Ablation of the leptin receptor in myeloid cells impairs pulmonary clearance of Streptococcus pneumoniae and alveolar macrophage bactericidal function

Abstract

Leptin is a pleiotropic hormone produced by white adipose tissue that regulates appetite and many physiological functions, including the immune response to infection. Genetic leptin deficiency in humans and mice impairs host defenses against respiratory tract infections. Since leptin deficiency is associated with obesity and other metabolic abnormalities, we generated mice that lack the leptin receptor (LepRb) in cells of the myeloid linage (LysM-LepRb-KO) to evaluate its impact in lean metabolically normal mice in a murine model of pneumococcal pneumonia. We observed higher lung and spleen bacterial burdens in LysM-LepRb-KO mice following an intratracheal challenge with Streptococcus pneumoniae. Although numbers of leukocytes recovered from bronchoalveolar lavage fluid did not differ between groups, we did observe higher levels of pulmonary IL-13 and TNFα in LysM-LepRb-KO mice 48 h post infection. Phagocytosis and killing of ingested S. pneumoniae were also impaired in alveolar macrophages (AMs) from LysM-LepRb-KO mice in vitro and were associated with reduced LTB4 and enhanced PGE2 synthesis in vitro. Pretreatment of AMs with LTB4 and the cyclooxygenase inhibitor, indomethacin, restored phagocytosis but not bacterial killing in vitro. These results confirm our previous observations in leptin-deficient ( ob/ob) and fasted mice and demonstrate that decreased leptin action, as opposed to metabolic irregularities associated with obesity or starvation, is responsible for the defective host defense against pneumococcal pneumonia. They also provide novel targets for therapeutic intervention in humans with bacterial pneumonia.

Keywords: Streptococcus pneumoniae; bacterial pneumonia; host defense; leptin receptor; lung.

Figures

Fig. 1.

Characterization of LysM-LepRb-KO mice. A: genetic deletion of LepRb from AMs by LysM-Cre is specific for cells of the myeloid lineage. B: body weights. C and D: blood glucose and serum leptin levels in control (open circles) and LysM-LepRb-KO (closed squares) mice at 8 wk of age. *P < 0.05 vs. control using the Student’s t-test. Lines indicate means ± SE. AM, alveolar macrophage; LepRb, long form of the leptin receptor; WT, weight.

Fig. 2.

Increased lung and spleen bacterial loads in LysM-LepRb-KO mice following intratracheal S. pneumoniae infection. Control (open circles) and LysM-LepRb-KO (closed squares) mice were infected with 5 × 104 CFUs of S. pneumoniae, and bacterial burdens were quantified in lungs and spleens harvested 24 h (A) and 48 h (B) post infection. *P < 0.05 compared with control using the Student’s t-test. Lines indicate means ± SE from 2 separate experiments. CFU, colony-forming unit; ND, not detected.

Fig. 3.

Leukocyte counts in BALF from control and LysM-LepRb-KO mice following intratracheal S. pneumoniae infection. Control (open circles) and LysM-LepRb-KO (closed squares) mice were infected with 5 × 104 CFUs of S. pneumoniae, and leukocytes were recovered from BALF 24 h (A) and 48 h (B) post infection. Lines indicate means ± SE from 2 separate experiments. BALF, bronchoalveolar lavage fluid; CFU, colony-forming unit; PMN, polymorphonuclear neutrophils.

Fig. 4.

Elevated IL-13 and TNF-α in lung homogenates from control and LysM-LepRb-KO mice 48 h following intratracheal S. pneumoniae infection. Lung homogenates were prepared from control (open circles) and LysM-LepRb-KO (closed circles) mice 24 h (A) and 48 h (B) post infection with 5 × 104 CFUs of S.pneumoniae. Cytokine concentrations in lung homogenates were measured using ELISA. *P < 0.05 compared with control to LysM-LepRb-KO mice using the Student’s t-test. Lines indicate means ± SE from 2 separate experiments. CFU, colony-forming unit.

Fig. 5.

Impaired phagocytosis and killing of S. pneumoniae in AMs from LysM-LepRb-KO mice. Phagocytosis (A) and intracellular killing (B) were assessed in AMs from wild-type (open circles) and LysM-LepRb-KO (closed squares) mice. Lines indicate means ± SE with at least 5 replicates per experiment. *P < 0.05 compared with control by Student’s t-test. AM, alveolar macrophage.

Fig. 6.

Reduced LTB4 and increased PGE2 in AMs from LysM-LepRb-KO mice and exogenous LTB4 and indomethacin restores phagocytosis of S. pneumoniae in AMs from LysM-LepRb-KO mice. A: AMs were stimulated with 1 × 108 CFUs of heat-killed S. pneumoniae overnight, and LTB4 and PGE2 were measured in cell culture media. B: phagocytosis of S. pneumoniae was assessed in AMs from control (dashed line) or LysM-LepRb-KO mice treated with media alone (closed squares), LTB4 (10 nM) (solid circles), INDO (10 µM) (closed triangles), or LTB4 (10 nM) and INDO (10 µM) (closed diamonds). Data are expressed as a percentage of the control. Lines indicate means ± SE with 4–5 replicates per experiment. *P < 0.05 compared with control by Student’s t-test or by ANOVA with different letters indicating differences between groups. AM, alveolar macrophage; CFU, colony-forming unit; INDO, indomethacin.

Fig. 7.

Reduced nitric oxide and ROI synthesis in AMs from LyzM-LepRb-KO mice. A: AMs stimulated for 24 h with LTA (10 g/ml) and IFN-γ (100 ng/ml) for nitrite production. B: AMs stimulated for 24 h with heat-killed S. pneumoniae for ROI synthesis. Lines indicate means ± SE with 4–5 replicates per experiment. *P < 0.05 compared with control by Student’s t-test. AM, alveolar macrophage; LTA, lipoteichoic acid; RFU, relative fluorescent unit.