interleukin-8 and Pulmonary-Aspergillosis

To study interactions of airborne pathogens, e.g. Aspergillus (A.) fumigatus with upper and lower respiratory tract epithelial and immune cells, we set up a perfused 3D human bronchial and small airway epithelial cell system. Culturing of normal human bronchial or small airway epithelial (NHBE, SAE) cells under air liquid interphase (ALI) and perfusion resulted in a significantly accelerated development of the lung epithelia associated with higher ciliogenesis, cilia movement, mucus-production and improved barrier function compared to growth under static conditions. Following the accelerated differentiation under perfusion, epithelial cells were transferred into static conditions and antigen-presenting cells (APCs) added to study their functionality upon infection with A. fumigatus. Fungi were efficiently sensed by apically applied macrophages or basolaterally adhered dendritic cells (DCs), as illustrated by phagocytosis, maturation and migration characteristics. We illustrate here that perfusion greatly improves differentiation of primary epithelial cells in vitro, which enables fast-track addition of primary immune cells and significant shortening of experimental procedures. Additionally, co-cultured primary DCs and macrophages were fully functional and fulfilled their tasks of sensing and sampling fungal pathogens present at the apical surface of epithelial cells, thereby promoting novel possibilities to study airborne infections under conditions mimicking the in vivo situation.

Topics: Antigen-Presenting Cells; Aspergillus fumigatus; Cell Culture Techniques; Dendritic Cells; Host-Pathogen Interactions; Humans; Immunophenotyping; Interleukin-8; Macrophages; Phagocytosis; Phenotype; Pulmonary Aspergillosis; Respiratory Mucosa

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses.

Topics: Alveolar Epithelial Cells; Area Under Curve; Aspergillus fumigatus; Biomarkers; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cell Line, Tumor; Complement Activation; Complement C3b; Complement Pathway, Mannose-Binding Lectin; Glycoproteins; Host-Pathogen Interactions; Humans; Immunity, Innate; Interleukin-8; Lectins; Lung; MAP Kinase Signaling System; Pneumonia; Predictive Value of Tests; Pulmonary Aspergillosis; ROC Curve; Up-Regulation

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

Arginine depletion via myeloid cell arginase is critically involved in suppression of the adaptive immune system during cancer or chronic inflammation. On the other hand, arginine depletion is being developed as a novel anti-tumor metabolic strategy to deprive arginine-auxotrophic cancer cells of this amino acid. In human immune cells, arginase is mainly expressed constitutively in PMNs. We therefore purified human primary PMNs from healthy donors and analyzed PMN function as the main innate effector cell and arginase producer in the context of arginine deficiency. We demonstrate that human PMN viability, activation-induced IL-8 synthesis, chemotaxis, phagocytosis, generation of ROS, and fungicidal activity are not impaired by the absence of arginine in vitro. Also, profound pharmacological arginine depletion in vivo via ADI-PEG20 did not inhibit PMN functions in a mouse model of pulmonary invasive aspergillosis; PMN invasion into the lung, activation, and successful PMN-dependent clearance of Aspergillus fumigatus and survival of mice were not impaired. These novel findings add to a better understanding of immunity during inflammation-associated arginine depletion and are also important for the development of therapeutic arginine depletion as anti-metabolic tumor therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Arginase; Arginine; Aspergillus fumigatus; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemotaxis, Leukocyte; Citrulline; Humans; Hydrolases; Immunity, Innate; Interleukin-8; Lung; Mice; Mice, Inbred C57BL; Neutrophils; Phagocytosis; Polyethylene Glycols; Primary Cell Culture; Pulmonary Aspergillosis; Reactive Oxygen Species; Respiratory Burst

Invasive fungal infections (IFIs) are a major cause of death in organ transplant patients. The murine hydrocortisone-mediated immunosuppression model of pulmonary aspergillosis is commonly used to characterise IFIs in these patients. However, this model does not take into account the effects of calcineurin inhibitors on transplant immunity to IFIs or the fungal calcineurin pathway, which is required for both virulence and antifungal drug resistance. To address these two issues, a new and clinically relevant transplant immunosuppression model of tacrolimus (FK506) and hydrocortisone-associated pulmonary aspergillosis was developed. We first characterised IFIs in 406 patients with a lung transplant. This showed that all of the patients with pulmonary aspergillosis were immunosuppressed with calcineurin inhibitors and steroids. Murine pharmacokinetic studies demonstrated that an ideal dose of 1 mg/kg/day of FK506 intraperitoneally produced blood trough levels in the human therapeutic range (5-12 ng/ml). There was increased mortality from pulmonary aspergillosis in a transplant-relevant immunosuppression model using both FK506 and hydrocortisone as compared with immunosuppression using hydrocortisone only. Lung histopathology showed neutrophil invasion and tracheobronchitis that was associated with reduced lung tumour necrosis factor-α (TNFα), JE (homologue of human MCP-1) and KC (homologue of human IL-8) at 24 hours, but increased lung TNFα, JE and KC at 48 hours when fungal burden was high. Furthermore, FK506 directly impaired fungal killing in alveolar macrophages in vitro, with FK506-mediated inhibition of the radial growth of Aspergillus fumigatus in vitro occurring at the low concentration of 5 ng/ml. Taken together, these findings show that the immunosuppressive activity of FK506 outweighs its antifungal activity in vivo. These observations demonstrate that FK506 impairs innate immune responses and leads to an incremental increase in susceptibility to IFIs when it is combined with steroids. This new and clinically relevant mouse model of invasive aspergillosis is a valuable addition to the further study of both fungal immunity and antifungal therapy in organ transplantation.

Topics: Animals; Aspergillus fumigatus; Chemokine CCL2; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Hydrocortisone; Immunosuppression Therapy; Immunosuppressive Agents; Injections, Intraperitoneal; Interleukin-8; Lung Transplantation; Male; Mice; Pneumonia; Pulmonary Aspergillosis; Risk Factors; Steroids; Survival Analysis; Tacrolimus; Tumor Necrosis Factor-alpha

It is not known whether antibiotic therapy for lung disease in cystic fibrosis (CF) has an influence on circulating polymorphonuclear neutrophil (PMN) function and apoptosis.. Blood PMNs were obtained from 14 CF patients before and after antibiotic treatment for an acute exacerbation, and from 10 healthy controls. PMNs were evaluated for production of reactive oxygen species (ROS) by spectrophotometry, of cytokines in the conditioned medium by ELISA, and apoptotic response by cytofluorimetry.. ROS and interleukin (IL)-8 were produced at higher levels by CF PMNs pre-therapy than control PMNs under basal conditions. IL-8 levels further increased after therapy. Early apoptotic response was higher in CF PMNs pre-therapy than in control PMNs, and this pattern did not change after antibiotic treatment.. Circulating PMNs are primed in CF acute patients. Further studies are needed to consider PMN-produced IL-8 as a biomarker to evaluate response to antibiotic therapy in CF patients.

Topics: Adolescent; Adult; Anti-Bacterial Agents; Antifungal Agents; Apoptosis; Biomarkers; Child; Cystic Fibrosis; Drug Monitoring; Female; Humans; Interleukin-8; Male; Neutrophils; Pilot Projects; Pneumonia, Bacterial; Pulmonary Aspergillosis; Reactive Oxygen Species; Young Adult