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