Additional file 1: Table S1. of Characteristics and longitudinal progression of chronic obstructive pulmonary disease in GOLD B patients

Baseline characteristics of patients categorised as GOLD C and D. (DOCX 17 kb

Additional file 3: Figure S2. of Blood and sputum eosinophils in COPD; relationship with bacterial load

A summary of the number of patients with blood / sputum eosinophil counts and qPCR data at exacerbation alone, or coupled to baseline measurements. PPM=potentially pathogenic microorganisms. (PPTX 51.6 kb

Additional file 1: of Blood and sputum eosinophils in COPD; relationship with bacterial load

Additional methods supplement. (DOCX 38.7 kb

Additional file 4: Figure S3. of Blood and sputum eosinophils in COPD; relationship with bacterial load

The blood eosinophil count at baseline and exacerbation in patients defined as PPM positive or PPM negative at exacerbation (PPM threshold value of 1x10 ). PPM=potentially pathogenic microorganisms. Dotted lines represent median values. (PPTX 274 kb

Additional file 6: Figure S4. of Blood and sputum eosinophils in COPD; relationship with bacterial load

The sputum neutrophil % at baseline and exacerbation in patients defined as PPM positive or PPM negative at exacerbation. PPM=potentially pathogenic microorganisms. Dotted lines represent median values. (PPTX 189 kb

Additional file 2: Figure S1. of Blood and sputum eosinophils in COPD; relationship with bacterial load

Outline of the methods used for the processing of sputum qPCR and differential cell count samples. (PPTX 64.1 kb

Cytotoxicity and efficacy of p38, PI3K and ROCK inhibition in macrophages.

<p><b>(A-F)</b> Alveolar macrophages (AM) from COPD patients or healthy controls were incubated with either vehicle (-), or incubated with (+) 1μM SCIO469, 1μM VX745, 100nM NVS-PI3K-2/3/5 or 200nM PF4950834 for 20 h, before cultures were assessed for apoptosis (A-C) by nuclear fragmentation, or necrosis (D-F). In all experiments, n = 4, there was no significant differences between groups.</p

COPD MDM have reduced phagocytosis of <i>S</i>. <i>pneumoniae</i> which is not modified by p38, PI3K or ROCK inhibition.

<p><b>(A-B)</b> MDM from healthy donors or patients with COPD were incubated with fluorescent beads (A) or fluorescently labelled <i>S</i>. <i>pneumoniae</i> (B) for 4h and phagocytosis measured by fluorimetry. Data are presented as individual data points and the line represents median *p<0.05 Mann-Whitney U test. <b>(C-D)</b> MDM from healthy donors or patients with COPD were pre-incubated with p38 inhibitors VX745 (C) or SCIO469 (B) for 1h prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data are presented as mean ± SEM for n = 10 healthy donors and n = 6 COPD. <b>(E-G)</b> MDM from healthy donors or patients with COPD were pre-incubated with the PI3K inhibitors NVS-PI3-2 (E), NVS-PI3-3 (F) or NVS-PI3-5 (G) for 2h prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data presented as mean ± SEM for n = 3 healthy and n = 3 COPD. <b>(H)</b> MDM from healthy donors or patients with COPD were pre-incubated for 2h with the ROCK inhibitor, PF4950834 prior to challenge with fluorescently labelled <i>S</i>. <i>pneumoniae</i> for 4h. Data are presented as mean ± SEM for n = 3 healthy and n = 3 COPD. In all experiments, no significant differences were observed in internalization.</p

p38, PI3K and ROCK inhibition modulates signalling in alveolar macrophages.

<p><b>(A-F)</b> COPD alveolar macrophages (AM) were pre-treated with the designated concentrations of SCIO469 (A) VX745 (B), NVS-PI3K-2/3/5 (C-E), or PF4950834 (F), were then challenged with <i>S</i>. <i>pneumoniae</i> for 6 h, before cells were lysed and probed for either p-HSP27 (A-B), p-AKT (C-E), or p-MLC (F). Plots are representative of three independent experiments and densitometry from all three experiments are shown, * = p<0.05, ** = p<0.01, ANOVA with Dunnetts post-test vs control.</p

p38, PI3K and ROCK inhibition modulates cytokine production in alveolar macrophages.

<p><b>(A-F)</b> COPD alveolar macrophages (AM) were pre-treated with the designated concentrations of SCIO469 or VX745 (A and D), NVS-PI3K-2/3/5 (B and E), or PF4950834 (C and F), before challenge with <i>S</i>. <i>pneumoniae</i> for 6 h. Supernatants were collected and levels of TNFα (A-C) and IL-6 (D-F) were measured by ELISA, n = 4, * = p<0.05, ANOVA with Dunnetts post-test vs control.</p