Assessment of bacterial exposure on phagocytic capability and surface marker expression of sputum macrophages and neutrophils in COPD patients.

Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte-derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non-typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never-smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10-fold more colony-forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD.

Airway host-microbiome interactions in chronic obstructive pulmonary disease.

BACKGROUND: Little is known about the interactions between the lung microbiome and host response in chronic obstructive pulmonary disease (COPD). METHODS: We performed a longitudinal 16S ribosomal RNA gene-based microbiome survey on 101 sputum samples from 16 healthy subjects and 43 COPD patients, along with characterization of host sputum transcriptome and proteome in COPD patients. RESULTS: Dysbiosis of sputum microbiome was observed with significantly increased relative abundance of Moraxella in COPD versus healthy subjects and during COPD exacerbations, and Haemophilus in COPD ex-smokers versus current smokers. Multivariate modeling on sputum microbiome, host transcriptome and proteome profiles revealed that significant associations between Moraxella and Haemophilus, host interferon and pro-inflammatory signaling pathways and neutrophilic inflammation predominated among airway host-microbiome interactions in COPD. While neutrophilia was positively correlated with Haemophilus, interferon signaling was more strongly linked to Moraxella. Moreover, while Haemophilus was significantly associated with host factors both in stable state and during exacerbations, Moraxella-associated host responses were primarily related to exacerbations. CONCLUSIONS: Our study highlights a significant airway host-microbiome interplay associated with COPD inflammation and exacerbations. These findings indicate that Haemophilus and Moraxella influence different components of host immune response in COPD, and that novel therapeutic strategies should consider targeting these bacteria and their associated host pathways in COPD.

The role of the liver X receptor in chronic obstructive pulmonary disease.

BACKGROUND: There is a need for novel anti-inflammatory therapies to treat COPD. The liver X receptor (LXR) is a nuclear hormone receptor with anti-inflammatory properties. METHODS: We investigated LXR gene and protein expression levels in alveolar macrophages and whole lung tissue from COPD patients and controls, the effect of LXR activation on the suppression of inflammatory mediators from LPS stimulated COPD alveolar macrophages, and the effect of LXR activation on the induction of genes associated with alternative macrophage polarisation. RESULTS: The levels of LXR mRNA were significantly increased in whole lung tissue extracts in COPD patients and smokers compared to non-smokers. The expression of LXR protein was significantly increased in small airway epithelium and alveolar epithelium in COPD patients compared to controls. No differences in LXR mRNA and protein levels were observed in alveolar macrophages between patient groups. The LXR agonist GW3965 significantly induced the expression of the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. In LPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10 and CCL5, whilst stimulating IL-10 production. CONCLUSIONS: GW3965 did not significantly suppress the production of TNFα, IL-1ß, or CXCL8. Our major finding is that LXR activation has anti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolar macrophages.

Ampicillin resistance in Haemophilus influenzae from COPD patients in the UK.

BACKGROUND: Haemophilus influenzae is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant H. influenzae strains, particularly ampicillin-resistant strains, including ß-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using ftsI sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of H. influenzae present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study H. influenzae strain switching and antibiotic sensitivity changes. PATIENTS AND METHODS: Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; H. influenzae was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 H. influenzae isolates were obtained. RESULTS: Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were ß-lactamase positive. The ß-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an ftsI mutation. Frequent H. influenzae strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern. CONCLUSION: We observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. H. influenzae strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.

The relationship between intranuclear mobility of the NF-kappaB subunit p65 and its DNA binding affinity.

It has been hypothesized that the main determinant of the intranuclear mobility of transcription factors is their ability to bind DNA. In the present study, we have extensively tested the relationship between the intranuclear mobility of the NF-kappaB subunit p65 and binding to its consensus target sequence. The affinity of p65 for this binding site is altered by mutation of specific acetylation sites, so these mutants provide a model system to study the relationship between specific DNA binding affinity and intranuclear mobility. DNA binding affinity was measured in vitro using an enzyme-linked immunosorbent assay-based method, and intranuclear mobility was measured using the fluorescence recovery after photobleaching technique on yellow fluorescent protein-tagged p65 constructs. A negative correlation was observed between DNA binding affinity and intranuclear mobility of p65 acetylation site mutants. However, moving the yellow fluorescent protein tag from the C terminus of p65 to the N terminus resulted in an increased mobility but did not significantly affect DNA binding affinity. Thus, all changes in DNA binding affinity produce alterations in mobility, but not vice versa. Finally, a positive correlation was observed between mobility and the randomness of the intranuclear distribution of p65. Our data are in line with a model in which the intranuclear mobility and distribution of a transcription factor are determined by its affinity for specific DNA sequences, which may be altered by protein-protein interactions.

Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain.

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.