Fig 1.
AOAH expression in alveolar macrophages is up-regulated upon LPS exposure in vitro and in vivo.
(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 Ccr2-/- 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+CD11blo 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.
Fig 2.
AOAH ameliorates LPS-induced lung injury and promotes recovery.
(A) Aoah-/- and Aoah+/+ mice were instilled with 200 μg LPS i.n. and their survival was monitored. Log-rank test was used. n = 28 (Aoah+/+) and 33 (Aoah-/-). (B) Body weight was measured daily in the mice that survived LPS instillation. Two-way ANOVA test was used. n = 26 (Aoah+/+) and 18 (Aoah-/-). (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 (Aoah+/+) and 18 (Aoah-/-). (D) On days 0, 1, 4, and 7 after 150 μg LPS i.n., Aoah+/+ and Aoah-/- 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. Aoah+/+ and Aoah-/- 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.
Fig 3.
Intranasal LPS induces prolonged lung inflammation in Aoah-/- mice.
(A) Aoah+/+ and Aoah-/- 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. Aoah-/- 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 Aoah+/+ (B) and Aoah-/- (C) mice 4 days after LPS i.n. (D) Geo MFI of MHC II on AMs from Aoah+/+ and Aoah-/- (Left panel). Histogram overlay of MHCII expression of Aoah+/+ and Aoah-/- 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) Aoah+/+ or Aoah-/- 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 Aoah+/+ 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.
Fig 4.
AOAH does not regulate HCl-induced lung inflammation.
Aoah+/+ and Aoah-/- mice were treated i.n. with 50 μl of 0.2 M HCl and followed for 4 days. Cells in BALF were counted and differentially analyzed. Aoah+/+and Aoah-/- mice developed similar degrees of alveolar inflammation and the inflammation resolved at similar rates. Combined data from 3 experiments. Two-way ANOVA test was used. n = 9.
Fig 5.
Persistent expression of neutrophil chemoattractants in Aoah-/- lung.
(A) Before and 1, 4, and 7 days after 150 μg LPS i.n., neutrophil chemoattractant MIP-2 (CXCL-2/3), KC (CXCL-1), IL-23, IL-17 and CXCL5 mRNA was determined in lung homogenates. Two-way ANOVA test was used. n = 8–9. (B) Seven days after 150 μg LPS i.n., CD45+ leukocytes and CD45- lung parenchymal cells were separated using MACS. MIP-2, KC, IL-23, IL-17 and CXCL5 mRNA expression was measured in both cell populations. Two-way ANOVA test was used. n = 4–5. (C, D) Seven days after PBS i.n. or 150 μg LPS i.n., AMs were isolated from Aoah+/+, Aoah-/- and Aoah-/-Tlr4-/- mice and either cultured alone or co-cultured with lung epithelial cell line MLE-12 for 6 hrs before MIP-2 and KC were measured in the media. One-way ANOVA test was used. n = 6. (E) Seven days after 150 μg LPS i.n., Aoah-/-Tlr4-/- AMs were harvested and co-cultured with MLE-12. Naïve Tlr4+/+ or Tlr4-/- AMs were added to the co-culture for 6 hrs. KC was measured in the culture media. Only Tlr4+/+ AMs promoted KC release. Student’s t test was used. n = 6. (F) Seven days after 150 μg LPS i.n., Aoah+/+ and Aoah-/-AMs were isolated and co-cultured with MLE-12 in the presence of anti-TNF-α, anti-IL-1β, both antibodies, or isotype control antibodies. KC in the culture media was measured by ELISA. Data were combined from 3 experiments. One-way ANOVA test was used. n = 8–11. (G) Seven days after 150 μg LPS i.n., AMs were isolated and RNA was extracted for qPCR. Student’s t test was used. n = 6–9. (H) Seven days after 150 μg LPS i.n., CD45- CD326+ lung epithelial cells were sorted using FACS and subjected to qPCR analysis. Student’s t test was used. n = 6–7. (I) Seven days after 150 μg LPS instillation, i.n., AMs were Isolated, allowed to adhere to plastic dishes and treated with 100 pg/ml LPS for 6 hrs before IL-6, TNF-α, MIP-2 and KC were measured in the culture media. Student’s t test was used to compare LPS-exposed AMs with control AMs of each strain of mice. n = 6.
Fig 6.
AOAH promotes the resolution of pulmonary inflammation induced by Gram-negative bacteria.
(A) Aoah+/+ and Aoah-/- mice were instilled i.n. with heat-inactivated 5 X 106 Klebsiella pneumoniae. 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.
Fig 7.
Chronic exposure to LPS leads to greater lung inflammation in Aoah-/- lungs.
Mice were treated with 10 μg LPS i.n. twice a week for 8 weeks. (A) Aoah-/- mice had more inflammatory cells in their BALF than Aoah+/+ mice. Two-way ANOVA was used. n = 6–8. (B) AOAH deficiency aggravated lung inflammation after chronic exposure to LPS. The images represent at least 80% of the whole sections. n = 6–8. Magnification, 100 X.
Fig 8.
AOAH promotes the resolution of acute lung inflammation/injury induced by LPS or Klebsiella pneumoniae.
(A) A working model. After LPS or Klebsiella pneumoniae 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.