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: 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 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

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

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

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