On dysregulated inflammation and airway host defense

Acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) are characterized by dysregulated inflammation of the airways. The increased influx of immune cells and the accumulation of cytokines lead to cell death, tissue destruction and impaired pulmonary function. Intracellular proteins like histones and granule proteins of neutrophils, DNA fibers act as Danger-Associated Molecular Patterns (DAMPs), further promoting tissue damage. As a result, the lungs of such patients are at an increased risk of infection due to impaired host defense functions. During inflammation, there is an increased expression of osteopontin (OPN), a highly anionic phosphoglycoprotein, in the airways and it is involved in cell recruitment, tissue remodeling, and repair. In this thesis we show that OPN can interact with many cationic proteins and peptides present in the extracellular milieu of the inflamed airways. In the first paper included in this thesis we show that OPN bound to extracellular histones have protective function against DAMPs-induced inflammation. In the second paper, we show that OPN binds to several common innate antibiotics and abrogate their antimicrobial activities. Taken together, these data suggest that OPN can modulate the host immune functions, thereby increasing the susceptibility of the patients with airway inflammatory diseases to acquire infections.Use of anti-inflammatory drugs like roflumilast is a common treatment strategy in COPD to ameliorate severe exacerbations. In the third paper we highlight the adverse effects of roflumilast, in a murine acute airway infection model. The findings suggest that use of this drug can impair host defense functions of immune cells, thereby increasing the susceptibility of COPD patients to bacterial pathogens. DNase I is used to clear the airways of CF patients from highly viscous, high molecular weight eDNA rich sputum. In the fourth paper of this thesis, we elucidated the molecular aspects of the fragmented DNA that are important to exhibit antimicrobial properties against the common CF lung pathogen, i.e. P. aeruginosa. The findings highlight a novel aspect of host defense that could be employed treating bacteria resistant against conventional antibiotics

DNA-fragmentation is a source of bactericidal activity against Pseudomonas aeruginosa

Pseudomonas aeruginosa airway infection is common in cystic fibrosis (CF), a disease also characterized by abundant extracellular DNA (eDNA) in the airways. The eDNA is mainly derived from neutrophils accumulating in the airways and contributes to a high sputum viscosity. The altered environment in the lower airways also paves the way for chronic P. aeruginosa infection. Here, we show that mice with P. aeruginosa airway infection have increased survival and decreased bacterial load after topical treatment with DNase. Furthermore, DNA from the sputum of CF patients showed increased bactericidal activity after treatment with DNase ex vivo. Both degraded DNA of neutrophil extracellular traps (NETs) and genomic DNA degraded by serum, acquired bactericidal activity against P. aeruginosa. In vitro, small synthetic DNA-fragments (<100 base pairs) but not large fragments nor genomic DNA, were bactericidal against Gram-negative but not Grampositive bacteria. The addition of divalent cations reduced bacterial killing, suggesting that chelation of divalent cations by DNA results in destabilization of the lipopolysaccharide (LPS) envelope. This is a novel antibacterial strategy where fragmentation of eDNA and DNA-fragments can be used to treat P. aeruginosa airway infection

Zoledronic Acid Targeting of the Mevalonate Pathway Causes Reduced Cell Recruitment and Attenuates Pulmonary Fibrosis

Background and aim: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreparable scarring of lung tissue, with most patients succumbing rapidly after diagnosis. The mevalonate pathway, which is involved in the regulation of cell proliferation, survival, and motility, is targeted by the bisphosphonate zoledronic acid (ZA). The aim of this study was to assess the antifibrotic effects of ZA and to elucidate the mechanisms by which potential IPF treatment occurs.Methods: A series of in vitro and in vivo models were employed to identify the therapeutic potential of ZA in treating IPF. In vitro transwell assays were used to assess the ability of ZA to reduce fibrotic-related immune cell recruitment. Farnesyl diphosphate synthase (FDPS) was screened as a potential antifibrotic target using a bleomycin mouse model. FDPS-targeting siRNA and ZA were administered to mice following the onset of experimentally-induced lung fibrosis. Downstream analyses were conducted on murine lung tissues and lung fluids including 23-plex cytokine array, flow cytometry, histology, Western blotting, immunofluorescent staining, and PCR analysis.Results: In vitro administration of ZA reduced myofibroblast transition and blocked NF-κB signaling in macrophages leading to impaired immune cell recruitment in a transwell assay. FDPS-targeting siRNA administration significantly attenuated profibrotic cytokine production and lung damage in a murine lung fibrosis model. Furthermore, ZA treatment of mice with bleomycin-induced lung damage displayed decreased cytokine levels in the BALF, plasma, and lung tissue, resulting in less histologically visible fibrotic scarring. Bleomycin-induced upregulation of the ZA target, FDPS, was reduced in lung tissue and fibroblasts upon ZA treatment. Confirmatory increases in FDPS immunoreactivity was seen in human IPF resected lung samples compared to control tissue indicating potential translational value of the approach. Additionally, ZA polarized macrophages towards a less profibrotic phenotype contributing to decreased IPF pathogenesis.Conclusion: This study highlights ZA as an expedient and efficacious treatment option against IPF in a clinical setting

Pseudomonas aeruginosa elastase cleaves a C-terminal peptide from human thrombin that inhibits host inflammatory responses

Pseudomonas aeruginosa is an opportunistic pathogen known for its immune evasive abilities amongst others by degradation of a large variety of host proteins. Here we show that digestion of thrombin by P. aeruginosa elastase leads to the release of the C-terminal thrombin-derived peptide FYT21, which inhibits pro-inflammatory responses to several pathogen-associated molecular patterns in vitro and in vivo by preventing toll-like receptor dimerization and subsequent activation of down-stream signalling pathways. Thus, P. aeruginosa ‘hijacks’ an endogenous anti-inflammatory peptide-based mechanism, thereby enabling modulation and circumvention of host responses.Published versio

Citrullination of extracellular histone H3.1 reduces antibacterial activity and exacerbates its proteolytic degradation

BACKGROUND: Cystic fibrosis (CF), involves excessive airway accumulation of neutrophils, often in parallel with severe infection caused by Pseudomonas aeruginosa. Free histones are known to possess bactericidal properties, but the degree of antibacterial activity exerted on specific lung-based pathogens is largely unknown. Neutrophils have a high content of peptidyl deiminase 4 (PADI4), which citrullinate cationic peptidyl-arginines. In histone H3.1, several positions in the NH2-terminal tail are subject to citrullination.METHODS: Full-length and segmented histone subunit H3.1 was investigated for bactericidal activity towards P. aeruginosa (strain PAO1). PADI4-induced citrullination of histone H3.1 was assessed for antibacterial activity towards P. aeruginosa. Next, the effect of neutrophil elastase (NE)-mediated proteolysis of histone H3.1 was investigated. Finally, PADI4, H3.1, and citrullinated H3.1 were examined in healthy control and CF patient lung tissues.RESULTS: Full-length histone H3.1 and sections of the histone H3.1 tail, displayed bactericidal activity towards P. aeruginosa. These antibacterial effects were reduced following citrullination by PADI4 or proteolysis by NE. Interestingly, citrullination of histone H3.1 exacerbated NE-mediated degradation. In CF lung tissue, citrullinated histone H3.1 and PADI4 immunoreactivity was abundant. Degraded histone H3.1 was detected in the sputum of CF patients but was absent in the sputum of healthy controls.CONCLUSIONS: Citrullination impairs the antibacterial activity of histone H3.1 and exacerbates its proteolytic degradation by NE. Citrullination is likely to play an important role during resolution of acute inflammation. However, in chronic inflammation akin to CF, citrullination may dampen host defense and promote pathogen survival, as exemplified by P. aeruginosa

Tartrate resistant acid phosphatase 5 (TRAP5) mediates immune cell recruitment in a murine model of pulmonary bacterial infection

Introduction: During airway infection, upregulation of proinflammatory cytokines and subsequent immune cell recruitment is essential to mitigate bacterial infection. Conversely, during prolonged and non-resolving airway inflammation, neutrophils contribute to tissue damage and remodeling. This occurs during diseases including cystic fibrosis (CF) and COPD where bacterial pathogens, not least Pseudomonas aeruginosa, contribute to disease progression through long-lasting infections. Tartrate-resistant acid phosphatase (TRAP) 5 is a metalloenzyme expressed by alveolar macrophages and one of its target substrates is the phosphoglycoprotein osteopontin (OPN).Methods: We used a knockout mouse strain (Trap5-/-) and BALB/c-Tg (Rela-luc)31Xen mice paired with siRNA administration or functional protein add-back to elucidate the role of Trap5 during bacterial infection. In a series of experiments, Trap5-/- and wild-type control mice received intratracheal administration of P.aerugniosa (Xen41) or LPS, with mice monitored using intravital imaging (IVIS). In addition, multiplex cytokine immunoassays, flow cytometry, multispectral analyses, histological staining were performed.Results: In this study, we found that Trap5-/- mice had impaired clearance of P. aeruginosa airway infection and reduced recruitment of immune cells (i.e. neutrophils and inflammatory macrophages). Trap5 knockdown using siRNA resulted in a decreased activation of the proinflammatory transcription factor NF-κB in reporter mice and a subsequent decrease of proinflammatory gene expression. Add-back experiments of enzymatically active TRAP5 to Trap5-/- mice restored immune cell recruitment and bacterial killing. In human CF lung tissue, TRAP5 of alveolar macrophages was detected in proximity to OPN to a higher degree than in normal lung tissue, indicating possible interactions.Discussion: Taken together, the findings of this study suggest a key role for TRAP5 in modulating airway inflammation. This could have bearing in diseases such as CF and COPD where excessive neutrophilic inflammation could be targeted by pharmacological inhibitors of TRAP5

OPN expression and co-localization with antimicrobial proteins in COPD lung tissues.

<p>(A) Multiple cells in the bronchiolar epithelium expressed OPN (brown-colored DAB). (I) Goblet cells, the inset shows OPN expression in the goblet cell mucus. (II) Bronchial submucosal glands (Br = bronchial epithelium. (III) OPN expression in some basal cells (arrowhead). (IV) Flask-shaped cells (arrowhead). Primary antibodies from non-immunized animals resulted in loss of labeling (not shown). Scale bars: (I) = 15 μm; (II) = 70 μm; (III, IV) = 20 μm. (B) Immunohistochemical staining for OPN and the AMPs lactoferrin, lysozyme, and SLPI performed on parallel sections of lung tissue obtained from a patients with COPD (GOLD stage IV). Immunoreactivity is visualized by a brown-colored DAB staining in the bronchiolar epithelium and also in the airway lumen of COPD lungs. OPN, lactoferrin, and SLPI are all detected both in bronchiolar epithelium (Ep) and in cellular debris and mucus of the lumen (Lu) while lysozyme is present only in the lumen and to a lesser extent in the airway epithelium. The latter is explained by its preferential expression in the submucosal glands of large airways (not shown). Cell nuclei are counterstained with Mayer’s hematoxylin (blue stain). The scale bar in the right panel of the bottom figure is 100 μm.</p

OPN impairs the bactericidal activity of AMPs.

<p>(A) Interference of OPN with the bactericidal activity of AMPs was investigated using viable counts with <i>S</i>. <i>pneumoniae</i> and <i>P</i>. <i>aeruginosa</i>. Bacteria were grown to mid-logarithmic phase and incubated with AMPs alone (3 μM) or AMPs pre-incubated with OPN at ratio of 1:1 for one hour at 37°C. OPN caused inhibition of the bactericidal activity of all AMPs investigated except for LL-37. The histograms represent mean and standard deviation from three separate experiments. Two-way ANOVA with Sidak’s multiple comparisons test was used for statistical analysis. *<i>P</i>≤0.05, and ****<i>P</i> ≤ 0.0001. (B) Scanning electron microscopy images shows the morphology of bacteria after incubation with AMPs alone or AMPs pre-incubated with OPN (1:1). The AMPs alone permeabilized the membrane and caused leakage of intracellular contents indicating killing of the bacteria, which was confirmed in a parallel viable counts assay. Upon co-incubation of AMPs and OPN, the bacteria remained intact, except in the case of LL-37 and to some extent in the cases of <i>P</i>. <i>aeruginosa</i> (OPN with lactoferrin, lysozyme, and midkine). The scale bars in bottom figures in the right panels are 5 μm.</p

OPN does not influence the muramidase and protease inhibitory functions of lysozyme and SLPI respectively.

<p>(A) Effect of OPN on muramidase activity of lysozyme. Lysozyme was pre-incubated with or without OPN at equimolar concentrations and fluorogenic substrate was added to the mixture and incubated for 1 h at 37°C. The lysozyme activity was determined by the development of fluorescence, which is represented as relative fluorescence units (RFU). (B) Neutrophil elastase inhibitory property of SLPI was investigated in presence of OPN. Equimolar concentrations of OPN and SLPI were pre-incubated for 1 h at 37°C. This mixture was incubated with human neutrophil elastase (NE) (0.05 U/ml) for 20 min at RT. The NE activity was determined by a chromogenic substrate solution by recording the absorbance at 405 nm. The above experiments suggest that OPN cannot influence the enzymatic activities of lysozyme and SLPI.</p