Line; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Heme Oxygenase-1; Homoserine; Humans; Interleukin-8; Lung; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Pseudomonas aeruginosa; Pseudomonas Infections; Quorum Sensing

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions.

Topics: Bacterial Proteins; Endopeptidases; Epithelial Cells; Flagella; Flagellin; Humans; Immune Evasion; Interleukin-8; Metalloendopeptidases; Pseudomonas aeruginosa; Pseudomonas Infections; Serine Endopeptidases; Virulence Factors

During the early developmental stage of salmonids, high mortality occurs largely as a result of pathogens. These cause low immune competence in fry, producing disease, decreasing production and finally leading to economic losses. Therefore, the aim of this study was to characterise the developmental stages in which rainbow trout acquires immune response capability when challenged with LPS from Pseudomona aeruginosa for 8 h, studying the hepcidin, cathelicidin-1 and IL-8. Total RNA was extracted from fry at 34, 42, 56 and 66 days post hatching (dph). Hepcidin and cathelicidin-1 transcripts were detected only at days 34 and 42, whereas the IL-8 transcript was detected from day 34 to day 66. To analyse the protein expression in the fry, polyclonal anti-peptide antibodies were generated in rabbit. These three immune sera demonstrated the ability to recognise the whole molecule in biological samples. Immunofluorescence showed that skin, gills and intestine mainly responded to the LPS challenge, indicating that these portals of pathogen entry are capturing LPS. This study constitutes a valuable approach, since it has the potential to identify molecules with biological activity that can be used to evaluate the status of fry in culture.

Topics: Animals; Antibodies; Antimicrobial Cationic Peptides; Biomarkers; Cathelicidins; Cells, Cultured; Fish Diseases; Fish Proteins; Gene Expression Regulation, Developmental; Hepcidins; Immunity, Innate; Interleukin-8; Lipopolysaccharides; Oncorhynchus mykiss; Pseudomonas aeruginosa; Pseudomonas Infections

The severity of cystic fibrosis (CF) is associated with classes of mutations in the CFTR gene (cystic fibrosis transmembrane regulator), physical environment and modifier genes interaction. The IL8 gene (interleukin 8), according to its respective polymorphisms, influences inflammatory responses. This study analyzed IL8 gene polymorphisms (rs4073, rs2227306 and rs2227307), by means of PCR/RFLP, and their association with pulmonary function markers and clinical severity scores in 186 patients with CF, considering the CFTR genotype. There was an association between rs2227307 and precocity of the disease. The severity of lung disease was associated with the following markers: transcutaneous arterial hemoglobin oxygen saturation (SaO2) (regardless of CFTR genotype, for the polymorphisms rs4073, rs2227306 and rs2227307); mucoid Pseudomonas aeruginosa (regardless of CFTR genotype, for the polymorphisms rs2227306 and rs2227307). Pulmonary function markers (SaO2 and spirometric variables) and clinical severity scores were also associated with IL8 gene polymorphisms. This study identified the IL8 gene, represented by rs4073 and rs2227306 polymorphisms, and particularly the rs2227307 polymorphism, as potentiating factors for the degree of variability in the severity of CF, especially in pulmonary clinical manifestation correlated with increased morbidity and mortality.

Topics: Adolescent; Child; Child, Preschool; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Female; Genetic Association Studies; Genotype; Hemoglobins; Humans; Inflammation; Interleukin-8; Lung Diseases; Male; Oxygen; Polymorphism, Single Nucleotide; Pseudomonas aeruginosa; Pseudomonas Infections; Severity of Illness Index

Pulmonary infections with Pseudomonas aeruginosa are a serious clinical problem and are often lethal. Because many strains of P. aeruginosa are resistant to antibiotics, therapeutic options are limited. Neutrophils play an important role in the host's early acute defense against pulmonary P. aeruginosa. Therefore, it is important to define the mechanisms by which P. aeruginosa interacts with host cells, particularly neutrophils.. Here, we report that pyocyanin, a membrane-permeable pigment and toxin released by P. aeruginosa, induces the death of wild-type neutrophils; its interaction with the mitochondrial respiratory chain results in the release of reactive oxygen species (ROS), the activation of mitochondrial acid sphingomyelinase, the formation of mitochondrial ceramide, and the release of cytochrome c from mitochondria. A genetic deficiency in acid sphingomyelinase prevents both the activation of this pathway and pyocyanin-induced neutrophil death. This reduced death, on the other hand, is associated with an increase in the release of interleukin-8 from pyocyanin-activated acid sphingomyelinase-deficient neutrophils but not from wild-type cells.. These studies identified the mechanisms by which pyocyanin induces the release of mitochondrial ROS and by which ROS induce neutrophil death via mitochondrial acid sphingomyelinase.. These findings demonstrate a novel mechanism of pyocyanin-induced death of neutrophils and show how this apoptosis balances innate immune reactions.

Topics: Animals; Cell Death; Cell Line; Ceramides; Cystic Fibrosis; Cytochromes c; HL-60 Cells; Humans; Interleukin-8; Jurkat Cells; Liver; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Rats; Reactive Oxygen Species; Sphingomyelin Phosphodiesterase

Glucocorticoids (GCs) are anti-inflammatory agents, but their use in cystic fibrosis (CF) is controversial. In CF, the early colonization with Pseudomonas aeruginosa is mainly due to nonmucoid strains that can internalize, and induce apoptosis in the epithelial cells. Uptake of P. aeruginosa by the epithelial cells and subsequent apoptosis may prevent colonization of P. aeruginosa in CF airways. In the airway epithelia, several other biological effects, including an anti-secretory role by decreasing intracellular Ca(2+) concentration have been described for this anti-inflammatory drug. However, the effects of GCs on the nonmucoid P. aeruginosa internalization and intracellular Ca(2+) in CF bronchial epithelial cells have not been evaluated.. We used cultured human CF bronchial airway epithelial cell (CFBE) monolayers to determine P. aeruginosa internalization, apoptosis, and intracellular Ca(2+)concentration in CF bronchial epithelial cells. Cells were treated with IL-6, IL-8, dexamethasone, betamethasone, or budesonide.. GCs in co-treatments with IL-6 reversed the effect of IL-6 by decreasing the internalization of P. aeruginosa in the CFBE cells. GCs decreased the extent of apoptosis in CFBE cells infected with internalized P. aeruginosa, and increased the intracellular Ca(2+) concentration.. These findings suggest that if internalization of P. aeruginosa reduces infection, GC therapy would increase the risk of pulmonary infection by decreasing the internalization of P. aeruginosa in CF cells, but GCs may improve airway hydration by increasing the intracellular Ca(2+) concentration. Whether the benefits of GC treatment outweigh the negative effects is questionable, and further clinical studies need to be carried out.

Topics: Anti-Inflammatory Agents; Apoptosis; Bronchi; Budesonide; Calcium; Cell Line; Cells, Cultured; Cystic Fibrosis; Dexamethasone; Epithelial Cells; Epithelium; Glucocorticoids; Humans; Interleukin-6; Interleukin-8; Pseudomonas aeruginosa; Pseudomonas Infections

Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods.. Lung inflammation was induced using the culture supernatants from two Pseudomonas aeruginosa clinical strains, VR1 and VR2, isolated from patients affected by cystic fibrosis and showing different phenotypes in terms of motility, colony characteristics and biofilm production as well as pyoverdine and pyocyanine release. More interesting, the strains differ also for the presence in supernatants of metalloproteases, a family of virulence factors with known pro-inflammatory activity. We have evaluated the benefit of using a mouse model, transiently expressing the luciferase reporter gene under the control of an heterologous IL-8 bovine promoter, to detect and monitoring lung inflammation.. In vivo imaging indicated that VR1 strain, releasing in its culture supernatant metalloproteases and other virulence factors, induced lung inflammation while the VR2 strain presented with a severely reduced pro-inflammatory activity. The bioluminescence signal was detectable from 4 to 48 h after supernatant instillation. The animal model was also used to test the anti-inflammatory activity of azithromycin (AZM), an antibiotic with demonstrated inhibitory effect on the synthesis of bacterial exoproducts. The inflammation signal in mice was in fact significantly reduced when bacteria grew in the presence of a sub-lethal dose of AZM causing inhibition of the synthesis of metalloproteases and other bacterial elements. The in vivo data were further supported by quantification of immune cells and cytokine expression in mouse broncho-alveolar lavage samples.. This experimental animal model is based on the transient transduction of the bovine IL-8 promoter, a gene representing a major player during inflammation, essential for leukocytes recruitment to the inflamed tissue. It appears to be an appropriate molecular read-out for monitoring the activation of inflammatory pathways caused by bacterial virulence factors. The data presented indicate that the model is suitable to functionally monitor in real time the lung inflammatory response facilitating the identification of bacterial factors with pro-inflammatory activity and the evaluation of the anti-inflammatory activity of old and new molecules for therapeutic use.

Topics: Animals; Azithromycin; Bronchoalveolar Lavage Fluid; Cattle; Cytokines; Diagnostic Imaging; Female; Humans; Interleukin-8; Mice, Inbred BALB C; Mice, Transgenic; Peptide Hydrolases; Phenotype; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Virulence Factors

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

Topics: Cell Line; Epithelial Cells; Gene Expression; Hemolysin Proteins; Humans; Interleukin-8; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pseudomonas aeruginosa; Pseudomonas Infections; Reproducibility of Results; Respiratory Mucosa; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 1; Toll-Like Receptor 2

Measures of ventilation distribution are promising for monitoring early lung disease in cystic fibrosis (CF). This study describes the cross-sectional and longitudinal impacts of pulmonary inflammation and infection on ventilation homogeneity in infants with CF.Infants diagnosed with CF underwent multiple breath washout (MBW) testing and bronchoalveolar lavage at three time points during the first 2 years of life.Measures were obtained for 108 infants on 156 occasions. Infants with a significant pulmonary infection at the time of MBW showed increases in lung clearance index (LCI) of 0.400 units (95% CI 0.150-0.648; p=0.002). The impact was long lasting, with previous pulmonary infection leading to increased ventilation inhomogeneity over time compared to those who remained free of infection (p<0.05). Infection with Haemophilus influenzae was particularly detrimental to the longitudinal lung function in young children with CF where LCI was increased by 1.069 units for each year of life (95% CI 0.484-1.612; p<0.001).Pulmonary infection during the first year of life is detrimental to later lung function. Therefore, strategies aimed at prevention, surveillance and eradication of pulmonary pathogens are paramount to preserve lung function in infants with CF.

Topics: Breath Tests; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Disease Progression; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Infant, Newborn; Interleukin-8; Longitudinal Studies; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Aspergillosis; Pulmonary Ventilation; Staphylococcal Infections; Staphylococcus aureus

Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage.. Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined.. IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B.. These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Topics: Adult; Antibodies, Bacterial; Biomarkers; Cathepsin B; Cathepsins; Cystic Fibrosis; Female; Humans; Immunoglobulin A; Immunoglobulin G; Interleukin-6; Interleukin-8; Kaplan-Meier Estimate; Leukocyte Elastase; Male; Multivariate Analysis; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Tumor Necrosis Factor, Type I; Sputum; Tumor Necrosis Factor-alpha; Young Adult

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.. The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.. We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.. The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.. Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.

Topics: Animals; Anti-Inflammatory Agents; Antifungal Agents; Blotting, Western; Cell Proliferation; Ceramides; Chromatography, Liquid; Cystic Fibrosis; Drug Carriers; Enzyme-Linked Immunosorbent Assay; Fatty Acids, Monounsaturated; Female; Humans; Interleukin-6; Interleukin-8; Male; Mice; Mice, Inbred CFTR; Nanoparticles; Pseudomonas aeruginosa; Pseudomonas Infections; Real-Time Polymerase Chain Reaction; Respiratory Mucosa; Respiratory Tract Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids

The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated.. Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared.. AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species.. The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Topics: Animals; Biofilms; Cell Line; Cystic Fibrosis; Epidemics; Epithelial Cells; Humans; Interleukin-8; Larva; Moths; Pancreatic Elastase; Phenotype; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Streptococcal Infections; Streptococcus; Virulence; Virulence Factors

Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa.. Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry.. MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa.. MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

Topics: Complement Activation; Complement System Proteins; Epithelial Cells; Epithelium, Corneal; Gene Expression Regulation; Hep G2 Cells; Humans; Interleukin-6; Interleukin-8; Keratitis; Mannose-Binding Lectin; Pseudomonas aeruginosa; Pseudomonas Infections

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3'-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3'-UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93.

Topics: 3' Untranslated Regions; Bronchi; Cell Line; Cystic Fibrosis; Down-Regulation; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; MicroRNAs; Minichromosome Maintenance Complex Component 7; NF-kappa B; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Messenger; Transcription, Genetic; Up-Regulation

The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation.. Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files.. Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration.. This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject.

Topics: Adult; Biomarkers; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Flagellin; Humans; Interleukin-8; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Insufficiency; Sampling Studies; Severity of Illness Index; Sputum

Monophosphoryl lipid A (MPLA) is a lipopolysaccharides (LPS) derivative associated with neutrophil-dependent anti-inflammatory outcomes in animal models of sepsis. Little is known about the effect of MPLA on neutrophil function. This study sought to test the hypothesis that MPLA would reduce release of cytotoxic mediators from neutrophils without impairing bacterial clearance. Neutrophils were isolated from whole blood of healthy volunteers. The effects of MPLA and LPS on autologous serum-opsonised Pseudomonas aeruginosa killing by neutrophils and phagocytosis of autologous serum-opsonised zymosan were examined. Neutrophil oxidative burst, chemotaxis, enzyme and cytokine release as well as Toll-like receptor 4 (TLR4) expression were assessed following exposure to LPS or MPLA. LPS, but not MPLA, induced significant release of superoxide and myeloperoxidase from neutrophils. However, MPLA did not impair neutrophil capacity to ingest microbial particles and kill P. aeruginosa efficiently. MPLA was directly chemotactic for neutrophils, involving TLR4, p38 mitogen-activated protein kinase and tyrosine and alkaline phosphatases. LPS, but not MPLA, impaired N-formyl-methionyl-leucyl phenylalanine-directed migration of neutrophils, increased surface expression of TLR4, increased interleukin-8 release and strongly activated the myeloid differentiation primary response 88 pathway. Phosphoinositide 3-kinase inhibition significantly augmented IL-8 release from MPLA-treated neutrophils. The addition of MPLA to LPS-preincubated neutrophils led to a significant reduction in LPS-mediated superoxide release and TLR4 surface expression. Collectively, these findings suggest that MPLA directs efficient chemotaxis and bacterial killing in human neutrophils without inducing extracellular release of cytotoxic mediators and suggest that MPLA warrants further attention as a potential therapeutic in human sepsis.

Topics: Alkaline Phosphatase; Humans; Interleukin-8; Lipid A; Lipopolysaccharides; Myeloid Differentiation Factor 88; Neutrophils; p38 Mitogen-Activated Protein Kinases; Phagocytosis; Phosphatidylinositol 3-Kinases; Protein Tyrosine Phosphatases; Pseudomonas aeruginosa; Pseudomonas Infections; Signal Transduction; Superoxides; Toll-Like Receptor 4

The statin family of cholesterol-lowering drugs is known to have pleiotropic properties which include anti-inflammatory and immunomodulatory effects. Statins exert their pleiotropic effects by altering expression of human immune regulators including pro-inflammatory cytokines. Previously we found that statins modulate virulence phenotypes of the human pathogen Pseudomonas aeruginosa, and sought to investigate if simvastatin could alter the host response to this organism in lung epithelial cells. Simvastatin increased the expression of the P. aeruginosa target genes KLF2, KLF6, IL-8 and CCL20. Furthermore, both simvastatin and P. aeruginosa induced alternative splicing of KLF6. The novel effect of simvastatin on wtKLF6 expression was found to be responsible for induction of the KLF6 regulated genes CCL20 and iNOS. Simvastatin also increased the adhesion of P. aeruginosa to host cells, without altering invasion or cytotoxicity. This study demonstrated that simvastatin had several novel effects on the pulmonary cellular immune response.

Topics: Alternative Splicing; Cell Line; Chemokine CCL20; Gene Expression Regulation; Humans; Immunity, Cellular; Interleukin-8; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Lung; Proto-Oncogene Proteins; Pseudomonas aeruginosa; Pseudomonas Infections; Simvastatin

Bone morphogenetic protein 4 (BMP4) is a multifunctional growth factor that belongs to the TGF-β superfamily. The role of BMP4 in lung diseases is not fully understood. Here, we demonstrate that BMP4 was upregulated in lungs undergoing lipopolysaccharide (LPS)-induced inflammation, and in airway epithelial cells treated with LPS or TNF-α. BMP4 mutant (BMP4(+/-) ) mice presented with more severe lung inflammation in response to LPS or Pseudomonas aeruginosa, and lower bacterial load compared with that in BMP4(+/+) mice. Knockdown of BMP4 by siRNA increased LPS and TNF-α-induced IL-8 expression in 16HBE human airway epithelial cells and in primary human bronchial epithelial cells. Similarly, peritoneal macrophages from BMP4(+/-) mice produced greater levels of TNF-α and keratinocyte chemoattractant (KC) upon LPS treatment compared with cells from BMP4(+/+) mice. Administration of exogenous BMP4 attenuated the upregulation of TNF-α, IL-8, or KC induced by LPS and/or TNF-α in airway epithelial cells, and peritoneal macrophages. Finally, partial deficiency of BMP4 in BMP4(+/-) mice protected the animals from restrictive lung function reduction upon chronic LPS exposure. These results indicate that BMP4 plays an important anti-inflammatory role, controlling the strength and facilitating the resolution of acute lung inflammation; yet, BMP4 also contributes to lung function impairment during chronic lung inflammation.

Topics: Animals; Bone Morphogenetic Protein 4; Cells, Cultured; Epithelial Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Lipopolysaccharides; Lung; Lung Injury; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad Proteins; Tumor Necrosis Factor-alpha

Cystic fibrosis (CF) lung disease is characterised by chronic Pseudomonas aeruginosa infection and leukocyte infiltration. Chemokines recruit leukocytes to sites of infection. Gene expression analysis identified the chemokine CCL18 as upregulated in CF leukocytes. We hypothesised that CCL18 characterises infection and inflammation in patients with CF lung disease. Therefore, we quantified CCL18 protein levels in the serum and airway fluids of CF patients and healthy controls, and studied CCL18 protein production by airway cells ex vivo. These studies demonstrated that CCL18 levels were increased in the serum and airway fluids from CF patients compared with healthy controls. Within CF patients, CCL18 levels were increased in P. aeruginosa-infected CF patients. CCL18 levels in the airways, but not in serum, correlated with severity of pulmonary obstruction in CF. Airway cells isolated from P. aeruginosa-infected CF patients produced significantly higher amounts of CCL18 protein compared with airway cells from CF patients without P. aeruginosa infection or healthy controls. Collectively, these studies show that CCL18 levels characterise chronic P. aeruginosa infection and pulmonary obstruction in patients with CF. CCL18 may, thus, serve as a potential biomarker and therapeutic target in CF lung disease.

Topics: Adolescent; Adult; Case-Control Studies; Chemokine CXCL1; Chemokine CXCL2; Chemokines, CC; Child; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Leukocytes; Male; Pseudomonas Infections; Sputum; Young Adult

Topics: Anthracenes; Bronchi; Cells, Cultured; Epithelial Cells; Flavonoids; Gene Expression; Humans; Interleukin-8; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Phosphorylation; Protein Kinase Inhibitors; Pseudomonas aeruginosa; Pseudomonas Infections; Pyrrolidines; RNA, Messenger; Thiocarbamates

Treatment of acute and chronic pulmonary infections caused by opportunistic pathogen Pseudomonas aeruginosa is limited by the increasing frequency of multidrug bacterial resistance. Here, we describe a novel adjunctive therapy in which administration of a mix of simple sugars-mannose, fucose, and galactose-inhibits bacterial attachment, limits lung damage, and potentiates conventional antibiotic therapy. The sugar mixture inhibits adhesion of nonmucoid and mucoid P. aeruginosa strains to bronchial epithelial cells in vitro. In a murine model of acute pneumonia, treatment with the sugar mixture alone diminishes lung damage, bacterial dissemination to the subpleural alveoli, and neutrophil- and IL-8-driven inflammatory responses. Remarkably, the sugars act synergistically with anti-Pseudomonas antibiotics, including β-lactams and quinolones, to further reduce bacterial lung colonization and damage. To probe the mechanism, we examined the effects of sugars in the presence or absence of antibiotics during growth in liquid culture and in an ex vivo infection model utilizing freshly dissected mouse tracheas and lungs. We demonstrate that the sugar mixture induces rapid but reversible formation of bacterial clusters that exhibited enhanced susceptibility to antibiotics compared with individual bacteria. Our findings reveal that sugar inhalation, an inexpensive and safe therapeutic, could be used in combination with conventional antibiotic therapy to more effectively treat P. aeruginosa lung infections.

Topics: Animals; Anti-Bacterial Agents; Bacterial Adhesion; Bronchi; Carbohydrates; Cells, Cultured; Cystic Fibrosis; Fucose; Galactose; Humans; Interleukin-8; Lung Injury; Male; Mannose; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Pneumonia, Bacterial; Polysaccharides; Pseudomonas aeruginosa; Pseudomonas Infections; Trachea

Cystic fibrosis (CF) is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which cause a massively proinflammatory phenotype in the CF airway. The chemical basis of the inflammation is hyperproduction of interleukin-8 (IL-8) by CF airway epithelial cells, based on both an intrinsic mutation-dependent mechanism and by infection. In infection-free, cultured CF lung epithelial cells, high levels of the microRNA (miR), miR-155, is responsible for hyperexpression of IL-8. However, whether infection-induced IL-8 expression in CF cells is also mediated by miR-155 is not known. We have hypothesized that miR-155 might be a general mediator of enhanced IL-8 expression in CF cells, either in response to other cytokine/chemokine mediators of inflammation, or after exposure to infectious agents. Here we find that a reduction in miR-155 accompanies suppression of IL-8 by either the anti-inflammatory cytokine IL-10 or by inhibition of ambient IL-1β with a neutralizing antibody. However, attempts to elevate IL-8 levels with either intact bacteria [viz. a mucoid strain of Pseudomonas aeruginosa (PA)], or lipopolysaccharide were unable to elevate miR-155 above its intrinsically high level in the absence of these agents. Instead, in response to PA infection, the CF cells modestly suppress the expression of miR-155, and express a novel set of miRs, including miR-215. We find that ex vivo CF lung epithelial cells also express high levels of both miR-155 and miR-215. The predicted module of infection-induced mRNA targets focuses on activation of the NFκB-signaling pathway, and on the proapoptotic p53-signaling pathway. We interpret these data to suggest that that CF lung epithelial cells respond to PA or bacterial cell products with a novel miR program that may carry with it serious challenges to survival.

Topics: Cell Line; Cystic Fibrosis; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Inflammation Mediators; Interleukin-10; Interleukin-1beta; Interleukin-8; Lung; MicroRNAs; Pseudomonas aeruginosa; Pseudomonas Infections

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa-induced IL-8 responses via the formation of caveolin-1-rich "signaling hubs" in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.

Topics: Animals; Caveolins; Cells, Cultured; Conjunctivitis, Bacterial; Epithelium, Corneal; Humans; Inflammation Mediators; Interleukin-8; Macrophage Migration-Inhibitory Factors; MAP Kinase Signaling System; Membrane Microdomains; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Primary Cell Culture; Protein Structure, Quaternary; Pseudomonas aeruginosa; Pseudomonas Infections

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.

Topics: Animals; Cell Line; Epithelial Cells; ErbB Receptors; Flagellin; Gene Expression Regulation; HEK293 Cells; Humans; Interleukin-8; Lung; Lung Diseases; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Mucin-1; Myeloid Differentiation Factor 88; NF-kappa B; Phosphorylation; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Signal Transduction; Toll-Like Receptor 5; Tumor Necrosis Factor-alpha

Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response.

Topics: Bacterial Proteins; Bacteriological Techniques; Cell Line; Culture Media, Conditioned; Cystic Fibrosis; Gene Expression; Humans; Inflammation Mediators; Interleukin-8; Metalloproteases; Neutrophils; Proteomics; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Interleukin-8A; RNA, Messenger; Systems Biology; Virulence

To determine whether repetitive airway Pseudomonas aeruginosa (Pa) infection results in lung inflammation and injury and, if so, whether these responses are affected by Muc1 mucin. Muc1 wild type (WT) and knockout (KO) mice were compared for body weights, lung inflammatory responses, and airspace enlargement using a chronic lung infection model system.. Mice were treated intranasally with Pa (10(7) CFU) on days 0, 4, 7 and 10. On day 14, body weights, inflammatory cell numbers in bronchoalveolar lavage fluid (BALF), and airspace enlargement were measured. Differences in inflammatory responses between groups were statistically analyzed by the Student's t test and ANOVA.. Muc1 WT mice exhibited mild degrees of both inflammation and airspace enlargement following repetitive airway Pa infection. However, Muc1 KO mice exhibited significantly decreased body weights, greater macrophage numbers in the BALF, and increased airspace enlargement compared with Muc1 WT mice.. This is the first report demonstrating that Muc1 deficiency can lead to lung injury during chronic Pa infection in mice. These results suggest that MUC1 may play a crucial role in the resolution of inflammation during chronic respiratory infections and that MUC1 dysfunction likely contributes to the pathogenesis of chronic inflammatory respiratory disease.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Disease Models, Animal; Interleukin-8; Lung Injury; Mice; Mice, Inbred C57BL; Mice, Knockout; Mucin-1; Pseudomonas aeruginosa; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Inflammation and infection are major determinants of disease severity and consequently, the quality of life and outcome for patients with cystic fibrosis (CF). Interleukin-1 beta (IL-1β) is a key inflammatory mediator. Secretion of biologically active IL-1β involves inflammasome-mediated processing. Little is known about the contribution of IL-1β and the inflammasomes in CF inflammatory disease. This study examines inflammasome-mediated IL-1β production in CF bronchial epithelial cell lines and human patients with CF.. Bronchial epithelial cell lines were found to produce negligible amounts of basal or stimulated IL-1β compared to hematopoeitic cells and they did not significantly upregulate caspase-1 activity upon inflammasome stimulation. In contrast, peripheral blood mononuclear cells (PBMCs) from both CF and healthy control subjects produced large amounts of IL-1β and strongly upregulated caspase-1 activity upon inflammasome stimulation. PBMCs from CF patients and controls displayed similar levels of caspase-1 activation and IL-1β production when stimulated with inflammasome activators. This IL-1β production was dependent on NF-κB activity and could be enhanced by priming with LPS. Finally, chemical inhibition of CFTR activity in control PBMCs and THP-1 cells did not significantly alter IL-1β or IL-8 production in response to P. aeruginosa.. Hematopoeitic cells appear to be the predominant source of inflammasome-induced pro-inflammatory IL-1β in CF. PBMCs derived from CF subjects display preserved inflammasome activation and IL-1β secretion in response to the major CF pathogen Pseudomonas aeruginosa. However, our data do not support the hypothesis that increased IL-1β production in CF subjects is due to an intrinsic increase in NF-κB activity through loss of CFTR function.

Topics: Analysis of Variance; Caspases; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Humans; Immunoblotting; Inflammasomes; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections

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

Topics: Animals; Cell Line; Female; Flagellin; Gene Expression Regulation; Humans; Interleukin-8; Mice; Mucin 5AC; Mucin-2; Mucus; Neuronal Apoptosis-Inhibitory Protein; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Signal Transduction; Toll-Like Receptor 5

In the clinical setting, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene enhance the inflammatory response in the lung to Pseudomonas aeruginosa (P. aeruginosa) infection. However, studies on human airway epithelial cells in vitro have produced conflicting results regarding the effect of mutations in CFTR on the inflammatory response to P. aeruginosa, and there are no comprehensive studies evaluating the effect of P. aeruginosa on the inflammatory response in airway epithelial cells with the ΔF508/ΔF508 genotype and their matched CF cell line rescued with wild-type (wt)-CFTR. CFBE41o- cells (ΔF508/ΔF508) and CFBE41o- cells complemented with wt-CFTR (CFBE-wt-CFTR) have been used extensively as an experimental model to study CF. Thus the goal of this study was to examine the effect of P. aeruginosa on gene expression and cytokine/chemokine production in this pair of cells. P. aeruginosa elicited a more robust increase in cytokine and chemokine expression (e.g., IL-8, CXCL1, CXCL2 and TNF-α) in CFBE-wt-CFTR cells compared with CFBE-ΔF508-CFTR cells. These results demonstrate that CFBE41o- cells complemented with wt-CFTR mount a more robust inflammatory response to P. aeruginosa than CFBE41o-ΔF508/ΔF508-CFTR cells. Taken together with other published studies, our data demonstrate that there is no compelling evidence to support the view that mutations in CFTR induce a hyperinflammatory response in human airway epithelial cells in vivo. Although the lungs of patients with CF have abundant levels of proinflammatory cytokines and chemokines, because the lung is populated by immune cells and epithelial cells there is no way to know, a priori, whether airway epithelial cells in the CF lung in vivo are hyperinflammatory in response to P. aeruginosa compared with non-CF lung epithelial cells. Thus studies on human airway epithelial cell lines and primary cells in vitro that propose to examine the effect of mutations in CFTR on the inflammatory response to P. aeruginosa have uncertain clinical significance with regard to CF.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Epithelial Cells; Humans; Interleukin-8; Lung; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Understanding the mechanisms underlying bacterial-driven inflammation and neutrophil recruitment is important to design better therapies for CF. CXCL8 is an important chemokine found elevated in the airways of CF patients that recruits neutrophil to sites of the inflammation.. Airway epithelial cells (AECs) expressing wild-type CFTR or CFTR∆F508 were challenged with Pseudomonas aeruginosa diffusible material (PsaDM) and the synthesis of CXCL8 was measured by quantitative real-time PCR and ELISA in absence or presence of MAPK inhibitors, TLR antagonists, glutathione and a NADPH oxidase inhibitor.. CFTR∆F508 AECs secrete more CXCL8 in response to PsaDM than their wild type counterpart, which can be reversed by addition of extracellular glutathione or incubating AECs at 27°C to favour folding and expression of CFTR at the cell membrane. Moreover, in CFTR∆F508 AECs, TLR2, TLR4 and TLR5 act redundantly to drive CXCL8 synthesis via the activation of NADPH oxidase.. These results demonstrate that NADPH oxidase is necessary for CXCL8 synthesis in response to TLRs activation by P. aeruginosa.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Gene Expression; Humans; Interleukin-8; MAP Kinase Signaling System; NADPH Oxidases; Pseudomonas aeruginosa; Pseudomonas Infections; Reactive Oxygen Species; Respiratory Mucosa; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptors

A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, micro-bleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from ΔF508 CFBE41o(-) bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, α(1)-antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o(-) cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.

Topics: Bronchoalveolar Lavage Fluid; Cell Line; Cystic Fibrosis; Epithelial Cells; ErbB Receptors; Heme; Hemoglobins; Humans; Interleukin-10; Interleukin-8; Leukocyte Elastase; Metalloendopeptidases; Myeloblastin; Peptide Hydrolases; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Serine Endopeptidases; Signal Transduction; Toll-Like Receptors

Intermittent viral exacerbations in patients with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with the majority of viral-related exacerbations in CF and require prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known.. Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). The need for dual oxidase 2, a component of NADPH oxidase, in RV-induced generation of H2O2 in CF cells was assessed using gene-specific siRNA.. Superinfection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2 in CF cells was sufficient for dispersal of planktonic bacteria from the biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and the interleukin-8 response in MPA- and RV-infected cells.. This study shows that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations.

Topics: Biofilms; Cell Differentiation; Cells, Cultured; Chemokines; Cystic Fibrosis; Epithelial Cells; Humans; Hydrogen Peroxide; Interleukin-8; Microscopy, Electron, Scanning; Picornaviridae Infections; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Rhinovirus; Superinfection; Viral Load

Pseudomonas fluorescens is an opportunistic indoor pathogen that can cause severe airway proinflammatory responses. Pulmonary epithelium, like other mucosal epithelial linings of the body, constitutes the first line of defense against airway microbial pathogens. Mucosal epithelial cells can be a sentinel of pathogenic bacteria via stimulation of specific cell surface receptors, including the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR). This study addressed the involvement of EGFR in airway epithelial pathogenesis by P. fluorescens. Human A549 pneumocytes showed prolonged production of proinflammatory interleukin-8 (IL-8) in response to infection with P. fluorescens, which was via the nuclear factor-kappa B (NF-κB) signaling pathway. Production of proinflammatory cytokine IL-8 was not mediated by P. fluorescens lipopolysaccharide, a representative TLR4 agonist, but was mediated through EGFR-linked signals activated by the opportunistic bacteria. Moreover, EGFR signals were involved in NF-κB signal-mediated production of proinflammatory cytokines. Along with persistent NF-κB activation, P. fluorescens enhanced the EGFR phosphorylation and subsequent activation of downstream mediators, including protein kinase B or extracellular-signal-regulated kinases 1/2. Blocking of EGFR-linked signals increased epithelial susceptibility to pathogen-induced epithelial cell death, suggesting protective roles of EGFR signals. Thus, airway epithelial exposure to P. fluorescens can trigger antiapoptotic responses via EGFR and proinflammatory responses via TLR4-independent NF-κB signaling pathway in human pneumocytes.

Topics: Apoptosis; Blotting, Western; Cell Line; Cell Separation; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; ErbB Receptors; Flow Cytometry; Humans; Immunoprecipitation; Interleukin-8; Microscopy, Confocal; NF-kappa B; Pseudomonas fluorescens; Pseudomonas Infections; Pulmonary Alveoli; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

The clinical consequence of chronic Pseudomonas aeruginosa colonization in cystic fibrosis (CF) varies between individuals for unknown reasons. Auto-antibodies against bactericidal/permeability increasing protein (BPI-ANCA) are associated with poor prognosis in CF. We hypothesize that there is a correlation between the presence of BPI-ANCA, the properties of the colonizing bacteria and the clinical conditions of the host. We compared isolates of P. aeruginosa from BPI-ANCA positive CF patients who have deteriorating lung disease with BPI-ANCA negative CF patients who are in stable clinical conditions. Epithelial cells (A549) and isolated polymorphonuclear granulocytes (PMNs) were stimulated with the isolates and cell death was analyzed with flow cytometry. We found that the ANCA associated strains in most cases showed pyocyanin negative phenotypes. These strains also induced less inflammatory response than the non-ANCA associated strains as shown by apoptosis and necrosis of epithelial cells and neutrophils. Our results suggest that colonization with strains of P. aeruginosa that induce a weak inflammatory response is associated with unfavorable outcome in CF. We speculate that inadequate control of pathogen proliferation through an insufficient inflammatory response results in a slowly increasing number of bacteria and accumulation of dying PMNs in the airways, contributing to progression in CF lung disease.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antimicrobial Cationic Peptides; Blood Proteins; Cell Death; Cell Line, Tumor; Cystic Fibrosis; Disease Progression; Humans; Immunoglobulin A; Interleukin-8; Lung Neoplasms; Necrosis; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Respiratory Mucosa

Following any acute irritation lung function declines rapidly. Reasons for pulmonary deterioration in humans had been attributed to the action of either interleukin-6 or interleukin-8 in the lungs.. The present study investigates the association between immune response and decline in lung function in a murine bacterial lung infection model.. Upon intratracheal inoculation of C57BL/6J mice with a sublethal dose of Pseudomonas aeruginosa lung function, cytokine, chemokine and cytometry in bronchoalveolar lavage fluid, bacterial counts and lung histology was assessed at 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, 96 and 120 h post inoculation.. Lung function measured by non-invasive head-out spirometry decreased most strongly between 6 and 10 h post inoculation and required up to 72 h to recover for selected parameters. CFU counts in the lungs peaked at 4h post inoculation with subsequent decline until at 24-48 h post inoculation background levels were reached. Cytokine and chemokine responses could be separated into an early pro-inflammatory phase (2-8h post inoculation; mainly tumor-necrosis factor α (TNFα) and interleukin-1α driven) and a late anti-inflammatory resolution phase (starting at 24h post inoculation; mainly interleukin-10 and interleukin-4 driven). Interleukin-6 levels correlated with the deterioration of lung function. Lung histology showed maximal changes in terms of inflammation and edema between 24 and 48 h post inoculation.. In summary, elevated interleukin-6, high local neutrophil counts and lung edema were found to be the most characteristic signs of the transient period of deterioration of lung function.

Topics: Animals; Bacterial Load; Bronchoalveolar Lavage Fluid; Catheterization; Edema; Inflammation; Interleukin-10; Interleukin-1alpha; Interleukin-4; Interleukin-6; Interleukin-8; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Respiratory Tract Infections; Trachea; Tumor Necrosis Factor-alpha

We investigated the influence of the type III effector, ExoS, on the host epithelial cell response to Pseudomonas aeruginosa infection, and we found that disruption of the exoS gene caused a significant increase in the amount of interleukin-8 (IL-8) in the culture medium of Caco-2 cells. We show that IL-8 was degraded in the culture medium following infection of the cells with the wild-type (PAO1), but not the exoS knock-out (the ΔexoS) strain. Purified ExoS protein itself did not degrade IL-8. We next show that IL-8 degradation by PAO1 was inhibited by the addition of serine protease inhibitors. These results strongly suggest that a bacterial serine protease that degrades IL-8 is expressed and secreted into the culture medium of Caco-2 cells infected with PAO1, and that the expression of this protein is repressed in cells infected with the ΔexoS strain. The PAO1 genome encodes 28 different protease genes, including two serine proteases: PA3535 and mucD. PA3535 and mucD gene knock-outs were constructed (ΔmucD and ΔPA3535), and ΔmucD but not ΔPA3535 showed reduced IL-8 degradation. To understand the significance of IL-8 degradation, we next evaluated neutrophil infiltration in lungs excised from mice intranasally infected with the P. aeruginosa strains. Increased neutrophil infiltration was observed in PAO1-infected mice, but not in ΔexoS- or ΔmucD-infected mice. Taken together, our results suggest that P. aeruginosa escapes from phagocytic killing due to IL-8 degradation following the secretion of the MucD serine protease, whose expression appears to be influenced by ExoS.

Topics: ADP Ribose Transferases; Animals; Bacterial Proteins; Bacterial Toxins; Caco-2 Cells; Female; Gene Knockout Techniques; Histocytochemistry; Host-Pathogen Interactions; Humans; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Serine Endopeptidases

IL-8 released from bronchial epithelial cells infected with Pseudomonas aeruginosa plays a crucial role in the chronic lung pathology of patients affected by cystic fibrosis. Novel anti-inflammatory approaches will benefit from a thorough understanding of the regulatory mechanisms involved in the transcription of this chemokine to identify potential pharmacological targets. We addressed this issue by investigating the role of phosphoproteins and transcription factors (TFs) on transcription of IL-8 gene in the human bronchial epithelial IB3-1, CuFi-1, and Calu-3 cells. P. aeruginosa increased the basal phosphorylation of the ERK1/2 pathway components 90-kDa ribosomal S6 kinase (RSK)1/2 and mitogen- and stress-activated kinase-2 and of the p38 MAPK pathway components p38α/δ/γ and heat shock protein 27 (HSP27). The involvement of these kinases in the expression of IL-8 gene was confirmed with pharmacological inhibitors of ERK1/2, RSK, p38, and HSP27 both at transcription and secretion levels. Transfection of TF decoy oligodeoxynucleotides, designed to interfere with the interaction of the TFs NF-κB, NF-IL6, AP-1, CREB, and CHOP with the corresponding consensus sequences identified in the IL-8 promoter, reduced the P. aeruginosa-dependent transcription of IL-8, suggesting their participation in the transcriptional machinery. Stimulation of IB3-1 cells with IL-1β led to a similar pattern of activation, whereas the pattern of phosphoproteins and of TFs modulated by TNF-α differentiated sharply. In conclusion, the results highlight a novel role for RSK1/2 and HSP27 phosphoproteins and of the cooperative role of the TFs NF-κB, NF-IL6, AP-1, CHOP, and CREB in P. aeruginosa-dependent induction of transcription of the IL-8 gene in human bronchial epithelial cells.

Topics: Bronchi; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Epithelial Cells; Gene Expression Regulation; HSP27 Heat-Shock Proteins; Humans; Interleukin-8; Phosphoproteins; Pseudomonas aeruginosa; Pseudomonas Infections; Real-Time Polymerase Chain Reaction; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Transcription, Genetic; Transfection

Airway epithelial cells contribute to the inflammatory response of the lung, and their innate immune response is primarily mediated via Toll-like receptor (TLR) signaling. Cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. We investigated the TLR-4 expression and the inflammatory profile (IL-8 and IL-6 secretion) in CF bronchial epithelial cell line CFBE41o- and its CF transmembrane ion condcutance regulator (CFTR)-corrected counterpart grown under air-liquid interface conditions after stimulation with lipopolysaccharide (LPS) from gram-negative bacteria. In CFTR-corrected cells, IL-8 and IL-6 secretions were constitutively activated but significantly increased after LPS stimulation compared with CFBE41o-. Blocking TLR-4 by a specific antibody significantly inhibited IL-8 secretion only in CFTR-corrected cells. Transfection with specific siRNA directed against TLR-4 mRNA significantly reduced the response to LPS in both cell lines. Fluorescence-activated cell sorter analysis revealed significantly higher levels of TLR-4 surface expression in CFTR-corrected cells. In histologic lung sections of patients with CF, the TLR-4 expression in the bronchial epithelium was significantly reduced compared with healthy control subjects. In CF the loss of CFTR function appears to decrease innate immune responses, possibly by altering the expression of TLR-4 on airway epithelial cells. This may contribute to chronic bacterial infection of CF airways.

Topics: Adolescent; Adult; Antibodies; Bronchi; Cell Line; Child; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Female; Gene Expression Regulation; Humans; Immunity, Innate; Interleukin-6; Interleukin-8; Lipopolysaccharides; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; RNA, Messenger; Signal Transduction; Toll-Like Receptor 4

In Cystic Fibrosis (CF), the absence of functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is key to better therapies for CF. In this manuscript, we have found that the presence of IL-17 in the airways of CF patients not only exacerbates inflammation through the recruitment of neutrophils via secretion of CXCL8, but also by priming airway epithelial cells lacking functional CFTR to increase response to the bacterial sensor NOD1. IL-17 stimulation of airway epithelial cells (AECs) lacking functional CFTR increased the expression of NOD1, NOD2, TLR4 and its own receptors IL-17RA and IL-17RC. Moreover, prior stimulation of AECs expressing the CFTRDeltaF508 mutant with IL-17 showed much greater CXCL8 secretion in response to a NOD1 agonist and Pseudomonas aeruginosa diffusible material. Taken together our results show that IL-17 primes AECs expressing CFTRDeltaF508 to increase host defence response to bacteria through the up-regulation of PRRs, and in particular of NOD1, and identifies another mechanism of action through which the CFTRDeltaF508 mutation leads to increase inflammation in response to bacterial ligands. Therefore preventing IL-17 function in CF may prove an important strategy in decreasing lung inflammation due to both direct and indirect effects.

Topics: Adolescent; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-17; Interleukin-8; Lung; Neutrophils; Nod1 Signaling Adaptor Protein; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Interleukin-17; Respiratory Mucosa; Toll-Like Receptor 4

Keratitis caused by Pseudomonas aeruginosa is a potentially vision-threatening condition that requires prompt treatment to prevent vision loss. The recognition of infectious agents by the Toll-like receptor (TLR) system initiates primary innate and later adaptive immune responses. In this study, in late cases of corneal P. aeruginosa infection, the expression of TLR2, 4, 5 and 9 mRNA were all upregulated. In early infection cases, only TLR9 mRNA expression was upregulated. In late cases, the protein expression of TLR2, 4, 5, 9 and pIkappaB-alpha were elevated. In early cases, only TLR9 and pIkappaB-alpha expression were upregulated. Concentrations of IL-6 and IL-8 increased in infected corneas, especially in late cases. Myeloperoxidase (MPO) activity suggested that polymorphonuclear leukocyte (PMN) numbers were higher in late than in early stages of infection. The delayed response of TLRs may explain why P. aeruginosa infection exacerbates rapidly at the early infection stage. This finding may have important implications for the treatment of innate immunologic responses to corneal infections.

Topics: Adult; Cornea; Female; Gene Expression Profiling; Humans; Interleukin-6; Interleukin-8; Keratitis; Male; Middle Aged; Neutrophils; Peroxidase; Pseudomonas aeruginosa; Pseudomonas Infections; Time Factors; Toll-Like Receptors; Up-Regulation; Young Adult

Probiotics reduce intestinal inflammation in, and Lactobacillus GG (LGG) reduces pulmonary exacerbation rate cystic fibrosis (CF) patients. We intended to determine the effect of a mixed probiotic preparation on pulmonary exacerbations and inflammatory characteristics of the sputum in CF patients.. A prospective pilot study of 10 CF patients with mild-moderate lung disease and Pseudomonas aeruginosa colonization, treated with probiotics for 6 months. Pulmonary function tests (PFT's), sputum cultures with semi-quantitative bacterial analysis, and sputum neutrophil count and interleukin-8 (IL-8) levels were compared to pre-treatment and post-treatment values. The rate of pulmonary exacerbations was compared to 2 years prior to the study.. The exacerbation rate was significantly reduced in comparison to the previous 2 years and to 6 months post-treatment (P = 0.002). PFT's have not changed at the end of treatment and during 6 months post-treatment. No change in sputum bacteria, neutrophil count, and IL-8 levels was observed.. Probiotics reduce pulmonary exacerbations rate in patients with CF. Probiotics may have a preventive potential for pulmonary deterioration in CF patients.

Topics: Adult; Cystic Fibrosis; Dietary Supplements; Disease Progression; Female; Humans; Interleukin-8; Male; Mutation; Neutrophils; Pilot Projects; Probiotics; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Sputum; Young Adult

Pseudomonas fluorescens is present in low number in the intestinal lumen and has been proposed to play a role in Crohn's disease (CD). Indeed, a highly specific antigen, I2, has been detected in CD patients and correlated to the severity of the disease. We aimed to determine whether P. fluorescens was able to adhere to human intestinal epithelial cells (IECs), induce cytotoxicity and activate a proinflammatory response.. Behaviour of the clinical strain P. fluorescens MFN1032 was compared to that of the psychrotrophic strain P. fluorescens MF37 and the opportunistic pathogen P. aeruginosa PAO1. Both strains of P. fluorescens were found to adhere on Caco-2/TC7 and HT-29 cells. Their cytotoxicity towards these two cell lines determined by LDH release assays was dose-dependent and higher for the clinical strain MFN1032 than for MF37 but lower than P. aeruginosa PAO1. The two strains of P. fluorescens also induced IL-8 secretion by Caco-2/TC7 and HT-29 cells via the AP-1 signaling pathway whereas P. aeruginosa PAO1 potentially used the NF-kappaB pathway.. The present work shows, for the first time, that P. fluorescens MFN1032 is able to adhere to IECs, exert cytotoxic effects and induce a proinflammatory reaction. Our results are consistent with a possible contribution of P. fluorescens in CD and could explain the presence of specific antibodies against this bacterium in the blood of patients.

Topics: Bacterial Adhesion; Caco-2 Cells; Cytotoxicity, Immunologic; Epithelial Cells; HT29 Cells; Humans; Interleukin-8; Intestines; NF-kappa B; Pseudomonas fluorescens; Pseudomonas Infections; Signal Transduction; Transcription Factor AP-1

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.

Topics: Acidosis; Animals; C-Reactive Protein; Cell Line; Chlorocebus aethiops; COS Cells; Escherichia coli Infections; Ficolins; Humans; Immunity, Innate; Interleukin-8; Lectins; Macromolecular Substances; Membrane Proteins; Monocytes; Pseudomonas Infections; Receptor Cross-Talk; Receptors, Cell Surface; Salmonella Infections; Signal Transduction; Staphylococcal Infections; U937 Cells; Up-Regulation

To assess the effects of Pseudomonas aeruginosa and Staphylococcus aureus infection on lower airway inflammation and clinical status in young children with cystic fibrosis (CF).. We studied 111 children age < 6 years who had 2 P aeruginosa-positive oropharyngeal cultures within 12 months. We examined bronchoalveolar lavage fluid (BALF) inflammatory markers (ie, cell count, differential, interleukin [IL]-8, IL-6, neutrophil elastase), CF-related bacterial pathogens, exotoxin A serology, and clinical indicators of disease severity.. Young children with CF with both upper and lower airway P aeruginosa infection had higher neutrophil counts, higher IL-8 and free neutrophil elastase levels, increased likelihood of positive exotoxin A titers, and lower Shwachman scores compared with those with positive upper airway cultures only. S aureus was associated with increased lower airway inflammation, and the presence of both P aeruginosa and S aureus had an additive effect on concentrations of lower airway inflammatory markers. BALF markers of inflammation were increased with the number of different bacterial pathogens detected.. Young children with CF who have upper and lower airway P aeruginosa infection have increased endobronchial inflammation and poorer clinical status compared with those with only upper airway P aeruginosa infection. The independent and additive effects of S aureus on inflammation support the significance of polymicrobial infection in early CF lung disease.

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Exotoxins; Female; Humans; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Lung; Male; Neutrophils; Oropharynx; Pseudomonas aeruginosa; Pseudomonas Infections; Severity of Illness Index; Staphylococcal Infections; Staphylococcus aureus

The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.

Topics: Adult; CCAAT-Enhancer-Binding Protein-beta; Cells, Cultured; Conjunctiva; Enzyme Activation; Female; Gene Expression Regulation; Humans; Interleukin-8; Male; Middle Aged; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Pseudomonas aeruginosa; Pseudomonas Infections; Transcription Factor RelA; Transcription, Genetic

Acid sphingomyelinase (ASMase) is a key enzyme in sphingolipid metabolism, which can be activated by various cellular stress mechanisms including bacterial pathogens. Activation of ASMase generates ceramide, which is important for innate immune response to eliminate infected pathogens. The current study reveals a defective ASMase pathway after Pseudomonas aeruginosa infection in both a cystic fibrosis (CF) bronchial epithelial cell line (IB3-1 cell) and in the lungs of CF transmembrane conductance regulator (CFTR) knockout (KO) mice as compared with S9 cells and wild-type C57BL/6 mice. ASMase activity and total ceramide levels significantly increased in S9 cells and C57BL/6 mice with P. aeruginosa infection, but not in IB3-1 cells and CFTR KO mice. The silencing of CFTR by CFTR RNAi in S9 cells significantly decreased ASMase activity after bacterial infection as compared with controls. This study also demonstrates that induction of ASMase is responsible for modulating the immune response to bacterial infection. Blocking ASMase activity with specific ASMase RNAi, an ASMase inhibitor, or an ASMase antibody in S9 cells significantly increased IL-8 levels with P. aeruginosa infection compared with controls. Reciprocally, adding exogenous bacterial sphingomyelinase to IB3-1 cells significantly decreased IL-8 levels compared with untreated cells. In addition, silencing of ASMase in S9 cells also significantly decreased bacterial internalization. Adding exogenous bacterial sphingomyelinase to IB3-1 cells reconstituted the cell death response to P. aeruginosa infection. This study demonstrates that the defective ASMase pathway in CF is a key contributor to the unabated IL-8 response with P. aeruginosa infection and to the compromised host response failing to eradicate bacteria.

Topics: Animals; Apoptosis; Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Interleukin-8; Mice; Mice, Inbred C57BL; Mice, Knockout; Pseudomonas aeruginosa; Pseudomonas Infections; RNA Interference; Signal Transduction; Sphingolipids; Sphingomyelin Phosphodiesterase

P. aeruginosa chronically colonizes the lung in CF patients and elicits a proinflammatory response. Excessive secretion of IL-6 and IL-8 by CF airway cells in response to P. aeruginosa infection in the CF airway is though to contribute to lung injury. Accordingly, the goal of this study was to test the hypothesis that Corr4a and VRT325, investigational compounds that increase DeltaF508-CFTR mediated Cl(-) secretion in human CF airway cells, reduce the pro-inflammatory response to P. aeruginosa.. IL-6 and IL-8 secretion by polarized CF human airway epithelial cells (CFBE41o-) were measured by multiplex analysis, and DeltaF508-CFTR Cl- secretion was measured in Ussing chambers. Airway cells were exposed to P. aeruginosa (PAO1 or PA14) and Corr4a or VRT325.. Corr4a and VRT325 increased DeltaF508-CFTR Cl(-) secretion but did not reduce either constitutive IL-6 or IL-8 secretion, or IL-6 and IL-8 secretion stimulated by P. aeruginosa (PA14 or PAO1).. Corr4a and VRT325 do not reduce the inflammatory response to P. aeruginosa in human cystic fibrosis airway epithelial cells.

Topics: Cell Line; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Piperazines; Pseudomonas Infections; Quinazolines

Prolonged macrolide antibiotic therapy at low doses improves clinical outcome in patients affected with diffuse panbronchiolitis and cystic fibrosis. Consensus is building that the therapeutic effects are due to anti-inflammatory, rather than anti-microbial activities, but the mode of action is likely complex. To gain insights into how the macrolide azithromycin (AZT) modulates inflammatory responses in airways, well-differentiated primary cultures of human airway epithelia were exposed to AZT alone, an inflammatory stimulus consisting of soluble factors from cystic fibrosis airways, or AZT followed by the inflammatory stimulus. RNA microarrays were conducted to identify global and specific gene expression changes. Analysis of gene expression changes revealed that the AZT treatment alone altered the gene profile of the cells, primarily by significantly increasing the expression of lipid/cholesterol genes and decreasing the expression of cell cycle/mitosis genes. The increase in cholesterol biosynthetic genes was confirmed by increased filipin staining, an index of free cholesterol, after AZT treatment. AZT also affected genes with inflammatory annotations, but the effect was variable (both up- and down-regulation) and gene specific. AZT pretreatment prevented the up-regulation of some genes, such as MUC5AC and MMP9, triggered by the inflammatory stimulus, but the up-regulation of other inflammatory genes, e.g., cytokines and chemokines, such as interleukin-8, was not affected. On the other hand, HLA genes were increased by AZT. Notably, secreted IL-8 protein levels did not reflect mRNA levels, and were, in fact, higher after AZT pretreatment in cultures exposed to the inflammatory stimulus, suggesting that AZT can affect inflammatory pathways other than by altering gene expression. These findings suggest that the specific effects of AZT on inflamed and non-inflamed airway epithelia are likely relevant to its clinical activity, and their apparent complexity may help explain the diverse immunomodulatory roles of macrolides.

Topics: Anti-Bacterial Agents; Azithromycin; Cell Cycle; Epithelial Cells; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; Lipid Metabolism; Lipids; Lung; Matrix Metalloproteinase 9; Mucin 5AC; Pseudomonas aeruginosa; Pseudomonas Infections

Both gastroesophageal reflux and airway colonization with Pseudomonas aeruginosa (P aeruginosa) are common in lung transplantation (LTx) recipients. There is mounting evidence that, due to their interaction with the epithelium, both may be involved in chronic allograft dysfunction/bronchiolitis obliterans syndrome (BOS) after LTx. We investigated whether gastric aspiration and airway colonization with P aeruginosa after LTx are associated.. In this retrospective, cross-sectional, case-control study, 24 stable double (SS) LTx recipients were included. Markers of gastroesophageal reflux (pepsin, bile acids) and airway inflammation (neutrophilia and interleukin-8 (IL-8)) were evaluated in bronchoalveolar lavage (BAL) samples of post-operatively colonized (n = 12) and non-colonized matched-control LTx recipients (n = 12).. BAL bile acid levels, but not pepsin levels, as well as neutrophilia and IL-8 protein levels were significantly elevated in colonized compared with non-colonized patients. Furthermore, bile acid levels, but not pepsin levels, correlated positively with BAL neutrophilia and IL-8 protein levels.. Bile acid aspiration and airway colonization by P aeruginosa after LTx seem to be associated. This relationship between reflux and airway colonization and their role in the development of chronic allograft dysfunction/BOS after LTx should be further elucidated; nevertheless, induction of IL-8-mediated neutrophilic airway inflammation may be a putative mechanism.

Topics: Adult; Bile Acids and Salts; Biomarkers; Bronchiolitis Obliterans; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cross-Sectional Studies; Female; Gastroesophageal Reflux; Humans; Inflammation; Interleukin-8; Lung Transplantation; Male; Middle Aged; Neutrophils; Pepsin A; Pneumonia, Bacterial; Postoperative Complications; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Aspiration; Respiratory System; Retrospective Studies

Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the DeltaF508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073-4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl- efflux was confirmed in MPB-07-treated IB3-1 and CuFi-1 cells. In conclusion, the DeltaF508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.

Topics: Anti-Inflammatory Agents; Bronchi; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Epithelial Cells; Gene Expression Regulation; Genistein; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; NF-kappa B; Protein Binding; Pseudomonas aeruginosa; Pseudomonas Infections; Quinolizines; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic

Neutrophil elastase (NE) activity is increased in many diseases. Other families of proteases, including cathepsins and matrix metalloproteases (MMPs), are also present at elevated levels in similar disease conditions. We postulated that NE could induce expression of cathepsins and MMPs in human macrophages. NE exposure resulted in macrophages, producing significantly greater amounts of cathepsin B and latent and active MMP-2. Cathepsin B and MMP-2 activities were decreased in Pseudomonas-infected NE knockout mice compared with wild-type littermates. We also demonstrate that NE can activate NF-kappaB in macrophages, and inhibition of NF-kappaB resulted in a reduction of NE-induced cathepsin B and MMP-2. Also, inhibition of TLR-4 or transfection of macrophages with dominant-negative IL-1R-associated kinase-1 resulted in a reduction of NE-induced cathepsin B and MMP-2. This study describes for the first time a novel hierarchy among proteases whereby a serine protease up-regulates expression of MMPs and cathepsins. This has important implications for therapeutic intervention in protease-mediated diseases.

Topics: Animals; Cathepsin B; Genes, Dominant; Humans; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Leukocyte Elastase; Lung; Macrophages; Matrix Metalloproteinase 2; Mice; Mice, Knockout; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Toll-Like Receptor 4; Transfection

Lower airway tract colonization may trigger neutrophil-mediated airway inflammation. We investigated whether transient airway colonization influences airway inflammation and pulmonary function after lung transplantation (LTx). In this retrospective study, stable LTx patients with consecutive broncho-alveolar lavages (BAL), the first colonized and the second noncolonized, were included to create a Pooled group (P, n=32) and a Gram Negative (GN, n=14), Gram Positive (GP, n=9) and Fungi (F, n=9) subgroup. Similarly, LTx patients with consecutive, noncolonized BAL samples were included as Control group (C, n=19). BAL analysis (cell counts, IL6, IL8) and forced expiratory volume (FEV(1)) were compared between groups. In the P group and the GN subgroup, colonized BAL samples showed a significant increase in total cells, neutrophilia and IL8-concentration and a significant decrease in FEV(1), even when compared with the matched samples of the C group. A significant increase in neutrophilia was observed in the GP subgroup, but no other significant difference was found either in the GP and F subgroup or the C group. IL6 levels were not significantly different between groups. Transient airway colonization, especially with Pseudomonas-like GN bacteria, in stable LTx patients may be associated with IL8-dependent neutrophilic airway inflammation and a, albeit subtle, decrease in FEV(1).

Topics: Adult; Bronchoalveolar Lavage Fluid; Cell Count; Female; Forced Expiratory Volume; Humans; Interleukin-6; Interleukin-8; Lung Transplantation; Male; Middle Aged; Neutrophils; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory System; Retrospective Studies

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.

Topics: Bronchi; Calcium; Calcium Signaling; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Enzyme-Linked Immunosorbent Assay; Flagellin; Humans; Inflammation; Interleukin-8; Microscopy, Confocal; Mutation; NF-kappa B; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa

Chronic Pseudomonas aeruginosa lung infection is the major reason for premature death in patients with cystic fibrosis (CF). Infected patients experience a progressive deterioration of the lung tissue caused by a persistent accumulation of PMNs. We investigated if the pulmonary accumulation of PMNs is reflected as a migration of PMNs through the blood in chronically infected CF patients.. Blood and sputum samples from 37 stable, chronically (CF+P) and 6 non-infected (CF-P) CF patients without exacerbations were compared using FACS, leukocyte counting, and ELISA. Within the CF+P patients, the blood parameters were compared to the lung function (FEV1 and FVC) and to the sputum. Similar measurements were performed on 15 chronically infected CF patients before and after elective antibiotic treatment.. In the CF+P patients the concentration of G-CSF in the sera and PMNs in the blood was increased and correlated to poor lung function. However, only the concentration of G-CSF in the sera was correlated to the concentration of TNF-alpha in the sputum. After the antibiotic treatment, the lung function was improved and the concentration of PMNs in the blood and G-CSF in the sera was reduced.. G-CSF in the sera may contribute to the pulmonary inflammation in CF patients with chronic P. aeruginosa lung infection by regulating the number of PMNs available for migration and may be considered as an indicator of clinical status.

Topics: Adolescent; Adult; Blood Cell Count; Cell Movement; Cystic Fibrosis; Female; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Humans; Inflammation; Interleukin-8; Male; Middle Aged; Neutrophils; Pneumonia; Pseudomonas Infections; Tumor Necrosis Factor-alpha

Considerable lung injury results from the inflammatory response to Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). The P. aeruginosa laboratory strain PAO1, an environmental isolate, and isolates from CF patients were cultured in vitro and outer membrane vesicles from those cultures were quantitated, purified, and characterized. Vesicles were produced throughout the growth phases of the culture and vesicle yield was strain-independent. Strain-dependent differences in the protein composition of vesicles were quantitated and identified. The aminopeptidase PaAP (PA2939) was highly enriched in vesicles from CF isolates. Vesicles from all strains elicited IL-8 secretion by lung epithelial cells. These results suggest that P. aeruginosa colonizing the CF lung may produce vesicles with a particular composition and that the vesicles could contribute to inflammation.

Topics: Bacterial Outer Membrane Proteins; Cystic Fibrosis; Electrophoresis, Gel, Two-Dimensional; Epithelial Cells; Humans; Interleukin-8; Lung; Mass Spectrometry; Pseudomonas aeruginosa; Pseudomonas Infections; Secretory Vesicles

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o(-), which is homozygous for the DeltaF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o(-). Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o(-) cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-kappaB-linked reporter gene and increased IL-8 protein production in CFTE29o(-) cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.

Topics: Adaptor Proteins, Signal Transducing; Antigens, Differentiation; Bacterial Proteins; Bronchoalveolar Lavage Fluid; Cell Line; CpG Islands; Cystic Fibrosis; Humans; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Lipopeptides; Lipopolysaccharides; Lipoproteins; Membrane Glycoproteins; Myeloid Differentiation Factor 88; NF-kappa B; Oligodeoxyribonucleotides; Oligopeptides; Pseudomonas Infections; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Interleukin-1; Respiratory Mucosa; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 9; Toll-Like Receptors; U937 Cells; Up-Regulation

Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) are inflammatory mediators involved in the neutrophil response to pulmonary bacterial colonization in cystic fibrosis (CF). The aim of this study was to investigate whether the LTB(4) and IL-8 levels in exhaled breath condensate (EBC) could be related to the type of bacterial colonization in CF patients. The pH level in EBC was analyzed as an estimate of airway acidification. Forty children were evaluated: 10 CF patients with P. aeruginosa, 10 CF patients with S. aureus, 10 not colonized CF patients, and 10 healthy children. LTB(4) and IL-8 in EBC were analyzed by specific enzyme immunoassay kits (EIA). The pH of EBC was measured with a pH-meter after deareation by bubbling with argon. Exhaled LTB(4) was higher in CF children with P. aeruginosa compared to those with S. aureus (P < 0.01), not colonized (P < 0.001), and healthy children (P < 0.01). Exhaled IL-8 was elevated in CF patients colonized by P. aeruginosa compared with other subgroups (vs. not colonized, P < 0.05; vs. healthy children, P < 0.001). IL-8 levels were higher in CF children with S. aureus than in healthy children (P < 0.05). There was an increase in IL-8 levels in not colonized CF patients compared with healthy children (P < 0.05). EBC pH was higher in healthy children compared to CF patients not colonized (P < 0.05). Our data suggest that EBC is suitable for evaluating neutrophil inflammatory mediators (LTB(4), IL-8, and pH) involved in the response to pulmonary bacterial colonization in CF children.

Topics: Biomarkers; Breath Tests; Child; Cystic Fibrosis; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Interleukin-8; Leukotriene B4; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Sputum; Staphylococcal Infections; Staphylococcus aureus

The Toll-like receptor 5 (TLR5) binding site has been predicted to be in the N terminus of the flagellin molecule. In order to better define the interaction between the N-terminal amino acids of Pseudomonas aeruginosa flagellin and TLR5, site-specific mutations were generated between residues 88 and 97 of P. aeruginosa PAK flagellin as well as outside of this region. The mutant flagellins were expressed in Escherichia coli BL21(plysS), purified by affinity chromatography, and passed through a polymyxin B column to remove contaminating lipopolysaccharide (LPS). Their ability to stimulate interleukin-8 (IL-8) release from A549 cells was examined. The cloned mutated genes were used to complement a PAK fliC mutant in order to test for effects on motility and on IL-8 release by purified flagellar preparations. All the mutations, single or double, in the predicted TLR5 binding region reduced IL-8 signaling to less than 95% of the wild-type flagellin levels, but the single mutation outside the binding region had no effect. Changes made at two amino acid sites resulted in loss/reduction of motility; however, changes made at single sites, i.e., Q83A, L88A, R90A, M91A, L94A, and Q97A, had no effect on motility. The mutated genes encoding two of the motile but poorly signaling flagellins had no compensatory mutations to allow motility. Thus, while it is speculated that pathogen-associated molecular patterns (PAMPs) have evolved in locations that are essential to maintain function, it appears that there is tolerance for at least single amino acid changes in the PAMP of P. aeruginosa flagellin. The purpose of flagellin glycosylation in P. aeruginosa is unknown. In order to examine its role, if any, in signaling an inflammatory response, we used whole flagella from the motile chromosomal mutant strains PAKrfbC and PAO1rfbC, which are defective in flagellin glycosylation. IL-8 release from A549 cells stimulated with nonglycosylated flagellar preparations (having less then 1 picogram of LPS/mug) was significantly reduced compared to their respective wild-type flagellar preparations, indicating a role of flagellar glycosylation in the proinflammatory action of Pseudomonas flagellin. The basis of the latter activity is unknown, since the glycosylation sites are found in the D3 domain of flagellins and the TLR5 binding site is located in the D1 domain. Thus, P. aeruginosa flagellin has evolved additional flagellar signaling mechanisms over that described for Salmonella flag

Topics: Amino Acid Sequence; Amino Acid Substitution; Cells, Cultured; Cloning, Molecular; Flagella; Flagellin; Glycosylation; Humans; Immunity, Innate; Interleukin-8; Molecular Sequence Data; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Recombinant Proteins; Toll-Like Receptor 5

Human beta-defensin (HBD)-1 and -2 are antimicrobial peptides present in the respiratory tract. Recent reports have indicated reduced activity of beta-defensins in cystic fibrosis, suggesting that beta-defensins may play an important role in the pathological process of chronic respiratory tract infection. Diffuse panbronchiolitis (DPB) is a progressive disease characterised by frequent episodes of superimposed infection, typically caused by Pseudomonas aeruginosa. The aim of this study was to elucidate the role of these antimicrobial peptides in this disease.. The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB.. High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1beta. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles.. These results indicate that beta-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.

Topics: Adult; Anti-Bacterial Agents; beta-Defensins; Bronchiolitis; Bronchoalveolar Lavage Fluid; Female; Humans; Immunohistochemistry; Interleukin-1; Interleukin-8; Macrolides; Male; Pseudomonas aeruginosa; Pseudomonas Infections

We report a rare case of a cute lymphoblasticleukemia (ALL) who developed dyspnea, neurological disturbance with illusions, pancytopenia, phagocytosis and coagulation disturbances following bacterial tonsillitis. The values of soluble interleukin-2 receptor (sIL-2R), IL-6 and IL-8 were also elevated. Her clinicolaboratory findings were similar to hemophagocytic lymphohistiocytosis (HLH), which is a cytokine disease induced by activated T cells and macrophages. Atypical HLH following bacterial tonsillitis should be kept in mind in leukemia patients.

Topics: Antigens, CD; Antineoplastic Agents; Blood Coagulation; Child; Dyspnea; Female; Histiocytosis, Non-Langerhans-Cell; Humans; Interleukin-6; Interleukin-8; Pancytopenia; Phagocytosis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Interleukin-2; Streptococcal Infections; Tonsillitis

Chronic Pseudomonas aeruginosa infection in cystic fibrosis (CF) leads to a damaging host inflammatory response. There are an increasing number of reports of P. aeruginosa cross-infection at CF centres. The clinical significance of acquisition of a transmissible strain for patients who already harbour P. aeruginosa is unclear. In this study, levels of inflammatory markers in clinically stable adult CF patients who harbour transmissible and sporadic strains of P. aeruginosa have been compared. Patients with CF and chronic P. aeruginosa infection were grouped into those who harbour a transmissible P. aeruginosa and those who harbour their own sporadic strains. Total white cell and differential counts, sputum neutrophil elastase (NE), interleukin (IL)-8, tumour necrosis factor (TNF)-alpha, plasma IL-6 and NE/alpha1-antitrypsin complexes, serum C-reactive protein, and urine TNF receptor 1 were all measured in clinically stable patients 4-6 weeks following completion of intravenous antibiotic therapy. The two groups (both n=20) were well matched for per cent predicted forced expiratory volume in one second, per cent predicted forced vital capacity and body mass index. There were no significant differences in levels of white cell counts or inflammatory markers between the two groups. At times of clinical stability, cystic fibrosis patients infected with transmissible Pseudomonas aeruginosa do not have a heightened inflammatory response above that of those harbouring sporadic strains.

Topics: Adult; alpha 1-Antitrypsin; Biomarkers; C-Reactive Protein; Case-Control Studies; Cell Count; Cystic Fibrosis; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Leukocyte Elastase; Male; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Tumor Necrosis Factor; Sputum; Tumor Necrosis Factor-alpha

To explore the effects of Zhikuofang, a TCM prescription, and Ofloxacin on the inflammation and cytostatics of the airway model of bronchiectasis.. The airway model of bronchiectasis (AMB) was set up and infused with Ps. Aeruginosa. A comparison between the effects of Zhikuofang and Of loxacin on the AMB was made.. Zhikuofang is better than Ofloxacin in following aspects: lowering the density of inflammation cells in blood, decreasing the volume of tracheal secretion and inhibiting the cytostatics (IL-8 and TNF-alpha) of the trachea tissue, but Ofloxacin is more effective in diminishing the amount of bacteria in trachea flushing liquor. There was no marked difference between them in their histopathy effects on the trachea.. Zhikuofang probably plays antiphlogistic and bacteriostatic effects by inhibiting the IL-8 and TNF-alpha, resisting secretion, decreasing the inflammation cells and resisting inflammation of trachea.

Topics: Animals; Anti-Infective Agents; Bronchiectasis; Bronchitis; Drug Combinations; Drugs, Chinese Herbal; Female; Interleukin-8; Male; Ofloxacin; Phytotherapy; Plants, Medicinal; Pseudomonas Infections; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Nasal polyps frequently appear in patients with cystic fibrosis (CF). The aims of this study were to focus on what problems (symptoms, endoscopic findings, and laboratory correlates) nasal polyps cause the CF patient, and how these correlate to the total health situation of this patient group.. The clinical histories, endoscopic investigations of the nasal cavity, and analyses of nasal lavage fluid of 44 patients with CF complicated with nasal polyposis have been compared with those of 67 CF control subjects. The patients were examined at annual control examinations (with pulmonary tests, working capacity, liver tests, and bacterial and blood tests) from 1995 to 1996 at Stockholm Cystic Fibrosis Center, Huddinge University Hospital. All patients were > 2 years of age. The endoscopic findings were related to the actual pulmonary function, inflammatory blood parameters, colonizing pathogens, antibodies (Staphylococcus aureus and Pseudomonas aeruginosa), and genotype.. The patients with nasal polyps differed with respect to chronic colonization of P aeruginosa in sputum samples and had a higher occurrence of serum antibodies against the same species. The two groups did not differ in pulmonary functions, inflammatory parameters, or genotype. The polyps found were mainly small (within the meatus media) and gave no significant increase in ongoing clinical symptoms such as rhinorrhea, nasal obstruction, or hyposmia. Neither was any significantly marked finding concerning the nose (mucosal swellings, secretion, etc.) made in the polyp patients. The patients with CF scored slightly lower in a smell identification test in comparison with the healthy control group. The nasal lavage fluid was analyzed (in 93 of the 111 patients) for the occurrence of P aeruginosa (by polymerase-chain reaction [PCR]), interleukin [IL]-5, IL-8, and lysozyme. The lysozyme and IL-8 content was equal in the two CF groups but increased in comparison with the healthy control group. P aeruginosa was not detected with PCR in any nasal lavage fluid. No measurable levels of IL-5 in the nasal lavage were found.. There was a higher frequency of chronic colonization of P aeruginosa in the lower respiratory tract in patients with nasal polyps. Otherwise, nonsevere nasal polyposis was not an indicator of lower respiratory tract morbidity in CF patients.

Topics: Adolescent; Adult; Antibodies, Bacterial; Child; Child, Preschool; Cystic Fibrosis; Endoscopy; Female; Humans; Interleukin-5; Interleukin-8; Male; Muramidase; Nasal Lavage Fluid; Nasal Polyps; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Risk Factors; Staphylococcal Infections

Our aim was to study the effect of lower airway infection on clinical parameters, pulmonary function tests, and inflammation in clinically stable infants and young children with cystic fibrosis (CF). To accomplish this goal, a prospective cohort of screened CF patients under 4 years of age were studied, using elective anesthesia and intubation for: passive respiratory mechanics (single breath occlusion passive deflation) and lung volumes (nitrogen washout), under neuromuscular blockade; and bronchoalveolar lavage (BAL) of 3 main bronchi for cytology, cytokine interleukin (IL)-8, and quantitative microbiology. There were 22 children studied, with a mean age of 23.2 months (6.7-44 months). A greater relative risk of lower airway pathogens was associated with prior respiratory admission (3.60, 95% confidence interval [CI] 2.87-4.51), history of asthma (1.75, 95% CI 1.52-2.03), and chronic symptoms (1.50, 95% CI 1.23-1.83), especially wheeze (1.88, 95% CI 1.61-2.19). Lower respiratory pathogens (> or = 10 cfu/ml BAL) were found in 14 out of 22, and greater than 10(5) cfu/ml in 8 out of 22 subjects. The level of pathogens in BAL (log10 cfu/ml) explained 78% of the variability in percent neutrophils and 34% of the variability in IL-8 levels. Pathogen level also correlated with pulmonary function tests of specific respiratory system compliance (r -0.49, p = 0.02) and functional residual capacity over total lung capacity (r 0.49, p = 0.03). We conclude that the presence of pathogens in the lower airways correlated with levels of inflammation, respiratory system compliance, and degree of air trapping.

Topics: Bronchoalveolar Lavage Fluid; Cell Count; Child, Preschool; Cohort Studies; Cystic Fibrosis; Female; Humans; Infant; Inflammation; Inflammation Mediators; Interleukin-10; Interleukin-8; Lung; Male; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mechanics; Respiratory Tract Infections

Ocular infection with Pseudomonas aeruginosa triggers extensive host inflammatory response and corneal damage. The purpose of present study was to investigate the gene expression of pro-inflammatory mediators interleukin (IL)-1 beta, IL-6, tumour necrosis factor-alpha (TNF-alpha),macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (KC) in the mouse eye challenged with P. aeruginosa. Scratched mouse corneas were infected with three phenotypes of P. aeruginosa individually. Total RNA was extracted from mouse eyes at 4 h, 8 h,16 h and 24 h post-challenge. Single stranded cDNA was synthesized from total RNA by reverse transcription and then subjected to polymerase chain reaction (PCR) using specific primers for IL-1 beta, IL-6, TNF-alpha, MIP-2 and KC. Results revealed three patterns of cytokines and chemokines expression in response to ocular infection with three phenotypes of P. aeruginosa. Ocular infection with the invasive strain induced the highest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA, followed by the infection with the cytotoxic strain. Ocular infection with the CLARE strain induced the lowest levels of IL-1 beta, IL-6, MIP-2 and KC mRNA. The expression of TNF-alpha mRNA was very low and irregular following P. aeruginosa challenge. These data indicate that over-expression of pro-inflammatory cytokines and chemokines may represent a vigorous immune response and therefore may contribute to corneal damage during P. aeruginosa infection.

Topics: Animals; Chemokine CXCL2; Chemokines; Cornea; Corneal Ulcer; Cytokines; Eye Infections, Bacterial; Female; Gene Expression; Interleukin-1; Interleukin-6; Interleukin-8; Mice; Mice, Inbred BALB C; Models, Animal; Monokines; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

A tendency toward excessive inflammation in cystic fibrosis (CF) patients often accompanies lung infections with Pseudomonas aeruginosa. We tested the cytokine response to P. aeruginosa in two pairs of human airway epithelial cell lines matched except for CF transmembrane conductance regulator activity. The 9/HTEo(-) CF-phenotypic cell line produced significantly more interleukin (IL)-8, IL-6, and granulocyte-macrophage colony-stimulating factor but not regulated on activation normal T cell expressed and secreted (RANTES) in response to Pseudomonas than the 9/HTEo(-) control line, and the differences widened over time. Similarly, a 16HBE cell line lacking transmembrane conductance regulator activity showed enhanced IL-8 and IL-6 responses compared with the control cell line. The pharmacology of the cytokine response also differed because dexamethasone reduced cytokine production to similar levels in the matched cell lines. The protracted proinflammatory cytokine response of the CF-phenotypic cell lines suggests that the limiting mechanisms of normal cells are absent or attenuated. These results are consistent with in vivo observations in patients with CF and suggest that our novel cell lines may be useful for further investigation of the proinflammatory responses in CF airways.

Topics: Bronchi; Cell Line; Cell Polarity; Chemokine CCL5; Cystic Fibrosis Transmembrane Conductance Regulator; Cytokines; Dexamethasone; Epithelial Cells; Glucocorticoids; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Oligonucleotides, Antisense; Pseudomonas Infections; Time Factors; Trachea

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.

Topics: Cell Line; Chemokine CCL5; Culture Media, Conditioned; Cytotoxicity, Immunologic; Epithelial Cells; Humans; Interleukin-1; Interleukin-8; Molecular Weight; Mutation; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory System; RNA, Messenger; Solvents; Tumor Necrosis Factor-alpha

Several recent reports have suggested that airway inflammation may precede infection and relate to an endogenous dysregulation of pro-inflammatory cytokines in cystic fibrosis (CF) airways. Evidence suggests that activation of the nuclear factor kappa B (NFkappaB), which regulates the inflammatory gene transcription, depends on the degradation of the inhibitory factor IkappaBalpha. We show that, in in situ human DeltaF508 CF bronchial tissues, inhibitor factor IkappaBalpha is not present in gland cells, although endogenous levels of chemokine IL-8 are high. These data are confirmed by studying cultured CF human bronchial gland cells, in which a lack of cytosolic IkappaBalpha and high levels of activated NFkappaB, concomitant with IL-8 overproduction (a 13-fold increase) are found when compared to non-CF bronchial gland cells. Interestingly, treatment of CF gland cells with the isoflavone genistein, a well known CFTR mutant Cl(-) channel stimulator, results in a significant decrease ( P < 0.001) in IL-8 production down to levels released by non-CF gland cells. The addition of genistein also reverses the effects of lipopolysaccharide (LPS) Pseudomonas-aeruginosa-induced nuclear translocation of NFkappaB by increasing IkappaBalpha protein level (65%) in CF gland cells. Our data indicate that the induction of IkappaBalpha protein in CF airway glandular epithelial cells may be a novel mechanism by which IL-8-mediated lung inflammatory events are markedly reduced in CF patients, at least at the airway glandular level.

Topics: Bronchi; Cells, Cultured; Cystic Fibrosis; Cytosol; DNA-Binding Proteins; Enzyme Inhibitors; Genistein; Humans; I-kappa B Proteins; Interleukin-8; NF-kappa B; NF-KappaB Inhibitor alpha; Protein-Tyrosine Kinases; Pseudomonas Infections; Respiratory Mucosa

Exacerbated inflammation is now recognized as an important component of cystic fibrosis (CF) airway disease. Whether inflammation is part of the basic defect in CF or a response to persistent infection remains controversial. We addressed this question using human fetal tracheal grafts in severe combined immunodeficient mice. This model yields histologically mature, and most importantly, naive CF and non-CF surrogate airways. Significant inflammatory imbalance was found in naive CF airway grafts, including a highly increased intraluminal interleukin 8 content (CF: 10.1 +/- 2.2 ng/ml; non-CF: 1.2 +/- 0.6 ng/ml; P < 0.05) and consistent accumulation of leukocytes in the subepithelial region (P < 0.001). CF airway grafts were not histologically affected until challenged with Pseudomonas aeruginosa, which provoked: (1) early (before 3 h) and massive leukocyte transepithelial migration, (2) intense epithelial exfoliation, and (3) rapid progression of bacteria toward the lamina propria. In non-CF grafts, these three sets of events were not observed before 6 h. Using a model of naive human airways, we thus demonstrate that before any infection, CF airways are in a proinflammatory state. After infection, the basal inflammatory imbalance contributes to exert severe damage to the mucosa, paving the way for bacterial colonization and subsequent steps of CF airway disease.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cystic Fibrosis; Fetal Tissue Transplantation; Fetus; Humans; Inflammation; Interleukin-8; Leukocytes; Mice; Mice, SCID; Microscopy, Electron; Pseudomonas Infections; Trachea; Transplantation, Heterologous

Endothelin (ET)-1 has been suggested to promote neutrophil adhesion to endothelium, migration to inflamed areas, and release of elastase. ET-1 might therefore play a role in the pathogenesis of bronchiectasis, a chronic inflammatory and infective airway disease which is still poorly understood. Thirty five patients with stable bronchiectasis (20 females, mean age+/-SD 49.1+/-15.0 yrs) and 18 control subjects (8 females, 49.4+/-11.3 yrs) were recruited prospectively. The ET-1 levels in serum and sputum were measured by commercially available enzyme linked immunosorbent assay (ELISA) kits. Patients with Pseudomonas aeruginosa in their sputum had a significantly higher serum level of ET-1 (median 25.8, interquartile range 13-43.9 pg x mL(-1)) than patients without P. aeruginosa (0, 0-10.5 pg x mL(-1); p=0.0004) and healthy control subjects (4.6, 0-16.3 pg x mL(-1); p=0.002). However, patients with and without P. aeruginosa infection had no significant difference in sputum ET-1 level (p=0.15). There was no correlation between serum or sputum ET-1 levels with the serum and sputum levels of the interleukin (IL)-1beta, IL-8 and tumour necrosis factor (TNF)-alpha; the number of bronchiectasis lung lobes; and spirometry. Serum ET-1 level correlated with 24 h sputum volume for the bronchiectasis patients (r=0.51, p=0.002). The results, therefore, suggest a significant pathogenic role for endothelin-1 among Pseudomonas aeruginosa-infected patients with bronchiectasis. Further studies should be performed to evaluate the clinico-pathological correlation and expression of endothelin-1 in bronchiectasis.

Topics: Bronchiectasis; Endothelin-1; Female; Humans; Interleukin-1; Interleukin-8; Leukocytes; Male; Middle Aged; Pancreatic Elastase; Prospective Studies; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Sputum; Tumor Necrosis Factor-alpha

To determine the effect of Pseudomonas aeruginosa exotoxin A (P-ExA) on cytokine production, we studied cytokine release induced by heat-killed P. aeruginosa (HKPA) in human whole blood in the presence or absence of P-ExA. P-ExA (0.01-1 microgram ml(-1)) caused a dose-dependent decrease in HKPA-induced production of tumor necrosis factor alpha (TNF), interleukin (IL-) 10, IL-6 and IL-8 (all P<0.05). P-ExA-induced inhibition of IL-10, IL-6 and IL-8 release was not dependent on reduced TNF concentrations, since the relative attenuation of the production of these cytokines was similar in the presence or absence of a neutralizing anti-TNF antibody. The effect of P-ExA on cytokine production may offer a disadvantage to the host with respect to clearance of the infection.

Topics: ADP Ribose Transferases; Bacterial Toxins; Cell Survival; Cytokines; Exotoxins; Hot Temperature; Humans; In Vitro Techniques; Interleukin-10; Interleukin-6; Interleukin-8; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Pseudomonas Infections; Tumor Necrosis Factor-alpha; Virulence; Virulence Factors

Various treatment regimens and difficulties with research design are encountered with cystic fibrosis (CF) because no standard diagnostic criteria exist for defining acute respiratory exacerbations. This study evaluated the role of serial monitoring of concentrations of selected cytokines and inflammatory mediators in serum and sputum as predictors of respiratory exacerbation, as useful outcome measures for CF, and to guide therapy. Interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), neutrophil elastase-alpha-1-protease inhibitor complex (NE complex), protein, and alpha-1-protease inhibitor (alpha-1-PI) were measured in serum and sputum collected from CF patients during respiratory exacerbations and periods of well-being. Levels of NE complex, protein, and alpha-1-PI in sputum rose during respiratory exacerbations and fell after institution of antibiotic therapy (P = 0.078, 0.001, and 0.002, respectively). Mean (+/- standard error of the mean) levels of IL-8 and TNF-alpha were extremely high in sputum (13,780 +/- 916 and 249.4 +/- 23.5 ng/liter, respectively) but did not change significantly with clinical deterioration of the patient (P > 0.23). IL-8 and TNF-alpha were generally undetectable in serum, and therefore these measures were unhelpful. Drop in forced expiratory volume in 1 s was the only clinical or laboratory parameter that was close to being a determinant of respiratory exacerbation (P = 0.055). This study provides evidence of intense immunological activity occurring continually within the lungs of adult CF patients. Measurement of cytokines and inflammatory mediators in CF sputum is not helpful for identifying acute respiratory exacerbations.

Topics: Acute Disease; Adolescent; Adult; alpha 1-Antitrypsin; Cystic Fibrosis; Disease Progression; Female; Humans; Inflammation Mediators; Interleukin-8; Leukocyte Elastase; Male; Pneumonia, Bacterial; Predictive Value of Tests; Pseudomonas Infections; Reproducibility of Results; Respiratory Function Tests; Sputum; Tumor Necrosis Factor-alpha

Airway infections initiated by the interaction of bacterial adhesins with carbohydrate receptors can be potentially prevented by nontoxic carbohydrate inhibitors. Intranasal inoculation of neonatal mice with Pseudomonas aeruginosa PAO1 caused pneumonia in 55% of control mice but in only 13% of mice inoculated 2 h after dextran inhalation (P<.001) and in 28% inoculated 4 h after dextran inhalation (P=.02). PAO1 adherence to epithelial cells was inhibited by 50% in the presence of dextran. Dextran was well distributed throughout the airways and stimulated tumor necrosis factor-alpha production in murine lungs but not interleukin-8 production by human epithelial cell lines. Phagocytosis of PAO1 was not affected by dextran nor was killing by human neutrophils diminished. Administration of dextran by aerosol may prevent murine pneumonia by impeding bacterial access to epithelial receptors and by stimulation of the immune functions of the epithelium.

Topics: Aerosols; Animals; Animals, Newborn; Bacterial Adhesion; Dextrans; Disease Models, Animal; Epithelial Cells; Interleukin-8; Lung; Mice; Mice, Inbred BALB C; Pneumonia, Bacterial; Polysaccharides; Pseudomonas Infections; Respiratory Therapy; Tumor Necrosis Factor-alpha

Recent studies suggest that inflammation plays a role in the pathogenesis of lung disease in cystic fibrosis (CF). The goal of the present study was to quantitatively compare bronchoalveolar lavage fluid (BALF) inflammation and its relation to bacterial infection, between children with CF and children with other chronic respiratory problems. Differential cell counts, immunoreactive interleukin 8 (IL-8), and quantitative bacterial cultures were done in BALF from 54 CF (median age 1.8 yr) and 55 control patients (median age 1.0 yr) who underwent bronchoscopy for clinical indications. Among infected CF patients, those with Pseudomonas aeruginosa did not have more inflammation than those without P. aeruginosa. The ratio of neutrophils or of IL-8 to bacteria in BALF was significantly greater for CF patients compared with control subjects, regardless of pathogen. Calculation of linear regression for either neutrophils or IL-8, as a function of bacterial quantity, yielded positive slopes for both CF and control patients, but with significant elevations for CF. We conclude that the inflammatory response to bacterial infection is increased or prolonged in CF compared with control patients, and that this increase is not necessarily due to pathogens specific for CF (e.g., P. aeruginosa). These data may provide further rationale for anti-inflammatory therapy early in CF.

Topics: Bronchoalveolar Lavage Fluid; Child; Child, Preschool; Colony Count, Microbial; Cystic Fibrosis; Female; Humans; Infant; Infant, Newborn; Inflammation Mediators; Interleukin-8; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Reference Values; Systemic Inflammatory Response Syndrome

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Topics: Acylation; Antimicrobial Cationic Peptides; Arabinose; Bacterial Proteins; Cells, Cultured; Cystic Fibrosis; Drug Resistance, Microbial; Humans; Infant; Interleukin-8; Lipid A; Lipopolysaccharides; Magnesium; Mutation; Palmitates; Peptides; Polymyxins; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory System; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Virulence

Chronic endobronchial inflammation and bacterial infection are the main causes of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive genetic disorder associated with improper function of chloride channels. Inflammation in CF lung is greatly amplified after Pseudomonas aeruginosa infection. In this study the relationship between P. aeruginosa status and inflammatory markers has been investigated. Seventeen CF children in acute lung exacerbation were examined. CF patients without P. aeruginosa infection were characterized by elevated activity of sputum elastase, reduced response of peripheral blood lymphocytes to PHA and significant resistance to the antiproliferative action of glucocorticoids. These parameters were normalized after antibiotic treatment. The patients with prolonged P. aeruginosa infection demonstrated extremely high levels of elastase activity and elevated amounts of sputum IL-8 and TNF-alpha. Although antibiotic treatment resulted in clinical improvement, it failed to suppress excessive immune response in the lung. The data indicate that CF patients with prolonged P. aeruginosa need the modified treatment, which should include immunomodulating drugs and protease inhibitors as well as antibacterial therapy.

Topics: Anti-Bacterial Agents; Cells, Cultured; Child; Cystic Fibrosis; Dexamethasone; Humans; Inflammation; Interleukin-8; Leukocyte Elastase; Lung Diseases; Lymphocyte Activation; Lymphocytes; Phytohemagglutinins; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum; Tumor Necrosis Factor-alpha; Vital Capacity

The neutrophil-dominated inflammation of the lung in cystic fibrosis (CF) has traditionally been seen as a physiological response to continuous opportunistic infection. Recent studies suggest, however, that regulation of the inflammatory response itself may be altered in CF. Neutrophil migration from the bloodstream involves alterations in surface expression of the adhesion molecules L-selectin and Mac-1 (CD11b/CD18). The aim of this study was to assess neutrophil adhesion molecule expression and responsiveness in CF. Neutrophils from chronic (n = 16) and acutely infected (n = 13) CF patients and 15 normal control subjects were directly assessed by Fluorescence-activated cell sorter (FACS) analysis for surface expression of L-selectin and CD11b before and after stimulation with interleukin 8 (IL-8) or f-Met-Leu-Phe (fMLP). Neutrophils from stable (n = 5) and acutely infected (n = 5) non-CF bronchiectasis patients were also assessed. Surface upregulation of CD11b was similar in all groups. Basal levels of L-selectin were also comparable among all groups, however, when stimulated, neutrophils from both stable and acutely infected CF patients shed significantly less L-selectin than those from control subjects (p < 0.05 and p < 0.01, respectively). This decreased responsiveness was not observed in either stable or acutely infected non-CF bronchiectasis patients. These results add to the accumulating evidence suggestive of a defective inflammatory response in CF.

Topics: Acute Disease; Adolescent; Adult; Aged; Bronchiectasis; CD11 Antigens; CD18 Antigens; Cell Movement; Cell Separation; Chemotaxis, Leukocyte; Chronic Disease; Cystic Fibrosis; Female; Flow Cytometry; Gene Expression Regulation; Humans; Interleukin-8; L-Selectin; Macrophage-1 Antigen; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Up-Regulation

We sought to identify which Pseudomonas aeruginosa products are involved initiating respiratory tract infection. Defined mutants derived from strain PAO i.e., PAOR1 (lasR),PAO-pmm (algC) (an LPS mutant), and AK1152 (which is Fla- and lacks functional pili), were significantly less virulent than PAO1 in a BALBc/ByJ neonatal mouse model of infection as measured by their abilities to cause acute pneumonia, bacteremia, and death. All three mutants were also less adherent to epithelial cells in an in vitro binding assay. PAOR1 and AK1152 were less able to elicit epithelial production of interleukin-8 than PAO1. LasR was found to be required for the optimal expression of neuraminidase under conditions of increased osmolarity, as might be present in certain pathological conditions. PAO-exsA::omega,, which lacks exoenzyme S expression, was fully virulent, causing at least as much pathology as PAO1. The expression of several P. aeruginosa virulence factors appears to be required to establish pulmonary infection in the neonatal mouse.

Topics: Animals; Animals, Newborn; Bacterial Adhesion; Bacterial Proteins; Cells, Cultured; Disease Models, Animal; DNA-Binding Proteins; Epithelial Cells; Fimbriae, Bacterial; Interleukin-8; Lipopolysaccharides; Lung; Mice; Mice, Inbred BALB C; Mutation; Phosphotransferases (Phosphomutases); Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Trans-Activators; Virulence

As collections of lower respiratory tract specimens from young children with cystic fibrosis (CF) are difficult, we determined whether oropharyngeal cultures predicted lower airway pathogens. During 1992-1994, 75 of 90 (83%) infants with CF diagnosed by neonatal screening had 150 simultaneous bronchoalveolar lavage (BAL) and oropharyngeal specimens collected for quantitative bacterial culture at a mean age of 17 months (range, 1-52). Ten children undergoing bronchoscopy for stridor served as controls. Total and differential cell counts and interleukin-8 concentrations were measured in BAL fluid. A subset of bacterial pathogens were typed by pulsed field gel electrophoresis. A non-linear relationship with inflammatory markers supported a diagnosis of lower airway infection when > or = 10(5) colony-forming units/ml were detected. This criterion was met in 47 (31%) BAL cultures from 37 (49%) children. Staphylococcus aureus (19%), Pseudomonas aeruginosa (11%), and Hemophilus influenzae (8%) were the major lower airway pathogens. In oropharyngeal cultures, S. aureus (47%), Escherichia coli (23%), H. influenzae (15%), and P. aeruginosa (13%) predominated. The sensitivity, specificity, and positive and negative predictive values of oropharyngeal cultures for pathogens causing lower respiratory infections were 82%, 83%, 41%, and 97%, respectively. When there was agreement between paired oropharyngeal and BAL cultures, genetic fingerprinting showed some strains of the same organism were unrelated. We conclude that oropharyngeal cultures do not reliably predict the presence of bacterial pathogens in the lower airways of young CF children.

Topics: Bacteriological Techniques; Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Escherichia coli Infections; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Interleukin-8; Male; Oropharynx; Pneumonia, Bacterial; Predictive Value of Tests; Pseudomonas Infections; Respiratory Tract Infections; Staphylococcal Infections

Bronchopulmonary disease in patients with cystic fibrosis (CF) is a paradigm of neutrophil-dominated airway inflammation. We hypothesized that proinflammatory cytokines contribute to a localized neutrophil-dominated inflammatory state as present in CF airways. In a cross-sectional study, we analyzed 63 sputum samples from 33 CF patients for concentrations of the cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), and granulocyte-colony stimulating factor (G-CSF) by means of enzyme-linked immunosorbent assay. Furthermore, the activity of neutrophil elastase (NE) in the sputum samples was determined using a specific chromogenic substrate. Compared to sputum samples from 10 healthy controls, there were significantly increased concentrations of IL-1 beta, IL-8 and TNF-alpha in the CF sputum samples. The concentration of IL-8 correlated significantly with NE activity in the CF sputum samples. In CF patients with airways chronically colonized with Pseudomonas aeruginosa, IL-8 concentrations in sputum were significantly enhanced. In glucocorticoid-treated patients, IL-1 alpha and G-CSF sputum concentrations were significantly lower when compared to levels in the other patients. These results show that there are high concentrations of proinflammatory cytokines in CF airways which may contribute to the localized neutrophil-dominated inflammatory state found clinically.

Topics: Adolescent; Adult; Child; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Female; Glucocorticoids; Granulocyte Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-8; Interleukins; Leukocyte Elastase; Lung; Male; Neutrophils; Pancreatic Elastase; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum; Tumor Necrosis Factor-alpha

Since 1973, the occurrence of respiratory tract infections due to P. aeruginosa has increased associated with the development of broad-spectrum penicillins. A clinical entity, diffuse panbronchiolitis (DPB) is a representative disease of chronic P. aeruginosa infections in Japan. In this paper, recent advances of research on pathogenesis and treatments of chronic P. aeruginosa lower respiratory tract infections in our department are reported. We examined sputum from patients with chronic P. aeruginosa infections under the electron microscope. Mucoid type of microcolonies were observed with fibrous matrix of exopolysaccharide. Neutrophils were found to be partially surrounding the microcolony in an attempt to defense. Debris was formed mainly by the destruction of the neutrophils. Most neutrophils were found full of phagocytized debris. These data support that instead of phagocytizing bacteria, neutrophils phagocytized debris and bacteria were not completely eradicated. This might be a factor in the pathogenesis of persistent colonization of P. aeruginosa. In the airways of patients with chronic airway diseases (CAD), neutrophils enhance the recruitment of more neutrophils through the production of neutrophil chemotactic factors such as interleukin-8 (IL-8) and LTB4, perpetuating a cycle of inflammation in the lung. We demonstrated increased levels of IL-8, a chemotactic cytokine, in bronchoalveolar lavage (BAL) fluid from patients with CAD associated with P. aeruginosa infections. We also documented a significant correlation between neutrophil numbers and IL-8 levels or IL-1 beta levels or neutrophil elastase levels in BAL fluids from patients with CAD. By immunohistochemical studies and in vitro data, three major sources of IL-8 in the airways of CAD patients were found to be alveolar macrophages, bronchial epithelial cells, and migrated neutrophils. In Japan, the clinical effectiveness of oral erythromycin (EM) for CAD, including DPB seems to be established, but its pharmacological mechanism remains unclear. In addition, we found a marked decrease of IL-8 levels in BAL fluid from two patients with CAD after treatment with EM. Therefore, we postulated that EM inhibited IL-8 production by stimulated respiratory cells. EM and Roxythromycin, suppressed IL-8 production in Pseudomonas-stimulated neutrophils in a dose-dependent manner. 1 alpha, 25-dihydroxy vitamin D3 also inhibited neutrophil-derived IL-8. Our data encourage the development of new anti-IL-8 a

Topics: Anti-Bacterial Agents; Bronchiolitis; Bronchoalveolar Lavage Fluid; Chronic Disease; Cytokines; Erythromycin; Humans; Interleukin-8; Pseudomonas aeruginosa; Pseudomonas Infections; Radiography; Respiratory Tract Infections

Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) concentrations were measured in faecal samples from nine patients with cystic fibrosis and nine healthy age matched controls. The patients were assessed with Shwachman score, apparent energy absorption, pancreatic enzyme dosage, simple spirometry, and presence of pseudomonal colonisation. Median (range) wet stool IL-8 and TNF-alpha concentrations in patients were 32,113 pg/g (21,656-178,128) and 3187 pg/g (368-17,611) respectively, compared with < 43.5 pg (IL-8)/g (< 22-4079) and 99 pg (TNF-alpha)/g (< 0.26-231) in controls. IL-8 concentration was negatively correlated with Shwachman score (r = -0.79) and pancreatic enzyme dosage (r = -0.77), but not with energy absorption. Seven patients were mature enough to cooperate with spirometry. Their IL-8 concentrations correlated with percentage predicted forced expiratory volume in one second (r = -0.78). IL-8 concentration was greater in four patients with, than five without, established pseudomonal colonisation: median difference 134,583 pg/g. TNF-alpha concentration was not correlated with measures of disease severity. Faecal IL-8 concentration might reflect the severity of pulmonary inflammation in cystic fibrosis and could provide an easily obtainable marker of disease activity.

Topics: Adolescent; Adult; Biomarkers; Child; Cystic Fibrosis; Feces; Humans; Interleukin-8; Lipase; Lung; Pancreatic Extracts; Pancrelipase; Pseudomonas Infections; Spirometry; Tumor Necrosis Factor-alpha

Pseudomonas aeruginosa infections are commonly observed in sepsis, burns, as well as cystic fibrosis (CF). Among the professional phagocytes neutrophils and monocytes are recruited by various chemotactic factors from the cellular environment. Although they provide the first line of host defense excessive neutrophil accumulation seems to be a major cause of pathogenesis during P. aeruginosa infection. Interleukin-8 (IL-8) represents one important chemoattractant for professional phagocytes. To evaluate IL-8 releasability by phagocytes in the context of P. aeruginosa infection and especially of CF, we stimulated human polymorphonuclear neutrophilic granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) as a source for monocytes with clinical P. aeruginosa isolates, with mucoid P. aeruginosa strain (CF3M) and its nonmucoid revertant (CF3), and with purified P. aeruginosa mucoid exopolysaccharide (alginate). A significant increase in IL-8 release as compared to unstimulated cells was observed after an incubation time of 90 min for PMN and after 60 min for PBMC which increased (PMN: up to 60-fold; PBMC: up to 40-fold) over time (up to 4 h). In contrast of PBMC, when PMN were studied, intracellular IL-8 exceeded the IL-8 release in unstimulated as well as in stimulated cells by up to 10-fold. All clinical P. aeruginosa isolates, independent of the clinical source, induced IL-8 release from human PBMC and PMN in a dose- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alginates; Chemotaxis; Humans; Interleukin-8; Phagocytes; Polysaccharides, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections

To evaluate of the role of interleukin-8 (IL-8), a chemotactic cytokine, in the continuous neutrophil accumulation in the airways of patients with chronic airway disease (CAD) and persistent Pseudomonas aeruginosa infection, we investigated the cell population, IL-8 levels, IL-1 beta levels, tumor necrosis factor (TNF) activities, and neutrophil elastase (NE) activities of bronchoalveolar lavage (BAL) fluids in 17 CAD patients (with P. aeruginosa infections [CAD+PA], n = 9; without any bacterial infections [CAD-PA], n = 8) and 8 normal volunteers. We found significant elevations of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from CAD patients, in the following rank order: CAD+PA > CAD-PA > normal volunteers. IL-1 beta/albumin ratios were elevated only in CAD+PA, while no TNF bioactivity was detected in BAL fluids. The neutrophil numbers correlated significantly with the IL-8/albumin ratios and NE/albumin ratios in the BAL fluids of CAD patients. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in BAL fluids, the mean reduction rate of NCF activities in CAD+PA patients was significantly higher than that in CAD-PA patients. We also evaluated the effects of low-dose, long-term erythromycin therapy in BAL fluids from three CAD+PA and two CAD-PA patients. Treatment with erythromycin caused significant reductions of neutrophil numbers, IL-8/albumin ratios, and NE/albumin ratios in BAL fluids from these patients. To elucidate the mechanism of erythromycin therapy, we also examined whether erythromycin suppressed IL-8 production by human alveolar macrophages and neutrophils in vitro. We demonstrated a moderate inhibitory effect of erythromycin on IL-8 production in Pseudomonas-stimulated neutrophils but not in alveolar macrophages. Our data support the view that persistent P. aeruginosa infection enhances IL-8 production and IL-8-derived NCF activity, causing neutrophil accumulation in the airways and the progressive lung injuries observed in patients with CAD. The clinical efficacy of erythromycin therapy for CAD patients might be partly mediated through a reduced IL-8 production, diminishing neutrophil accumulation and NE release in the airways.

Topics: Adult; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chronic Disease; Erythromycin; Female; Humans; Interleukin-1; Interleukin-8; Leukocyte Elastase; Male; Neutrophils; Pancreatic Elastase; Pseudomonas Infections; Respiratory Tract Diseases; Tumor Necrosis Factor-alpha

Polymorphonuclear leukocytes (PMNL) in bronchoalveolar lavage (BAL) fluid of cystic fibrosis patients express increased levels of receptor for the Fc portion of IgA (Fc alpha R), similar to those found on PMNL stimulated with FMLP in vitro. Since tumor necrosis factor-alpha (TNF alpha) is an activator of PMNL and is found at inflammatory sites, its effects on Fc alpha R expression and IgA-mediated PMNL functions were investigated. Exposure of PMNL to TNF alpha increased surface expression of Fc alpha R 2- to 3-fold, increased superoxide production in response to aggregated IgA 5-fold, and increased phagocytosis of IgA aggregates 3- to 4-fold. Interleukin-8 did not increase Fc alpha R expression and did not enhance IgA-mediated superoxide generation. IgA-dependent PMNL killing of Pseudomonas aeruginosa was enhanced when PMNL were pretreated with TNF alpha. Thus, interactions between phagocytic host defense mechanisms and mucosal IgA may be enhanced by TNF alpha in the inflammatory milieu of the cystic fibrosis lung.

Topics: Adolescent; Adult; Cystic Fibrosis; Humans; Immunoglobulin A; Immunoglobulin G; Interleukin-8; Neutrophils; Phagocytosis; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Fc; Superoxides; Tumor Necrosis Factor-alpha

The effect of treatment with interleukin-8 (IL-8), a neutrophil-activating cytokine, was investigated in normal and neutropenic mice infected with a lethal dose of Pseudomonas aeruginosa, Klebsiella pneumoniae, or Plasmodium berghei. Intraperitoneal (i.p.) IL-8 treatment was associated with accelerated death when IL-8 was administered shortly before i.p. infection with P. aeruginosa or shortly after i.p. infection with P. aeruginosa and K. pneumoniae. Histopathological analyses demonstrated a tendency to more severe organ lesions in IL-8-treated mice. Only nonneutropenic mice that received IL-8 shortly before the infectious challenge and at the site of infection were protected by IL-8. Whether IL-8 is protective of or detrimental to the survival of infection appeared to depend on the presence of bacteria at the injection site and on the presence of neutropenia. IL-8 may be an important participant in the cascade of interacting cytokines that is induced by the lethal infectious challenge.

Topics: Animals; Bacterial Infections; Brain; Female; Interleukin-8; Klebsiella Infections; Klebsiella pneumoniae; Leukocyte Count; Malaria; Mice; Neutropenia; Peritoneal Cavity; Plasmodium berghei; Pseudomonas Infections

Patients suffering from serious bacterial infection present to the hospital after early inflammatory events, such as release of tumor necrosis factor (TNF), have been initiated. The role of other cytokines, such as interleukin-8 (IL-8), a neutrophil chemoattractant and activator, in the pathophysiology of human sepsis is not well characterized, and there are only limited data on IL-6. We studied serial concentrations of TNF, IL-6 (involved in the acute-phase response), and IL-8 in plasma and leukocyte levels of mRNA for these cytokines in patients with localized and septicemic Pseudomonas pseudomallei infection on admission to the hospital and during a prolonged recovery phase (up to 30 days). Of 18 patients, 8 had detectable plasma IL-8 and all had raised plasma IL-6 concentrations. In patients who died median initial concentration of IL-8 (167 pg/ml; range, 97 to 362 pg/ml) and IL-6 (4,800 pg/ml; range, 60 to 9,245 pg/ml) in plasma were higher than those in survivors (P less than 0.008 and P = 0.007, respectively). Septic patients who survived and patients with localized disease had similar cytokine levels. Plasma IL-8 and IL-6 concentrations were elevated throughout the inpatient period of recovery. Circulating leukocytes contained mRNA for IL-8 but not for IL-6 and TNF, and they may secrete IL-8. An elevated plasma IL-6 concentration (greater than 1,000 pg/ml) had 75% mortality) was the best predictor of mortality in P. pseudomallei sepsis. Fifty percent of patients with detectable plasma IL-8 concentrations died. In contrast, plasma TNF bioactivity did not relate to outcome; 75% of patients who did never had detectable plasma TNF activity.

Topics: Adult; Aged; Burkholderia pseudomallei; Female; Humans; Interleukin-6; Interleukin-8; Leukocytes; Male; Middle Aged; Pseudomonas Infections; RNA, Messenger; Sepsis; Tumor Necrosis Factor-alpha

Topics: Bacterial Infections; Blood Bactericidal Activity; Chemotactic Factors; Exotoxins; Humans; Interleukin-8; Leukotriene B4; Neutrophils; Pseudomonas Infections; Staphylococcal Infections