Table 1.
Demographics of participants in AM experiments.
Table 2.
Demographics of participants in MDM experiments.
Fig 1.
Cytotoxicity and efficacy of p38, PI3K and ROCK inhibition in macrophages.
(A-F) 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.
Fig 2.
p38, PI3K and ROCK inhibition modulates signalling in alveolar macrophages.
(A-F) 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 S. pneumoniae 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.
Fig 3.
p38, PI3K and ROCK inhibition modulates cytokine production in alveolar macrophages.
(A-F) 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 S. pneumoniae 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.
Fig 4.
COPD alveolar macrophages have reduced phagocytosis of S. pneumoniae, which is not modified by p38, PI3K or ROCK inhibition.
(A) Alveolar macrophages (AM) from COPD patients or healthy controls were challenged with S. pneumoniae (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-D) 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.
Fig 5.
p38, PI3K or ROCK inhibition does not affect early-phase bacterial killing in alveolar macrophages.
(A) Alveolar macrophages (AM) from healthy donors or COPD patients were challenged with S. pneumoniae (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-D) 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.
Fig 6.
COPD MDM have reduced phagocytosis of S. pneumoniae which is not modified by p38, PI3K or ROCK inhibition.
(A-B) MDM from healthy donors or patients with COPD were incubated with fluorescent beads (A) or fluorescently labelled S. pneumoniae (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. (C-D) 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 S. pneumoniae for 4h. Data are presented as mean ± SEM for n = 10 healthy donors and n = 6 COPD. (E-G) 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 S. pneumoniae for 4h. Data presented as mean ± SEM for n = 3 healthy and n = 3 COPD. (H) MDM from healthy donors or patients with COPD were pre-incubated for 2h with the ROCK inhibitor, PF4950834 prior to challenge with fluorescently labelled S. pneumoniae 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.
Fig 7.
COPD MDM have reduced phagocytosis of H. influenzae, which is not modified by p38, PI3K or ROCK inhibition.
(A) MDM from healthy donors or COPD patients were challenged with fluorescently labelled H. influenzae 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-C) 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 S.pneumoniae for 4h. Data are presented as mean ± SEM for n = 10 healthy donors and n = 6 COPD. (D-F) 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 S.pneumoniae for 4h. Data presented as mean ± SEM for n = 3 healthy and n = 3 COPD. (G) MDM from healthy donors or patients with COPD were pre-incubated for 2h with the ROCK inhibitor, PF4950834 prior to challenge with fluorescently labelled H. influenzae 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.
Fig 8.
Inhibition of ROCK, but not p38 or PI3K pathways, increases efferocytosis in COPD alveolar and monocyte-derived macrophages.
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. (A-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. (C-H) 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.