Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice

Abstract

Background: Pyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear.

Methods: C57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence.

Results: The expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-α2 (IL-1α2) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-α± (TNF-α±), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS.

Conclusions: This study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Figure 1

Alveolar macrophage pyroptosis occurs in vivo after LPS stimulation. Mice were treated with YVAD (6.5 mg/kg, in 1% DMSO in PBS) or DEVD (6.5 mg/kg, in 1% DMSO in PBS) by IP injection 1 h before LPS (20 kg/mg) administration. Alveolar macrophages were isolated from mice after 16 h of LPS administration. Proteins were obtained and caspase-1 P10 and cleaved caspase-3, NLRP3 inflammation activation were analyzed by Western blotting. *P < 0.05 vs. the sham group; †P < 0.05 vs. the LPS + vehicle group; ‡P < 0.05 vs. the LPS + YVAD group. Results are representative of three separate independent experiments.

Figure 2

Alveolar macrophage pyroptosis occurs ex vivo after LPS stimulation. Alveolar macrophages isolated from mice were stimulated for 5 h with or without LPS (500 ng/ml) and ATP (5 mmol) added during the last hour of culture, in the absence or presence of 50 μmol of YVAD or DEVD. (a) Cells were stained with FLICA, and pyroptotic cells were detected by flow cytometry. Caspase-1 speck (b) and ASC (c) cells were analyzed by immunofluorescence. *P < 0.05 vs. the control group; †P < 0.05 vs. the LPS/ATP group. ‡P < 0.05 vs. the lipopolysaccharide/ATP + YVAD group. Results are representative of three separate independent experiments.

Figure 3

Mice were treated with YVAD (6.5 mg/kg, in 1% DMSO in PBS) or DEVD (6.5 mg/kg, in 1% DMSO in PBS) by IP injection 1 h before LPS (20 kg/mg) administration. (a) Changes in the histology of lung injury after 16 h of LPS administration (H and E, ×400). The arrowheads show alveolar macrophages; (b) The lung injury score; (c)The total number of alveolar macrophages in bronchoalveolar lavage fluid;(d) Water content of lung; (e)The total protein concentration in bronchoalveolar lavage fluid. *P < 0.05 vs. the sham group; †P < 0.05 vs. the LPS + vehicle group; ‡P < 0.05 vs. the LPS + YVAD group. n = 6 mice/group. Results are representative of three separate independent experiments.

Figure 4

The influences of YVAD and DEVD treatment of LPS-induced cytokine levels in plasma and bronchoalveolar lavage fluid. After YVAD and DEVD treatment and LPS injection at predetermined time points, serum and bronchoalveolar lavage fluid samples were prepared and tested for levels for IL-18, IL-1β, TNF-α and HMGB1 in serum (a–d) and bronchoalveolar lavage fluid (e–h). *P < 0.05 vs. the sham group; †P < 0.05 vs. the LPS + vehicle group. ‡P < 0.05 vs. the LPS + YVAD group. n = 6 mice/group. All data shown are representative of at least three separate independent experiments.