Simvastatin decreases lipopolysaccharide-induced pulmonary inflammation in healthy volunteers

Abstract

Rationale: Simvastatin inhibits inflammatory responses in vitro and in murine models of lung inflammation in vivo. As simvastatin modulates a number of the underlying processes described in acute lung injury (ALI), it may be a potential therapeutic option.

Objectives: To investigate in vivo if simvastatin modulates mechanisms important in the development of ALI in a model of acute lung inflammation induced by inhalation of lipopolysaccharide (LPS) in healthy human volunteers.

Methods: Thirty healthy subjects were enrolled in a double-blind, placebo-controlled study. Subjects were randomized to receive 40 mg or 80 mg of simvastatin or placebo (n = 10/group) for 4 days before inhalation of 50 microg LPS. Measurements were performed in bronchoalveolar lavage fluid (BALF) obtained at 6 hours and plasma obtained at 24 hours after LPS challenge. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) was measured in monocyte-derived macrophages.

Measurements and main results: Pretreatment with simvastatin reduced LPS-induced BALF neutrophilia, myeloperoxidase, tumor necrosis factor-alpha, matrix metalloproteinases 7, 8, and 9, and C-reactive protein (CRP) as well as plasma CRP (all P < 0.05 vs. placebo). There was no significant difference between simvastatin 40 mg and 80 mg. BALF from subjects post-LPS inhalation induced a threefold up-regulation in nuclear NF-kappaB in monocyte-derived macrophages (P < 0.001); pretreatment with simvastatin reduced this by 35% (P < 0.001).

Conclusions: Simvastatin has antiinflammatory effects in the pulmonary and systemic compartment in humans exposed to inhaled LPS.

Figure 1.

Study design. Subjects were randomized to receive 40 mg simvastatin (n = 10), 80 mg simvastatin (n = 10), or placebo (n = 10) for 4 days before LPS inhalation. Bronchoaveolar lavage (BAL) was undertaken at 6 hours after lipopolysaccharide (LPS) inhalation and plasma was collected before subjects took the Day 4 medication under direct observation of the study team, 1 hour before LPS inhalation and at 24 hours after LPS inhalation.

Figure 2.

Effect of simvastatin on lipopolysaccharide (LPS)-induced neutrophil recruitment. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS, and supernatants were analyzed for (A) myeloperoxidase and (B) neutrophil elastase:α-1-antitrypsin complex (NE: α-1-AT). Open bars = placebo; solid bars = simvastatin. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Data are medians (IQR). * P < 0.05 versus placebo.

Figure 2.

Effect of simvastatin on lipopolysaccharide (LPS)-induced neutrophil recruitment. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS, and supernatants were analyzed for (A) myeloperoxidase and (B) neutrophil elastase:α-1-antitrypsin complex (NE: α-1-AT). Open bars = placebo; solid bars = simvastatin. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Data are medians (IQR). * P < 0.05 versus placebo.

Figure 3.

Effect of simvastatin on neutrophil apoptosis. Healthy human volunteers inhaled 50 μg lipopolysaccharide (LPS). Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF)was obtained 6 hours after inhalation of LPS. Neutrophil apoptosis analyzed by flow cytometry for BAL cells labeled with either annexin V (AV) or propidium iodide (PI). Cells labeled with neither AV nor PI (AV⁻/PI⁻) are viable, cells labeled with AV only (AV⁺/PI⁻) indicate early apoptosis, cells labeled with AV and PI (AV⁺/PI⁺) indicate those in late stage apoptosis, and cells labeled with PI only (AV⁻/PI⁺) indicate necrotic cells. Data are shown as individual points; the horizontal lines represent mean values. Data are available for 9 subjects in the placebo group and 15 subjects in the simvastatin group. *P < 0.05 versus placebo.

Figure 4.

Simvastatin inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α concentration. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS and supernatants were analyzed for (A) TNF-α and (B) IL-1β. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data are medians (interquartile range). *P < 0.05 versus placebo.

Figure 4.

Simvastatin inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α concentration. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS and supernatants were analyzed for (A) TNF-α and (B) IL-1β. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data are medians (interquartile range). *P < 0.05 versus placebo.

Figure 5.

Simvastatin inhibits lipopolysaccharide (LPS)-induced matrix metalloproteinase (MMP) concentration. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS and supernatants were analyzed for (A) MMP-7, (B) MMP-8, and (C) MMP-9. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data for MMP-7 are medians (interquartile range). *P < 0.05 versus placebo.

Figure 5.

Simvastatin inhibits lipopolysaccharide (LPS)-induced matrix metalloproteinase (MMP) concentration. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS and supernatants were analyzed for (A) MMP-7, (B) MMP-8, and (C) MMP-9. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data for MMP-7 are medians (interquartile range). *P < 0.05 versus placebo.

Figure 5.

Simvastatin inhibits lipopolysaccharide (LPS)-induced matrix metalloproteinase (MMP) concentration. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. Bronchoaveolar lavage fluid (BALF) was obtained 6 hours after inhalation of LPS and supernatants were analyzed for (A) MMP-7, (B) MMP-8, and (C) MMP-9. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data for MMP-7 are medians (interquartile range). *P < 0.05 versus placebo.

Figure 6.

Simvastatin inhibits lipopolysaccharide (LPS)-induced pulmonary and systemic C-reactive protein (CRP) production. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. CRP was measured in bronchoaveolar lavage fluid (BALF) obtained at (A) 6 hours and in plasma obtained at (B) 24 hours after LPS inhalation. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data for BALF CRP are medians (interquartile range). *P < 0.05 versus placebo.

Figure 6.

Simvastatin inhibits lipopolysaccharide (LPS)-induced pulmonary and systemic C-reactive protein (CRP) production. Healthy human volunteers inhaled 50 μg LPS. Simvastatin or placebo was administered for 4 days before LPS inhalation. CRP was measured in bronchoaveolar lavage fluid (BALF) obtained at (A) 6 hours and in plasma obtained at (B) 24 hours after LPS inhalation. Data are shown for the 10 subjects in the placebo group and the 20 subjects in the combined simvastatin group. Open bars = placebo; solid bars = simvastatin. Data for BALF CRP are medians (interquartile range). *P < 0.05 versus placebo.

Figure 7.

Simvastatin pretreatment reduces nuclear NF-κB translocation. Healthy human volunteers inhaled 50 μg lipopolysaccharide (LPS). Simvastatin or placebo was administered for 4 days before LPS inhalation. Monocyte-derived macrophages (MDMs) were pretreated with 5 mM simvastatin or medium control for 4 hours before stimulation with pooled bronchoaveolar lavage fluid (BALF) from subjects who had been pretreated with statin or placebo. Cells were lysed at 1 hour, and relative nuclear concentrations of the p65 subunit of NF-κB were measured. Data represent duplicate readings from MDMs from three separate volunteers, *P = 0.0001 for control versus placebo BALF, **P = 0.03 for placebo BALF versus simvastatin BALF, #P = 0.03 for placebo BALF versus simvastatin + placebo BALF.