Moraxella catarrhalis acquisition, airway inflammation and protease-antiprotease balance in chronic obstructive pulmonary disease

<p>Abstract</p> <p>Background</p> <p><it>Moraxella catarrhalis </it>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 <it>M. catarrhalis </it>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 <it>M. catarrhalis</it>; (2) Explore protease-antiprotease balance in colonization and exacerbation due to <it>M. catarrhalis</it>. 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 <it>M. catarrhalis </it>as compared to colonization.</p> <p>Methods</p> <p>Thirty-nine consecutive COPD patients with 76 acquisitions of a new strain of <it>M. catarrhalis </it>over a 6-year period were identified in a prospective study. Seventy-six pre-acquisition sputum supernatant samples, obtained just before acquisition of <it>M catarrhalis</it>, 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.</p> <p>Results</p> <p>IL-8, TNF-α and NE were significantly elevated after acquisition of <it>M. catarrhalis</it>, 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).</p> <p>Conclusion</p> <p>Acquisition of <it>M. catarrhalis </it>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.</p

Outer Membrane Protein P6 of Nontypeable Haemophilus influenzae Is a Potent and Selective Inducer of Human Macrophage Proinflammatory Cytokines

Interactions of nontypeable Haemophilus influenzae (NTHI) with human macrophages contribute to the pathogenesis of NTHI-induced infection in humans. However, the immunologic mechanisms that initiate and perpetuate NTHI-mediated macrophage responses have not been well explored. Outer membrane protein (OMP) P6 is a conserved lipoprotein expressed by NTHI in vivo that possesses a Pam(3)Cys terminal motif, characteristic of immunoactive bacterial lipoproteins associated with Toll-like receptor signaling. We theorized that OMP P6 is a potent immunomodulator of human macrophages. To test this hypothesis, we purified OMP P6 as well as OMP P2, the predominant NTHI outer membrane protein, and lipooligosaccharide (LOS), the specific endotoxin of NTHI, from NTHI strain 1479. Human blood monocyte-derived macrophages, purified from healthy donors, were incubated with each outer membrane constituent, and cytokine production of macrophage supernatants interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), IL-10, IL-12, and IL-8 was measured. OMP P6 selectively upregulated IL-10, TNF-α, and IL-8. While OMP P6 (0.1 μg/ml for 8 h) elicited slightly greater concentrations of IL-10, it resulted in over ninefold greater concentrations of TNF-α and over fourfold greater concentrations of IL-8 than did OMP P2. OMP P6 at doses as low as 10 pg/ml was still effective at induction of macrophage IL-8, while OMP P2 and LOS were not. OMP P6 of NTHI is a specific trigger of bacteria-induced human macrophage inflammatory events, with IL-8 and TNF-α as key effectors of P6-induced macrophage responses

Impaired Innate COPD Alveolar Macrophage Responses and Toll-Like Receptor-9 Polymorphisms

<div><p>Background</p><p>Dysfunctional innate responses of alveolar macrophages to nontypeable <i>Haemophilus influenzae</i>, <i>Moraxella catarrhalis</i> and <i>Streptococcus pneumoniae</i> contribute to morbidity in chronic obstructive pulmonary disease (COPD). Our earlier studies discovered impaired COPD alveolar macrophage responses to Toll-like receptor (TLR) ligands of nontypeable <i>H</i>. <i>influenzae</i> and provide rationale for further evaluation of TLR signaling. While the role of TLR single nucleotide polymorphisms is increasingly recognized in inflammatory diseases, TLR single nucleotide polymorphisms in COPD have only recently been explored. We hypothesized that specific TLR polymorphisms are associated with dysfunctional innate immune COPD alveolar macrophage responses and investigated polymorphisms of TLR2(Arg753Gln), TLR4(Thr399Ile; Asp299Gly), and TLR9(T1486C; T1237C).</p><p>Methods</p><p>DNA was purified from cells of 1) healthy nonsmokers (n = 20); 2) COPD ex-smokers (n = 83); 3) COPD active smokers (n = 93). DNA amplifications (polymerase chain reaction) were performed for each SNP. Alveolar macrophages from each group were incubated with nontypeable <i>H</i>. <i>influenzae</i>, <i>M</i>. <i>catarrhalis</i> and <i>S</i>. <i>pneumoniae</i>. Cytokine induction of macrophage supernatants was measured and the association with TLR single nucleotide polymorphism expression was determined.</p><p>Results</p><p>No significant inter-group differences in frequency of any TLR SNP existed. However both TLR9 single nucleotide polymorphisms were expressed in high frequency. Among COPD ex-smokers, diminished IL-8 responsiveness to nontypeable <i>H</i>. <i>influenzae</i>, <i>M</i>. <i>catarrhalis</i> and <i>S</i>. <i>pneumoniae</i> was strongly associated with carriage of TLR9(T1237C) (p = 0.02; p = 0.008; p = 0.02), but not TLR9(T1486C). Carriage of TLR9(T1237C), but not TLR9(T1486C), correlated with diminished FEV₁%predicted (p = 0.037).</p><p>Conclusion</p><p>Our results demonstrate a notable association of TLR9(T1237C) expression with dysfunctional innate alveolar macrophage responses to respiratory pathogens and with severity of COPD.</p></div

Single nucleotide polymorphisms of TLR’s.

<p>PCR-based RFLP’s were performed with genomic DNA from each participant. Representative agarose gels of shown for: TLR9 (T1237C) (Panel A), TLR9 (T1486C) (Panel B), TLR4 (Thr399Ile) (Panel C), TLR4 (Asp299Gly) (Panel D) and TLR2 (Arg753Gln) (Panel E). Numbers in left margin of each gel correspond to DNA ladder base pairs. Lanes with restriction fragments for heterozygotes and homozygotes of each SNP are distinguishable from restriction fragments for wildtypes. Representative patient samples are shown in each lane of each panel.</p

Demographic characteristics: Bronchoscopy participants.

<p>^a p≤0.05—nonsmokers vs. COPD ex-smokers</p><p>^b p≤0.05—nonsmokers vs. COPD active smokers</p><p>^c p≤0.05 –COPD ex-smokers vs. COPD active smokers</p><p>^# GOLD classification is as detailed in the Global Initiative for Chronic Obstructive Lung Disease [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134209#pone.0134209.ref024" target="_blank">24</a>].</p><p>Data are expressed as mean±SEM.</p

TLR9 SNPs and FEV₁%predicted.

<p>Correlation of FEV₁%predicted and expression of TLR9 wildtype (dark shading) compared with (T1237C) (light shading) is shown in left panel. Correlation of FEV₁%predicted and expression of TLR9 wildtype (dark shading) compared with TLR9 (T1486C) (striped shading) is shown in right panel. Results are given for all COPD participants.</p

Alveolar macrophage IL-8 (pg/ml) induction of alveolar macrophages expressing wildtype (w/t), TLR9 (T1237C) and TLR9 (T1486C).

<p>*p<0.05- TLR9 SNP vs. w/t</p><p>IL-8 values are expressed as median [IQR] and correspond with data of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134209#pone.0134209.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134209#pone.0134209.g003" target="_blank">3</a>.</p

Association of TLR9 SNPs and bacterial induction of COPD ex-smoker alveolar macrophage IL-8.

<p>Supernatant IL-8 concentrations of alveolar macrophages, obtained from ex-smokers with COPD, incubated with nontypeable <i>Haemophilus influenzae</i> 11P6H1 (Panels A), <i>Moraxella catarrhalis</i> 6P29B1 (Panels B) and <i>Streptococcus pneumoniae</i> 25P55S1 (Panels C), were measured at 6 hours. Analysis is shown for TLR9 wildtype (dark shading) compared with TLR9 (T1237C) (light shading) on left panels. Analysis for TLR9 wildtype (dark shading) compared with TLR9 (T1486C) (striped shading) is shown in right panels. Results are shown as box plots for each group. Each box encompasses the 25^th to 75^th interquartile range, with the horizontal line in each box representing median values. Each vertical bar encompasses the 10^th to 90^th percentile ranges. Values correspond with data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134209#pone.0134209.t006" target="_blank">Table 6</a>.</p

Demographic characteristics: Total study participants.

<p>^a p≤0.05—nonsmokers vs. ex-smokers</p><p>^b p≤0.05—nonsmokers vs. active smoker</p><p>^c p≤0.05—ex-smokers vs. active smokers</p><p>^# GOLD classification is as detailed in the Global Initiative for Chronic Obstructive Lung Disease [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134209#pone.0134209.ref024" target="_blank">24</a>].</p><p>Data are expressed as mean±SEM.</p

Impaired Alveolar Macrophage Response to Haemophilus Antigens in Chronic Obstructive Lung Disease

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