Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates.
We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons.
These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.
The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
In COPD patients, mortality risk is influenced by age, severity of respiratory disease, and comorbidities. With an unbiased statistical approach we sought to identify clusters of COPD patients and to examine their mortality risk.
Stable COPD subjects (n = 527) were classified using hierarchical cluster analysis of clinical, functional and imaging data. The relevance of this classification was validated using prospective follow-up of mortality.
The most relevant patient classification was that based on three clusters (phenotypes). Phenotype 1 included subjects at very low risk of mortality, who had mild respiratory disease and low rates of comorbidities. Phenotype 2 and 3 were at high risk of mortality. Phenotype 2 included younger subjects with severe airflow limitation, emphysema and hyperinflation, low body mass index, and low rates of cardiovascular comorbidities. Phenotype 3 included older subjects with less severe respiratory disease, but higher rates of obesity and cardiovascular comorbidities. Mortality was associated with the severity of airflow limitation in Phenotype 2 but not in Phenotype 3 subjects, and subjects in Phenotype 2 died at younger age.
We identified three COPD phenotypes, including two phenotypes with high risk of mortality. Subjects within these phenotypes may require different therapeutic interventions to improve their outcome.
Strains of nontypeable Haemophilus influenzae show enormous genetic heterogeneity and display differential virulence potential in different clinical settings. The igaB gene, which encodes a newly identified IgA protease, is more likely to be present in the genome of COPD strains of H. influenzae than in otitis media strains. Analysis of igaB and surrounding sequences in the present study showed that H. influenzae likely acquired igaB from Neisseria meningitidis and that the acquisition was accompanied by a ∼20 kb genomic inversion that is present only in strains that have igaB. As part of a long running prospective study of COPD, molecular typing of H. influenzae strains identified a clonally related group of strains, a surprising observation given the genetic heterogeneity that characterizes strains of nontypeable H. influenzae. Analysis of strains by 5 independent methods (polyacrylamide gel electrophoresis, multilocus sequence typing, igaB gene sequences, P2 gene sequences, pulsed field gel electrophoresis) established the clonal relationship among the strains. Analysis of 134 independent strains collected prospectively from a cohort of adults with COPD demonstrated that ∼10% belonged to the clonal group. We conclude that a clonally related group of strains of nontypeable H. influenzae that has two IgA1 protease genes (iga and igaB) is adapted for colonization and infection in COPD. This observation has important implications in understanding population dynamics of H. influenzae in human infection and in understanding virulence mechanisms specifically in the setting of COPD.
Antibiotics, along with oral corticosteroids, are standard treatments for acute exacerbations of chronic obstructive pulmonary disease (AECOPD). The ultimate aims of treatment are to minimize the impact of the current exacerbation, and by ensuring complete resolution, reduce the risk of relapse. In the absence of superiority studies of antibiotics in AECOPD, evidence of the relative efficacy of different drugs is lacking, and so it is difficult for physicians to select the most effective antibiotic. This paper describes the protocol and rationale for MAESTRAL (moxifloxacin in AECBs [acute exacerbation of chronic bronchitis] trial; www.clinicaltrials.gov: NCT00656747), one of the first antibiotic comparator trials designed to show superiority of one antibiotic over another in AECOPD. It is a prospective, multinational, multicenter, randomized, double-blind controlled study of moxifloxacin (400 mg PO [ per os] once daily for 5 days) vs amoxicillin/clavulanic acid (875/125 mg PO twice daily for 7 days) in outpatients with COPD and chronic bronchitis suffering from an exacerbation. MAESTRAL uses an innovative primary endpoint of clinical failure: the requirement for additional or alternate treatment for the exacerbation at 8 weeks after the end of antibiotic therapy, powered for superiority. Patients enrolled are those at high-risk of treatment failure, and all are experiencing an Anthonisen type I exacerbation. Patients are stratified according to oral corticosteroid use to control their effect across antibiotic treatment arms. Secondary endpoints include quality of life, symptom assessments and health care resource use.
AECOPD; moxifloxacin; amoxicillin/clavulanic acid; clinical trial design; exacerbation; antibiotic
Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke–induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.
Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death in the world by 2020. It is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, most commonly cigarette smoke. Among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate. One possible explanation is that bacterial colonization of smoke-damaged airways, most commonly with nontypeable Haemophilus influenzae (NTHi), perpetuates airway injury and inflammation. Furthermore, COPD has also been identified as an independent risk factor for lung cancer irrespective of concomitant cigarette smoke exposure. In this article, we review the role of NTHi in airway inflammation that may lead to COPD progression and lung cancer promotion.
COPD; NTHi; inflammation
Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies.
Haemophilus influenzae generally colonizes the human upper respiratory tract. When isolated from the lower respiratory tract, this opportunistic pathogen is associated with inflammatory conditions such as pneumonia and exacerbations of chronic obstructive pulmonary disease (COPD). Here we show that one of the adaptations made by H. influenzae isolated from the lower respiratory tract is increased resistance to the bactericidal effect of antibody and complement. To define the mechanism for increased resistance, mutants were screened to identify the complete set of genes required to inhibit killing by antibody and complement. These included multiple genes that all contribute to biosynthesis of the organism's surface oligosaccharide (lipooligosaccharide), which is targeted by bactericidal antibody. Our results also revealed a novel function for additional genes that maintain the lipid asymmetry of the surface membrane and thereby limit recognition of the pathogen by anti-oligosaccharide antibodies.
We designed and tested three PilA-derived vaccine candidates in a chinchilla model of ascending nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM). Delivery of antiserum directed against each immunogen conferred varying degrees of protection. Presentation of a B-cell epitope derived from the OMP P5 adhesin at the N-terminus of recombinant soluble PilA protein (as opposed to the C-terminus), resulted in a protective chimeric immunogen that combined epitopes from two distinct NTHI adhesins (type IV pili and OMP P5). Incorporating protective epitopes derived from two NTHI adhesins/virulence determinants into a single pediatric vaccine candidate to prevent OM has multiple potential inherent advantages.
chinchilla; otitis media; COPD
Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.
Acute exacerbations contribute to the morbidity and mortality associated with chronic obstructive pulmonary disease (COPD). This proof-of-concept study evaluates whether intermittent pulsed moxifloxacin treatment could reduce the frequency of these exacerbations.
Stable patients with COPD were randomized in a double-blind, placebo-controlled trial to receive moxifloxacin 400 mg PO once daily (N = 573) or placebo (N = 584) once a day for 5 days. Treatment was repeated every 8 weeks for a total of six courses. Patients were repeatedly assessed clinically and microbiologically during the 48-week treatment period, and for a further 24 weeks' follow-up.
At 48 weeks the odds ratio (OR) for suffering an exacerbation favoured moxifloxacin: per-protocol (PP) population (N = 738, OR 0.75, 95% confidence interval (CI) 0.565-0.994, p = 0.046), intent-to-treat (ITT) population (N = 1149, OR 0.81, 95% CI 0.645-1.008, p = 0.059), and a post-hoc analysis of per-protocol (PP) patients with purulent/mucopurulent sputum production at baseline (N = 323, OR 0.55, 95% CI 0.36-0.84, p = 0.006).
There were no significant differences between moxifloxacin and placebo in any pre-specified efficacy subgroup analyses or in hospitalization rates, mortality rates, lung function or changes in St George's Respiratory Questionnaire (SGRQ) total scores. There was, however, a significant difference in favour of moxifloxacin in the SGRQ symptom domain (ITT: -8.2 vs -3.8, p = 0.009; PP: -8.8 vs -4.4, p = 0.006). Moxifloxacin treatment was not associated with consistent changes in moxifloxacin susceptibility. There were more treatment-emergent, drug related adverse events with moxifloxacin vs placebo (p < 0.001) largely due to gastrointestinal events (4.7% vs 0.7%).
Intermittent pulsed therapy with moxifloxacin reduced the odds of exacerbation by 20% in the ITT population, by 25% among the PP population and by 45% in PP patients with purulent/mucopurulent sputum at baseline. There were no unexpected adverse events and there was no evidence of resistance development.
ClinicalTrials.gov number, NCT00473460 (ClincalTrials.gov).
Moraxella catarrhalis causes approximately 10% of exacerbations in chronic obstructive pulmonary disease (COPD) and also colonizes the lower airway in stable patients. Little is known about the effects of colonization by M. catarrhalis on airway inflammation and protease-antiprotease balance, and how these changes compare to those seen during exacerbations. Since COPD is a progressive inflammatory disease, elucidating the effects of bacterial colonization and exacerbation on airway inflammation is relevant to understanding disease progression in COPD. Our aims were (1) Analyze changes in airway inflammation in colonization and exacerbation of COPD due to M. catarrhalis; (2) Explore protease-antiprotease balance in colonization and exacerbation due to M. catarrhalis. Our hypothesis were (1) Acquisition of a new strain of M. catarrhalis in COPD increases airway inflammation from baseline and alters the protease-antiprotease balance towards a more proteolytic environment; (2) These changes are greater during exacerbations associated with M. catarrhalis as compared to colonization.
Thirty-nine consecutive COPD patients with 76 acquisitions of a new strain of M. catarrhalis over a 6-year period were identified in a prospective study. Seventy-six pre-acquisition sputum supernatant samples, obtained just before acquisition of M catarrhalis, and 76 acquisition samples (34 were associated with exacerbation, 42 with colonization) were analyzed for IL-8, TNF-α, Neutrophil Elastase (NE) and Secretory leukocyte protease inhibitor (SLPI). Changes were compared in paired samples from each patient.
IL-8, TNF-α and NE were significantly elevated after acquisition of M. catarrhalis, compared to pre-acquisition samples (p =< 0.001 for all three). These changes were present in colonization (p = 0.015 for IL-8; p =< 0.001 for TNF-α and NE) as well as in exacerbation (p =< 0.001 for all three), compared to pre-acquisition levels. SLPI was significantly lower after acquisition (p =< 0.001), in colonization (p =< 0.001) as well as in exacerbation (p = 0.004), compared to pre-acquisition levels. SLPI levels correlated negatively with NE levels (R2 = 0.07; p = 0.001).
Acquisition of M. catarrhalis in COPD causes increased airway inflammation and worsening protease-antiprotease imbalance during exacerbations and also in colonization, even in the absence of increased symptoms. These effects could contribute to progression of airway disease in COPD.
Moraxella catarrhalis is a common cause of respiratory tract infection in the setting of chronic obstructive pulmonary disease (COPD). Adults with COPD acquire and clear strains of M. catarrhalis from the respiratory tract continuously and develop strain-specific protection following clearance of a strain. In previous work, we identified Hag/MID (Moraxella immunoglobulin D-binding protein), a large multifunctional surface protein that acts as an adhesin and hemagglutinin, as a target of antibody responses in adults with COPD after clearance of M. catarrhalis. The goal of the present study was to characterize the domains of Hag/MID to which humans make antibodies, including both systemic and mucosal antibody responses. Analysis of recombinant peptide constructs, which spanned the M. catarrhalis strain O35E Hag/MID protein, with well-characterized serum and sputum samples revealed that most adults with COPD made antibodies directed toward a region of the molecule bounded by amino acids 706 to 863. Serum immunoglobulin G (IgG) and IgA purified from sputum both recognized the same domain. Some flanking sequence of this fragment was necessary for the epitope(s) in this region to maintain its conformation to bind human antibodies. These results reveal that humans consistently generate both systemic and mucosal antibody responses to an immunodominant region of the Hag/MID molecule, which was previously shown to overlap with several biologically relevant domains, including epithelial cell adherence, IgD binding, collagen binding, and hemagglutination.
In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.
Exacerbations are a major cause of morbidity and mortality in chronic obstructive pulmonary disease. Exacerbations can be of bacterial, viral or mixed etiology, with bacteria involved in 50% of exacerbations. Consequently, current management of exacerbations frequently involves the use of antibiotics. The paper by Puhan et al published this month in BMC Medicine examines the benefit of antibiotics in placebo-controlled trials in mild to moderate outpatient exacerbations. The authors use a meta-analytic approach and rightly conclude that more trials are needed in this area. However, the heterogeneity of chronic obstructive pulmonary disease patients and exacerbations and the limited end-points in past trials do not allow firm conclusions to be drawn about antibiotic use in outpatient exacerbations based on this meta-analysis. Future trials need to take into account this heterogeneity as well as incorporate novel end-points to address this important issue.
Our understanding of the etiology, pathogenesis and consequences of acute exacerbations of chronic obstructive pulmonary disease (COPD) has increased substantially in the last decade. Several new lines of evidence demonstrate that bacterial isolation from sputum during acute exacerbation in many instances reflects a cause-effect relationship. Placebo-controlled antibiotic trials in exacerbations of COPD demonstrate significant clinical benefits of antibiotic treatment in moderate and severe episodes. However, in the multitude of antibiotic comparison trials, the choice of antibiotics does not appear to affect the clinical outcome, which can be explained by several methodological limitations of these trials. Recently, comparison trials with nontraditional end-points have shown differences among antibiotics in the treatment of exacerbations of COPD. Observational studies that have examined clinical outcome of exacerbations have repeatedly demonstrated certain clinical characteristics to be associated with treatment failure or early relapse. Optimal antibiotic selection for exacerbations has therefore incorporated quantifying the risk for a poor outcome of the exacerbation and choosing antibiotics differently for low risk and high risk patients, reserving the broader spectrum drugs for the high risk patients. Though improved outcomes in exacerbations with antibiotic choice based on such risk stratification has not yet been demonstrated in prospective controlled trials, this approach takes into account concerns of disease heterogeneity, antibiotic resistance and judicious antibiotic use in exacerbations.
COPD; exacerbation; bronchitis; antibiotics
airway epithelial cells; bacterial adherence; interleukin-8; nuclear factor-κB; p38 mitogen-activated protein kinase
Rationale: Interactions of nontypeable Haemophilus influenzae (NTHI) with macrophages are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the immunologic mechanisms that mediate NTHI-macrophage inflammation are poorly understood. Outer membrane protein (OMP) P6 and lipooligosaccharide (LOS) of NTHI are potent immunomodulators. We theorized that alveolar macrophages in COPD possess fundamental immune defects that permit NTHI to evade host responses.
Objective: To test this hypothesis, we obtained human alveolar and blood macrophages from exsmokers with COPD, exsmokers without COPD, and nonsmokers.
Methods: Alveolar and blood macrophages from each donor were incubated with purified LOS and OMP P6 and with OMP P2 and the total outer membrane preparation (0.1–1 μg/ml).
Measurements: Supernatants (24 h) were assayed for IL-1β, TNF-α, IL-10, IL-12, and IL-8 by multianalyte multiplexed flow cytometry.
Results: Comparative induction of COPD and non-COPD alveolar macrophages by LOS and OMP P6 revealed diminished IL-8, TNF-α, and IL-1β responses of COPD alveolar macrophages (p ⩽ 0.03 for each). COPD alveolar macrophages also had diminished responses to total outer membrane (p ⩽ 0.03 for each). In contrast, COPD blood macrophages had no significant differences among donor groups in IL-8, TNF-α, or IL-1β responsiveness to NTHI antigens. Diminished IL-12 responses of COPD blood macrophages to NTHI antigens, compared with nonsmokers, could not be independently dissociated from group differences in age and pack-years.
Conclusions: These findings support a paradigm of defective immune responsiveness of alveolar macrophages, but not blood macrophages, in COPD.
chronic obstructive pulmonary disease; macrophage, alveolar; nontypeable Haemophilus influenzae; phagocytosis
Rationale: Inflammation is now recognized as an integral part of the pathogenesis of chronic obstructive pulmonary disease (COPD). In contrast to the sterile airways of normal lungs, bacterial pathogens are often isolated from the airways in stable COPD. This “colonization” of the tracheobronchial tree, currently believed to be innocuous, could serve as an inflammatory stimulus, independent of current tobacco smoke exposure.
Objective: To test the hypothesis that bacterial colonization is associated with airway inflammation in stable COPD.
Methods: Bronchoscopy with bronchoalveolar lavage (BAL) was performed in three groups of subjects: 26 ex-smokers with stable COPD (COPD), 20 ex-smokers without COPD (ex-smokers), and 15 healthy nonsmokers (nonsmokers). Quantitative bacterial cultures, cell counts, chemokine, cytokine, proteinase/antiproteinase, and endotoxin levels in the BAL fluid were compared.
Results: Potentially pathogenic bacteria were recovered at ⩾ 100 cfu/ml in 34.6% of COPD, 0% of ex-smokers, and in 6.7% of nonsmokers (p = 0.003). All values are expressed as median (interquartile range). Subjects with colonized COPD had significantly greater relative (12.0 [28.4] vs. 3.0 [7.8]%, p = 0.03) and absolute (4.98 [5.26] × 104/ml vs. 3.04 [2.82] × 104/ml, p = 0.02) neutrophil counts, interleukin 8 (33.8 [189.8] vs. 16.9 [20.1] pg/ml, p = 0.005), active matrix metalloproteinase-9 (2.16 [4.30] vs. 0.84 [0.99] U/ml, p = 0.03), and endotoxin (36.0 [72.6] vs. 3.55 [7.17] mEU/ml, p = 0.004) levels in the BAL than the subjects with noncolonized COPD. These inflammatory constituents of BAL were also significantly elevated in subjects with colonized COPD when compared with ex-smokers and nonsmokers.
Conclusions: Bacterial colonization is associated with neutrophilic airway lumen inflammation in ex-smokers with COPD and could contribute to progression of airway disease in COPD.
bacterial colonization; chronic obstructive pulmonary disease; neutrophilic inflammation
Haemophilus influenzae is an important cause of otitis media in children and lower respiratory infection in adults with chronic obstructive pulmonary disease (COPD). Patients with COPD experience periodic exacerbations that are associated with acquisition of new bacterial strains. However, not every strain acquisition is associated with exacerbation. To test the hypothesis that genetic differences among strains account for differences in pathogenic potential, a microarray consisting of 4,992 random 1.5- to 3-kb genomic fragments of an exacerbation strain was constructed. Competitive hybridization was performed using six strains associated with exacerbation as well as five strains associated with asymptomatic colonization. Seven sequences that were absent in all five colonization strains and present in at least two exacerbation strains were identified. One such sequence was a previously unreported gene with high homology to the meningococcal immunoglobulin A (IgA) protease gene, which is distinct from the previously described H. influenzae IgA protease. To assess the distribution of the seven sequences among well-characterized strains of H. influenzae, 59 exacerbation strains and 73 asymptomatic colonization strains were screened by PCR for the presence of these sequences. The presence or absence of any single sequence was not significantly associated with exacerbations of COPD. However, logistic regression and subgroup analysis identified combinations of the presence and absence of genes that are associated with exacerbations. These results indicate that patterns of genes are associated with the ability of strains of H. influenzae to cause exacerbations of COPD, supporting the concept that differences in pathogenic potential are based in part on genomic differences among infecting strains, not merely host factors.
Rationale: Moraxella catarrhalis is frequently present in the sputum of adults with chronic obstructive pulmonary disease (COPD). Little is known about the role of M. catarrhalis in this common disease. Objective: To elucidate the burden of disease, the dynamics of carriage, and immune responses to M. catarrhalis in COPD. Methods: Prospective cohort study of 104 adults with COPD in an outpatient clinic at the Buffalo Veterans Affairs Medical Center. Measurements: Clinical information, sputum cultures, molecular typing of isolates, and immunoassays to measure antibodies to M. catarrhalis. Main Results: Over 81 months, 104 patients made 3,009 clinic visits, 560 during exacerbations. Molecular typing identified 120 episodes of acquisition and clearance of M. catarrhalis in 50 patients; 57 (47.5%) of the acquisitions were associated with clinical exacerbations. No instances of simultaneous acquisition of a new strain of another pathogen were observed. The duration of carriage of M. catarrhalis was shorter with exacerbations compared with asymptomatic colonization (median, 31.0 vs. 40.4 days; p = 0.01). Reacquisition of the same strain was rare. The intensity of the serum IgG response was greater after exacerbations than asymptomatic colonization (p = 0.009). Asymptomatic colonization was associated with a greater frequency of a sputum IgA response than exacerbation (p = 0.009). Conclusions: M. catarrhalis likely causes approximately 10% of exacerbations of COPD, accounting for approximately 2 to 4 million episodes annually. The organism is cleared efficiently after a short duration of carriage. Patients develop strain-specific protection after clearance of M. catarrhalis from the respiratory tract.
chronic bronchitis; mucosal immunity; respiratory tract infection
Rationale: Airway infection with Haemophilus influenzae causes airway inflammation, and isolation of new strains of this bacteria is associated with increased risk of exacerbations in patients with chronic obstructive pulmonary disease (COPD). Objective: To determine whether strains of H. influenzae associated with exacerbations cause more inflammation than strains that colonize the airways of patients with COPD. Methods: Exacerbation strains of H. influenzae were isolated from patients during exacerbation of clinical symptoms with subsequent development of a homologous serum antibody response and were compared with colonization strains that were not associated with symptom worsening or an antibody response. Bacterial strains were compared using an in vivo mouse model of airway infection and in vitro cell culture model of bacterial adherence and defense gene and signaling pathway activation in primary human airway epithelial cells. Results: H. influenzae associated with exacerbations caused more airway neutrophil recruitment compared with colonization strains in the mouse model of airway bacterial infection. Furthermore, exacerbation strains adhered to epithelial cells in significantly higher numbers and induced more interleukin-8 release after interaction with airway epithelial cells. This effect was likely mediated by increased activation of the nuclear factor-κB and p38 mitogen-activated protein kinase signaling pathways. Conclusions: The results indicate that H. influenzae strains isolated from patients during COPD exacerbations often induce more airway inflammation and likely have differences in virulence compared with colonizing strains. These findings support the concept that bacteria infecting the airway during COPD exacerbations mediate increased airway inflammation and contribute to decreased airway function.
bacterial adhesion; interleukin-8; NF-κB; neutrophil infiltration; p38 mitogen-activated protein kinases
Moraxella catarrhalis is an important human mucosal pathogen causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Little is known about the mucosal antibody response to M. catarrhalis in adults with COPD. In this study, 10 pairs of well-characterized sputum supernatant samples from adults with COPD who had acquired and subsequently cleared M. catarrhalis from their respiratory tracts were studied in detail in an effort to begin to elucidate potentially protective immune responses. Flow cytometry analysis was used to study the distribution of immunoglobulin isotypes in paired preacquisition and postclearance sputum samples. The results showed that immunoglobulin A (IgA) is the predominant M. catarrhalis-specific immunoglobulin isotype and that the sputum IgA contains a secretory component, indicating that it is locally produced at the mucosal site. Most patients made new sputum IgA responses to the adhesins UspA1 and Hag, along with the surface protein UspA2. A smaller proportion of patients made new sputum IgA responses to the iron-regulated proteins TbpB and CopB and to lipooligosaccharide. These results have important implications in understanding the mucosal immune response to M. catarrhalis in the setting of COPD and in elucidating the elements of a protective immune response.