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

Demographics of participants in AM experiments.

<p>Demographics of participants in AM experiments.</p

Inhibition of ROCK, but not p38 or PI3K pathways, increases efferocytosis in COPD alveolar and monocyte-derived macrophages.

<p>Alveolar (AM) or monocyte-derived macrophages (MDM) were incubated with PKH-26 stained apoptotic neutrophils for 90 min, before efferocytosis was assessed by flow cytometry. (<b>A-B)</b> Pooled vehicle data for AM, (A) n = 4–9, *** = p<0.001, Mann-Whitney and MDM (B), n = 7–12, ** = p<0.01, Student t-test. <b>(C-H)</b> Healthy or COPD AM (C, E and G) were pre-treated with vehicle (-) or 1μM SCIO469, 1μM VX745 (C), 100nM NVS-PI3K-2/3/5 (E), or 200nM PF4950834 (G) (+). MDM (D, F and H) were treated with vehicle (-) or compounds at the designated dose (+), ns = non significant, * = p<0.05, Wilcoxon matched pairs test.</p

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

<p><b>(A)</b> Alveolar macrophages (AM) from COPD patients or healthy controls were challenged with <i>S</i>. <i>pneumoniae</i> (Spn) at a multiplicity of infection (MOI) of 10. 4 h post challenge, the number of viable intracellular bacteria was determined. Data presented as median ± IQR, n = 10/14 healthy/COPD, *** = p<0.001, Mann-Whitney U test. <b>(B-D)</b> Healthy or COPD AM were treated with vehicle (-) or the designated doses of SCIO469, VX745 (B), NVS-PI3K-2/3/5, (C) or PF4950834 (D) before challenge with Spn at MOI 10. 4 h post challenge, numbers of viable internalized bacteria were determined, n = 3–5, data shown as paired vehicle and compound data for each donor, ns = non-significant, paired t- test.</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

p38, PI3K or ROCK inhibition does not affect early-phase bacterial killing in alveolar macrophages.

<p><b>(A)</b> Alveolar macrophages (AM) from healthy donors or COPD patients were challenged with <i>S</i>. <i>pneumoniae</i> (Spn) at a multiplicity of infection of 10. 2 h after challenge non-internalised bacteria were washed off, and antibiotics added. At the designated time post-antimicrobials persisting viable bacteria were measured. <b>(B-D)</b> COPD AM were pre-treated with vehicle or the designated inhibitor before being challenged with Spn at MOI of 10. At the designated time post-antimicrobials persisting viable bacteria were measured. In all experiments, n = 4, with no significant differences between any groups at any time point, Friedman test.</p

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

<p><b>(A)</b> MDM from healthy donors or COPD patients were challenged with fluorescently labelled <i>H</i>. <i>influenzae</i> for 4h and phagocytosis measured by fluorimetry. Data are presented as individual data points and the line represents median where **p<0.01 Mann-Whitney U test. <b>(B-C)</b> MDM from healthy donors or patients with COPD were pre-incubated with p38 inhibitors VX745 (B) or SCIO469 (C) for 2h 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>(D-F)</b> MDM from healthy donors or patients with COPD were pre-incubated with the PI3K inhibitors NVS-PI3-2 (D), NVS-PI3-3 (E) or NVS-PI3-5 (F) 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>(G)</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>H</i>. <i>influenzae</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 internalisation between vehicle and any concentration of compound for either healthy or COPD MDM.</p