Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia

Abstract

Fatty acid binding protein 4 (FABP4), an intracellular lipid chaperone and adipokine, is expressed by lung macrophages, but the function of macrophage-FABP4 remains elusive. We investigated the role of FABP4 in host defense in a murine model of Pseudomonas aeruginosa pneumonia. Compared with wild-type (WT) mice, FABP4-deficient (FABP4^-/-) mice exhibited decreased bacterial clearance and increased mortality when challenged intranasally with P. aeruginosa. These findings in FABP4^-/- mice were associated with a delayed neutrophil recruitment into the lungs and were followed by greater acute lung injury and inflammation. Among leukocytes, only macrophages expressed FABP4 in WT mice with P. aeruginosa pneumonia. Chimeric FABP4^-/- mice with WT bone marrow were protected from increased mortality seen in chimeric WT mice with FABP4^-/- bone marrow during P. aeruginosa pneumonia, thus confirming the role of macrophages as the main source of protective FABP4 against that infection. There was less production of C-X-C motif chemokine ligand 1 (CXCL1) in FABP4^-/- alveolar macrophages and lower airway CXCL1 levels in FABP4^-/- mice. Delivering recombinant CXCL1 to the airways protected FABP4^-/- mice from increased susceptibility to P. aeruginosa pneumonia. Thus, macrophage-FABP4 has a novel role in pulmonary host defense against P. aeruginosa infection by facilitating crosstalk between macrophages and neutrophils via regulation of macrophage CXCL1 production.-Liang, X., Gupta, K., Rojas Quintero, J., Cernadas, M., Kobzik, L., Christou, H., Pier, G. B., Owen, C. A., Çataltepe, S. Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Keywords: CXCL1; host defense; innate immunity.

Figure 1

FABP4^−/− mice demonstrate increased mortality and decreased bacterial clearance in P. aeruginosa pneumonia. A) C57BL/6 WT and FABP4^−/− mice were i.n. infected with 2 × 10⁹ CFUs P. aeruginosa, and survival was monitored ≤3 d, n = 15 mice/group (P = 0.0012). B–D) Bacterial burdens in the lung (B), spleen (C), and blood (D) were assessed 24 h after infection (n = 5/group). Data are presented as means ± sem. *P < 0.05, **P < 0.01.

Figure 2

Pseudomonas aeruginosa pneumonia results in worse ALI in FABP4^−/− mice. C57BL/6 WT and FABP4^−/− mice were i.n. infected with 2 × 10⁸ CFUs P. aeruginosa, and ALI was assessed 24 h later (n = 5–9/group). A) Representative hematoxylin and eosin–stained control and P. aeruginosa–infected (Pa) lungs 24 h after infection are shown. Scale bars, 50 µm. B) Lung injury scores were determined by a senior pathologist (L.K.) blinded to the genotypes and treatment, based on the presence of interstitial inflammation, alveolar inflammation, pleuritis, bronchitis, and vasculitis. C–G) Wet-to-dry lung-weight ratios (C) and BAL albumin levels (D) 24 h after infection are shown. TNF-α (E), IL-1β (F), and IL-6 (G) protein levels in BALF obtained 6 or 24 h after infection from P. aeruginosa–infected WT and FABP4^−/− mice were measured by ELISA. Data are presented as means ± sem. *P < 0.05, **P < 0.01.

Figure 3

FABP4^−/− mice have impaired early neutrophil accumulation in the lung during P. aeruginosa pneumonia. C57BL/6 WT and FABP4^−/− mice were i.n. infected with 2 × 10⁸ CFUs on live P. aeruginosa (n = 8–12/group). BALF samples were collected 6 and 24 h after infection. A–F) Total cells (A), neutrophils (B), and macrophages (C) were quantified in BALF samples. In another cohort of P. aeruginosa–infected mice (n = 5/group), lung homogenates were analyzed by flow cytometry and total number of leukocytes (CD45⁺) (D), neutrophils (CD45⁺CD11b⁺Ly6C⁺Ly6G⁺) (E), and macrophages (CD45⁺CD11c⁺SiglecF⁺MHCII^int) (F) were determined. G–I) Terminal blood collection was performed by cardiac puncture 6 and 24 h after infection from WT and FABP4^−/− mice with P. aeruginosa pneumonia for complete blood cell and differential blood counts (n = 5–8/group). Data are presented as means ± sem. **P < 0.01.

Figure 4

FABP4 is expressed in macrophages in P. aeruginosa–infected lungs. A, B) Immunohistochemistry for FABP4 (A) and double-immunofluorescence analysis for F4/80 and FABP4 (B) was performed on P. aeruginosa–infected FABP4^−/− and WT murine lung sections 24 h after infection. C) Double-immunofluorescence analysis for F4/80 and FABP4 was performed on cytospins of BALF harvested from control and P. aeruginosa–infected WT (Pap) mice 6 h after infection. Representative images are shown (n = 3–5/group).

Figure 5

Plasma FABP4 levels are increased during P. aeruginosa pneumonia in mice. WT mice were given i.n. PBS (n = 5) or 2 × 10⁸ CFUs of live P. aeruginosa (n = 10; Pa). A) Blood was collected 24 h later, and plasma FABP4 levels were measured using ELISA. B) Fat pads from the left epididymis of control and P. aeruginosa–infected mice were dissected and weighed (n = 6/group). Data are presented as means ± sem. *P < 0.05, ***P < 0.001.

Figure 6

Macrophage-derived FABP4 provides protection in P. aeruginosa pneumonia. WT and FABP4^−/− recipient mice were irradiated. BM was isolated from WT or FABP4^−/− donor mice, and 2 × 10⁶ BM cells were injected into tail veins of the recipient mice. Mice were infected i.n. with P. aeruginosa 8–10 wk after BM transplant. A) Kaplan-Meier survival plots are shown for FABP4 chimeric mice [WT recipients with WT BM cells (WT → WT, n = 26), WT recipients with FABP4^−/− BM cells (FABP4^−/− → WT, n = 28), FABP4^−/− recipients with FABP4^−/− BM cells (FABP4^−/− → FABP4^−/−, n = 9), and FABP4^−/− recipients with WT BM cells (WT → FABP4^−/−, n = 16)]. Combined survival curves were compared by Mantel-Cox log-rank test. B) Lung bacterial burdens of WT recipients with FABP4^−/− BM cells and FABP4^−/− recipients with WT BM cells were assessed 24 h after infection. C) Plasma FABP4 levels of WT recipients with FABP4^−/− BM cells and FABP4^−/− recipients with WT BM cells were measured 24 h after infection. Data are means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Macrophage-FABP4 is a key regulator of CXCL1 production during P. aeruginosa pneumonia. A) Alveolar macrophages were isolated from P. aeruginosa–infected WT and FABP4^−/− mice 6 h after infection. Relative mRNA levels of CXCL1, CXCL2, CXCL5, and CCL3 were determined by real-time RT-PCR. Means ± sem from 2 independent experiments are shown. B) Alveolar macrophages were isolated from WT and FABP4^−/− mice and were treated with 100 ng/ml P. aeruginosa LPS for 6 h. Relative mRNA levels of CXCL1, CXCL2, CXCL5, and CCL3 were determined by real-time RT-PCR. Means ± sem from 2 independent experiments are shown. C) CXCL1 protein levels in BALF from P. aeruginosa–infected WT and FABP4^−/− mice 6 or 24 h after infection were measured by ELISA (n = 5–8). D) CXCL2 protein levels in BALF from P. aeruginosa–infected WT and FABP4^−/− mice 6 or 24 h after infection were measured by ELISA (n = 5–8). Data are means ± sem. *P < 0.05, **P < 0.01.

Figure 8

Airway CXCL1 reconstitution protects FABP4^−/− mice from increased susceptibility to P. aeruginosa pneumonia. PBS or recombinant CXCL1 protein was instilled i.n. 30 min before P. aeruginosa infection. A) BALF from P. aeruginosa–infected mice was collected 6 h after infection, and CXCL1 levels were measured. B) Kaplan-Meier survival plots are shown for 13 mice/group (P = 0.02). C) Lung bacterial burdens were measured 24 h after infection. D–F) BALF was performed at 6 and 24 h postinfection and total cells (D), neutrophils (E), and macrophages (F) were enumerated. Data are means ± sem. *P < 0.05, **P < 0.01.