Aspirin reduces lipopolysaccharide-induced pulmonary inflammation in human models of ARDS

Abstract

Rationale: Platelets play an active role in the pathogenesis of acute respiratory distress syndrome (ARDS). Animal and observational studies have shown aspirin's antiplatelet and immunomodulatory effects may be beneficial in ARDS.

Objective: To test the hypothesis that aspirin reduces inflammation in clinically relevant human models that recapitulate pathophysiological mechanisms implicated in the development of ARDS.

Methods: Healthy volunteers were randomised to receive placebo or aspirin 75 or 1200 mg (1:1:1) for seven days prior to lipopolysaccharide (LPS) inhalation, in a double-blind, placebo-controlled, allocation-concealed study. Bronchoalveolar lavage (BAL) was performed 6 hours after inhaling 50 µg of LPS. The primary outcome measure was BAL IL-8. Secondary outcome measures included markers of alveolar inflammation (BAL neutrophils, cytokines, neutrophil proteases), alveolar epithelial cell injury, systemic inflammation (neutrophils and plasma C-reactive protein (CRP)) and platelet activation (thromboxane B2, TXB2). Human lungs, perfused and ventilated ex vivo (EVLP) were randomised to placebo or 24 mg aspirin and injured with LPS. BAL was carried out 4 hours later. Inflammation was assessed by BAL differential cell counts and histological changes.

Results: In the healthy volunteer (n=33) model, data for the aspirin groups were combined. Aspirin did not reduce BAL IL-8. However, aspirin reduced pulmonary neutrophilia and tissue damaging neutrophil proteases (Matrix Metalloproteinase (MMP)-8/-9), reduced BAL concentrations of tumour necrosis factor α and reduced systemic and pulmonary TXB2. There was no difference between high-dose and low-dose aspirin. In the EVLP model, aspirin reduced BAL neutrophilia and alveolar injury as measured by histological damage.

Conclusions: These are the first prospective human data indicating that aspirin inhibits pulmonary neutrophilic inflammation, at both low and high doses. Further clinical studies are indicated to assess the role of aspirin in the prevention and treatment of ARDS.

Trial registration number: NCT01659307 Results.

Keywords: ARDS; Innate Immunity; Neutrophil Biology.

Conflict of interest statement

Competing interests: None declared.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Figures

Figure 1

CONSORT diagram of healthy volunteer trial. BAL, bronchoalveolar lavage; LPS, lipopolysaccharide.

Figure 2

Aspirin does not significantly reduce bronchoalveolar lavage (BAL) interleukin (IL)-8 in the healthy volunteer model of lipopolysaccharide (LPS)-induced lung injury, but does reduce neutrophil recruitment to the alveolar space. Healthy human subjects were randomised to placebo (n=13) or aspirin (n=20) for 7 days prior to LPS inhalation (50 μg) and underwent BAL 6 hours later. (A) IL-8 was measured in BAL fluid, but was not significantly reduced, p=0.11 (B) Neutrophils in BAL fluid were counted on cytospin and were reduced by over a third in the aspirin-treated group (estimated difference in median=0.8×105/mL, 95% CIs 0.06 to 2.1; *p=0.03 for aspirin vs placebo).

Figure 3

Aspirin reduces neutrophil proteases in the bronchoalveolar lavage (BAL) fluid of healthy volunteers who have inhaled lipopolysaccharide (LPS). BAL as in figure 2A was analysed for neutrophil granular enzymes. (a) Aspirin significantly reduced BAL MMP-8 (difference in median 2.2 ng/mL, 95% CIs 0.15 to 4.45; *p=0.04 for aspirin (n=20) vs. placebo (n=13)). (B) Aspirin significantly reduced BAL MMP-9 (difference in medians 14.2 ng/mL, 95% CIs 1.0 to 29.8; *p=0.04 for aspirin (n=20) vs. placebo (n=13)). (C) Aspirin did not significantly reduce MPO (n=20) versus control (n=13) (difference in median 12.4 ng/mL, 95% CIs −6.6 to 52.6; p=0.19). (D) Aspirin (n=20) did not statistically significantly reduce BAL neutrophil elastase compared with placebo (n=13) (median difference 4.5 ng/mL, 95% CIs −3.0 to 26.4; p=0.30).

Figure 4

Aspirin does not reduce alveolar leak nor markers of epithelial injury/activation in bronchoalveolar lavage (BAL) in healthy volunteers who have inhaled lipopolysaccharide. BAL as in figure 2 was analysed for (A) albumin as a marker of alveolar barrier integrity and (B) IgG:total protein ratio. Neither was affected by aspirin pretreatment. For albumin, difference in medians was 9.7 mg/L, 95% CIs −23.5 to 41.2. For IgG:total, protein difference in medians was 0.02, 95% CIs −0.01 to 0.04. (C) Aspirin did not affect BAL SP-D, a marker of type II alveolar epithelial cell activation; difference in medians 8.4 ng/mL, 95% CIs −0.5 to 19.5. (D) Aspirin did not reduce BAL MMP-7, an epithelial cell-derived protease in the alveolar space; difference in medians 0.2 ng/mL, 95% CIs −0.7 to 1.0). n=13 for placebo, n=20 for aspirin for all measures.

Figure 5

The effect of aspirin on peripheral neutrophil count. (A) Peripheral neutrophil count in healthy volunteers 6 hours after lipopolysaccharide (LPS) inhalation is not significantly reduced by aspirin, difference in medians 1.47×106/mL, 95% CIs −0.1 to 3.4, p=0.07. (B) Neutrophil extracellular trap (NET) formation in blood 6 hours after LPS inhalation was not statistically significantly reduced in the aspirin-treated cohort (n=20) versus placebo (n=13), difference in medians 0.9, 95% CIs −0.1 to 2,1; p=0.09. Data are expressed as multiples of mean optical density (OD) values obtained for NETs in normal blood.

Figure 6

The effect of aspirin on bronchoalveolar lavage (BAL) and plasma thromboxane B2 (TXB2) after lipopolysaccharide inhalation (A) Plasma thromboxane B2 was reduced by aspirin treatment (n=12 placebo, n=20 aspirin). Difference in medians 3077 pg/mL, 95% CIs 755 to 7850; *p=0.003. (B) BAL thromboxane B2 was significantly reduced in the aspirin-treated cohort (n=20) compared with placebo (n=13). One outlier from the placebo group is not shown within the range of the graph (value >7000 pg/mL), but is included in statistical analysis presented. Difference in medians 41 pg/mL, 95% CIs 14.7 to 64.3; *p=0.003. (BAL TXB2 is also significantly reduced in response to aspirin with this outlier excluded.)

Figure 7

Aspirin significantly reduces neutrophilic inflammation in the human ex vivo lung perfusion model. Human lungs were ventilated and perfused ex vivo. Blood was preincubated with aspirin (n=5) or placebo (n=6) for 1 hour before adding to the lung perfusate and simultaneously injuring the lung with lipopolysaccharide (LPS) (6 mg). Bronchoalveolar lavage (BAL) was carried out 4 hours after injury. (A) Aspirin significantly reduces BAL neutrophilia. Neutrophils in BAL fluid were counted in cytospins. *p=0.03 for aspirin (n=5) versus placebo (n=6), MWU test. (B) Ventilated perfused isolated human lungs that were injured ex vivo with LPS had reduced injury as measured by lung injury score when treated with aspirin (n=5) versus placebo (n=6). y axis, AU, arbitrary units), *p=0.05 MWU test. (C) Illustrative histological sections of lung are shown. (i) and (iii) are placebo-treated lungs at low and high power, respectively; (ii) and (iv) are aspirin-treated lungs at low and high power. Scale bar represents 100 μm. Alveolar septal wall thickening, neutrophil recruitment and proteinaceous debris staining are less marked in the aspirin than the placebo-treated group. Quantification of lung injury score was carried out in a blinded manner to counter any potential bias in image selection.