Acyloxyacyl hydrolase promotes the resolution of lipopolysaccharide-induced acute lung injury.

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection

AOAH ameliorates LPS-induced lung injury and promotes recovery.

<p>(A) <i>Aoah</i>^<i>-/-</i> and <i>Aoah</i>^<i>+/+</i> mice were instilled with 200 μg LPS i.n. and their survival was monitored. Log-rank test was used. n = 28 (<i>Aoah</i>^<i>+/+</i>) and 33 (<i>Aoah</i>^<i>-/-</i>). (B) Body weight was measured daily in the mice that survived LPS instillation. Two-way ANOVA test was used. n = 26 (<i>Aoah</i>^<i>+/+</i>) and 18 (<i>Aoah</i>^<i>-/-</i>). (C) The mice that survived were also quantitatively assessed for signs of illness (weight loss, ruffled fur, ocular discharge, rapid shallow breathing, and lethargy). Data were combined from 5 experiments. Two-way ANOVA test was used. n = 26 (<i>Aoah</i>^<i>+/+</i>) and 18 (<i>Aoah</i>^<i>-/-</i>). (D) On days 0, 1, 4, and 7 after 150 μg LPS i.n., <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> mouse BALF was collected and the protein concentration was determined. Two-way ANOVA test was used. n = 6–10. (E) In separate experiments, mice were injected with Evans Blue 1 hr before euthanasia. The extravascular dye in the lungs was extracted and measured. Two-way ANOVA test was used. n = 3–6. (F) Before and 1, 4, and 7 days after 150 μg LPS i.n., MPO activity in lung homogenates was measured. Two-way ANOVA test was used. n = 8–9. (G) Hematoxylin-Eosin stained sections of paraformaldehyde-fixed lungs are shown. <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> mice were treated with PBS or 150 μg LPS, i.n. One, four and seven days later, their lungs were removed, fixed, sectioned and stained. The images represent at least 75% of whole sections. Original magnification X 40; insets, X 400. n = 3–4.</p

AOAH expression in alveolar macrophages is up-regulated upon LPS exposure <i>in vitro</i> and <i>in vivo</i>.

<p>(A) AMs and PMs were purified by letting the alveolar or peritoneal cells adhere to tissue culture plates, then they were cultured untreated (controls) or treated with 10 ng/ml LPS, 1 μg/ml Pam3CSK4, 10 μg/ml Poly I:C or 1 μg/ml Pam3CSK4 + 10 μg/ml Poly I:C for 18 hrs. AOAH mRNA was measured by using quantitative real-time PCR. The expression levels of control AMs were set to 1 and the relative expression levels of AOAH in treated AMs, control and treated PMs were calculated. Each group of treated AMs was compared with control AMs using one-way ANOVA. n = 8–10. AOAH expression in LPS-treated PMs was compared with that in untreated control PMs using Student’s t test. n = 6. (B) Mice were instilled i.n. with 50 μl PBS, 200 μg LPS in 50 μl PBS or 50 μl 0.2M HCl, AMs were isolated 18 hours later and purified, and AOAH mRNA was measured. LPS induced AOAH mRNA expression whereas HCl did not. The AOAH expression levels of AMs from PBS i.n. mice were set to 1. Data were combined from 3 experiments. One-way ANOVA test was used. n = 6–8. AOAH expression in AMs from PBS or LPS instilled <i>Ccr2</i>^<i>-/-</i> mice were compared using Student’s t test. n = 7–8. (C) Mice were instilled i.n. with LPS-FITC or LPS as a control and BAL was performed to obtain alveolar cells 18 hours later. The cells were stained and subjected to FACS. Gating strategy: Alveolar cells were divided into 4 groups according to their surface expression of CD11c, Ly6G and CD11b: CD11c⁺CD11b^lo AMs, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺Ly6G^- mono-macrophages, and CD11b^-Ly6G^- lymphocytes. (D) The geometric mean florescence intensity of FITC (Geo MFI) was measured in cells from mice instilled with LPS-FITC (Green bars) or LPS (Black bars). AMs have high autofluorescence. n = 6.</p

Intranasal LPS induces prolonged lung inflammation in <i>Aoah</i>^<i>-/-</i> mice.

<p>(A) <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> mice were untreated or treated with 10 μg LPS i.n. and 1, 4, and 7 days later their BALF was harvested. Total cell numbers were counted. The differential analysis of cells was performed by using cytospin and Wright-Giemsa staining. <i>Aoah</i>^<i>-/-</i> mice have prolonged immune cell infiltration in their airspaces in response to LPS i.n. Two-way ANOVA test was used. n = 6–11. (B and C) Representative flow cytometric plots of neutrophils (Ly6G+) in BALF from <i>Aoah</i>^<i>+/+</i> (B) and <i>Aoah</i>^<i>-/-</i> (C) mice 4 days after LPS i.n. (D) Geo MFI of MHC II on AMs from <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> (Left panel). Histogram overlay of MHCII expression of <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> AM (right panel). (E) CD86 expression on AMs. (F) CD11c expression on AMs. Combined data from 3 experiments (D-E). Two-way ANOVA test was used. n = 6–11. (G) <i>Aoah</i>^<i>+/+</i> or <i>Aoah</i>^<i>-/-</i> mice were treated with 10 μg LPS i.n. Their lungs were perfused and lavaged. Total RNA was extracted from lung homogenates, reverse transcribed, and mRNA abundance was measured by using quantitative real-time PCR. The mRNA levels in <i>Aoah</i>^<i>+/+</i> lungs (PBS, i.n., day 0) were set to 1, and the relative expression of other groups was calculated. Data were combined from 3 experiments. Two-way ANOVA test was used. n = 6–11.</p

AOAH promotes the resolution of acute lung inflammation/injury induced by LPS or <i>Klebsiella pneumoniae</i>.

<p>(A) A working model. After LPS or <i>Klebsiella pneumoniae</i> are instilled i.n., AMs take up LPS or Gram-negative bacteria and degrade/inactive LPS. When AOAH is missing, AMs release bioactive LPS that can directly stimulate primed AMs to release cytokines and chemokines. Proinflammatory cytokines can further act on lung epithelial cells to produce more neutrophil chemoattactants, thus preventing neutrophil clearance and inflammation resolution. (B) AOAH deficiency has distinct consequences in different tissue compartments: prolonged macrophage tolerance in the peritoneum and persistent inflammation in the lung.</p

AOAH promotes the resolution of pulmonary inflammation induced by Gram-negative bacteria.

<p>(A) <i>Aoah</i>^<i>+/+</i> and <i>Aoah</i>^<i>-/-</i> mice were instilled i.n. with heat-inactivated 5 X 10⁶ <i>Klebsiella pneumoniae</i>. BALF was harvested for immune cell analysis before instillation and one and four days afterward. (B) The AM surface markers were measured using flow cytometry. (C) The lungs were excised and homogenized for cytokine and chemokine mRNA measurement. Data were combined from 2 experiments. Two-way ANOVA was used. n = 4–7.</p