Acyloxyacyl hydrolase promotes pulmonary defense by preventing alveolar macrophage tolerance

Abstract

Although alveolar macrophages (AMs) play important roles in preventing and eliminating pulmonary infections, little is known about their regulation in healthy animals. Since exposure to LPS often renders cells hyporesponsive to subsequent LPS exposures ("tolerant"), we tested the hypothesis that LPS produced in the intestine reaches the lungs and stimulates AMs, rendering them tolerant. We found that resting AMs were more likely to be tolerant in mice lacking acyloxyacyl hydrolase (AOAH), the host lipase that degrades and inactivates LPS; isolated Aoah-/- AMs were less responsive to LPS stimulation and less phagocytic than were Aoah+/+ AMs. Upon innate stimulation in the airways, Aoah-/- mice had reduced epithelium- and macrophage-derived chemokine/cytokine production. Aoah-/- mice also developed greater and more prolonged loss of body weight and higher bacterial burdens after pulmonary challenge with Pseudomonas aeruginosa than did wildtype mice. We also found that bloodborne or intrarectally-administered LPS desensitized ("tolerized") AMs while antimicrobial drug treatment that reduced intestinal commensal Gram-negative bacterial abundance largely restored the innate responsiveness of Aoah-/- AMs. Confirming the role of LPS stimulation, the absence of TLR4 prevented Aoah-/- AM tolerance. We conclude that commensal LPSs may stimulate and desensitize (tolerize) alveolar macrophages in a TLR4-dependent manner and compromise pulmonary immunity. By inactivating LPS in the intestine, AOAH promotes antibacterial host defenses in the lung.

Fig 1. Aoah^-/- mice are more susceptible to pulmonary infection with Pseudomonas aeruginosa.

(A) Mice were infected with 3 × 10⁶ PA i.n. and their body weights were measured daily for one week. None of the mice died. Data were combined from 3 experiments. n = 10. In another experiment, the lungs and blood were obtained for bacterial load analysis 48 h after infection. No bacteria or fewer than 75 CFUs were recovered from total blood. n = 6 or 7. (B-D) Mice were instilled i.n. with 1 × 10⁷ heated inactivated (HIA) PA. Control mice received PBS i.n. Five h later, cytokines or chemokines in the BALF were analyzed using ELISA. Data were combined from 2 experiments. n = 5 (B). Total cell numbers in BALF were counted. BALF immune cells were identified using FACS: CD11b⁺Ly6G⁺ neutrophils, Ly6G^-CD11c⁺CD11b^lo SiglecF⁺ alveolar macrophages (AMs, red), Ly6G^-CD11c^loCD11b⁺Ly6C⁺ mono-macrophages (monocyte-derived macrophages, cyan), and Ly6G^-CD11c^-CD11b^-SSC^loFSC^lo lymphocytes (black) (C). BALF immune cell numbers were shown (D). n = 3–6. (A, B and D) Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.

Fig 2. The innate immune responses to LPS of both AMs and alveolar epithelial cells are decreased in Aoah^-/- mice in vivo.

(A) Aoah^+/+ and Aoah^−/− mice were instilled with 10 μg LPS i.n. or PBS i.n. as controls. Five h later, the concentrations of inflammatory cytokines and chemokines in BALF were determined using ELISA. (B) Cells in BALF were counted and analyzed using FACS. (A, B) Data were combined from 4 experiments. n = 7–16. Mann-Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3. Aoah^-/- alveolar macrophages have reduced innate responses to LPS in vitro.

(A) AMs in BALF were allowed to adhere to plastic plates. Then they were treated with PBS or 10 ng/ml LPS in vitro for 2 h. The mRNAs were measured using quantitative real-time PCR. Data were combined from 2 or 3 experiments, n = 5–9. (B) AMs were treated with PBS or 10 ng/ml LPS in vitro for 6 h and the released cytokines or chemokines were measured in the culture media. Data were combined from 2 or 3 experiments, n = 5–8. (C) AMs were isolated and treated with PBS or 10 ng/ml LPS in vitro for 2 h. The mRNAs were measured using quantitative real-time PCR. The expression levels in the Aoah^+/+ PBS group were set to 1 and the relative expression levels of genes in the other groups were calculated. Data were combined from 2 or 3 experiments, n = 5–9. (D) AMs were cultured in RPMI 1640 containing 10% mouse serum in low-adherence plates. FITC-E. coli K12 at a ratio of 50 bacteria/cell or PBS was added. After 2 h incubation, cells were collected. After the extracellular FITC was quenched by trypan blue, the geometric mean fluorescence intensity (Geo MFI) of FITC was measured in AMs using FACS. Data were combined from 2 experiments, n = 5 or 6. (E) Mice were instilled i.n. with 2 × 10⁷ FITC-E. coli K12. Two h later, BALF cells were stained with anti-CD11c Ab and subjected to FACS analysis. AMs were gated as CD11c^hi cells and the Geo MFI of FITC was measured. Data were combined from 2 experiments, n = 5–7. (A-E) Mann-Whitney test was used. *, P < 0.05; **, P < 0.01.

Fig 4. Aoah^-/- alveolar macrophages have metabolic changes characteristic of tolerant monocytes.

(A) AMs were stained with Mitotracker Green and analyzed using flow cytometry. Histogram overlay of representative Aoah^+/+ and Aoah^-/- AMs (left panel). Geometric mean florescence intensity (Geo MFI) of Mitotracker Green on AMs was measured (right panel). Data were combined from 3 experiments, n = 8. (B) AMs were treated with PBS or 10 ng/ml LPS in vitro for 2 h. mRNA was measured using qPCR. The unstimulated expression levels of Aoah^+/+ (PBS) were set to 1 and the relative expression levels of genes in other groups were calculated. Data were combined from 2 experiments, n = 3–9. (C) AMs were cultured in RPMI 1640 containing 0.5% FBS and treated with PBS or 10 ng/ml LPS for 24 h. Lactate in the culture media was measured. Data were combined from 2 experiments, n = 7. (D) AMs were cultured in RPMI containing 5% FBS and treated with PBS or 10 ng/ml LPS for 24 h. ECARs were accessed using the Seahorse technology. Glycolysis was measured after the addition of 10 mM glucose. Data were combined from 2 experiments, n = 5–6. (A-D) Mann-Whitney test and Two-way ANOVA test (D, ECAR) were used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5. Aoah^-/- AMs express more MHC class II and costimulatory molecules.

(A and B) Aoah^+/+ and Aoah^-/- AMs were collected, treated with PBS or 1 ng/ml LPS for 6 h before the cell-surface expression of Ia, CD86 and SIRP-α was measured using FACS (A). The Geo MFI of Ia, CD86 and SIRPα was measured (B). (C) Aoah^+/+ and Aoah^-/- AMs were collected, treated with PBS or 1 ng/ml LPS for 2 h before MHC II, CIITA and DM mRNA was measured. (A-C) Data were combined from 2 experiments, n = 5–9. Mann-Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 6. Circulating LPS inhibits pulmonary innate responses in Aoah^+/+ mice.

(A-C) Aoah^+/+ mice were treated with 50 μg LPS s.c. (footpad injection). Four days later, in some experiments, lung LPS was measured, n = 5 (A). In other experiments, 10 μg LPS was instilled i.n. and 5 h later, BALF cytokine levels were measured using ELISA. Data were combined from 3 experiments. n = 8–12 (B). Four days later after LPS s.c. injection, Aoah^+/+ mouse AMs were explanted and treated with PBS or 10 ng/ml LPS for 6 h ex vivo. The culture media were collected for cytokine and chemokine ELISA. Data were combined from 2 experiments. n = 5 or 6 (C). (D, E) PBS or 50 μg LPS was given intrarectally on day 0, 2 and 4 to Aoah^+/+ mice. On day 8, in some experiments, lung LPS was measured, n = 6 (D). In other experiments, AMs were explanted and stimulated with 10 ng/ml LPS. After 6 h treatment, TNF-α, IL-6, KC and MIP-2 were measured in culture media using ELISA (E). Data were combined from 2 experiments. n = 7 or 8. (A–E) The Mann-Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 7. Intestinal LPS regulates pulmonary innate responses; TLR4 signaling is required for AM tolerance.

(A) Aoah^+/+ and Aoah^−/− mice were co-housed and treated with neomycin (1 g/L) in drinking water for 3 weeks and the lung LPS was measured. n = 6 or 7. (B) Five h after 10 μg LPS was instilled i.n., BALF cytokine levels were measured using ELISA. Data were combined from 2 or 3 experiments. n = 7–15. (C) AMs were isolated from naive Aoah^+/+, Aoah^-/-, Aoah^+/+TLR4^-/-, and Aoah^-/-Tlr4^-/- mice. Then they were treated with PBS or 10 ng/ml Pam3CSK4 for 6 h. TNF-α, KC and MIP-2 were measured in the culture medium. Data were combined from two experiments. n = 5 or 6. (A–C) Mann-Whitney test was used. *, P < 0.05; **, P < 0.01; ***, P < 0.001.