Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice

Abstract

The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections.

Keywords: Streptococcus pneumoniae; bacterial pneumonia; host defense; lung; microsomal prostaglandin E synthase; prostaglandins.

Fig. 1.

Prostanoid biosynthesis begins with the activation of cytosolic phospholipase A₂ (cPLA₂) after infection. After liberation from tissue phospholipids, arachidonic acid (AA) is oxygenated by cyclooxygenase enzymes (COX-1 and COX-2), resulting in the formation of prostaglandin H₂ (PGH₂). Subsequent metabolism by terminal synthase enzymes thromboxane synthase (TXS), prostaglandin F synthase (PGFS), microsomal prostaglandin E₂ synthase-1 (mPGES-1), prostaglandin D₂ synthase (PGDS), and prostaglandin I₂ synthase (PTGIS) converts PGH₂ to thromboxane (TXA₂), prostaglandin F_2α (PGF_2α), prostaglandin E₂ (PGE₂), prostaglandin D₂ (PGD₂), and prostacyclin (PGI₂), respectively. TXB₂ and 6-keto-PGF_1α are stable metabolites of TXA₂ and PGI₂, respectively.

Fig. 2.

Reduced survival and increased pulmonary and spleen bacterial burdens in mPGES-1 KO mice after S. pneumoniae infection. A: wild-type (WT) and mPGES-1 KO mice were infected with 5 × 10⁴ CFUs of the serotype 3 6303 strain of S. pneumoniae, and survival was monitored for 10 days. B: in a separate group of mice, bacterial burdens were quantified in lungs and spleens harvested 24 and 48 h after infection. C: WT and mPGES-1 KO mice were infected with 50,000 CFUs of the D39 serotype 2 strain of S. pneumoniae, and lung and spleen bacterial burdens were assessed 24 h later. Survival curves, representing n = 20 from 4 separate experiments, were evaluated with a log-rank test. *P < 0.05 comparing WT to KO by Student's t-test. Bars represent means ± SE of n = 5–11 mice per group from 3 separate experiments.

Fig. 3.

Leukocyte counts in bronchoalveolar lavage fluid recovered from mice after S. pneumoniae challenge. WT and mPGES-1 KO mice were infected with 5 × 10⁴ CFUs of S. pneumoniae. Twenty-four (A) and forty-eight (B) hours later, leukocytes were recovered by bronchoalveolar lavage, and monocyte/macrophage (Mono/Mac), neutrophil (PMN), and lymphocyte (lymph) counts were determined. Bars represent means ± SE of n = 8–15 mice per group from 3 separate experiments.

Fig. 4.

Redistribution of prostanoids in mPGES-1 KO mice after S. pneumoniae infection. WT and mPGES-1 KO mice were infected with 5 × 10⁴ CFUs of S. pneumoniae. A and B: lung homogenates were assayed for PGE₂ (A) and TXB₂ and 6-keto-PGF_1α (B). AMs obtained from WT and mPGES-1 KO mice were infected with S. pneumoniae. C: supernatants harvested at 2 h were assayed for PGE₂, TXB₂, and 6-keto-PGF_1α. Bars represent means ± SE of n = 3–10 mice per group from 2 separate experiments, and AM stimulation in vitro was done with n = 3 with 3–5 replicates per experiment. *P <0.05 compared with WT by Student's t-test.

Fig. 5.

Cytokine production in mPGES-1 KO mice after S. pneumoniae infection. WT and mPGES-1 KO mice were infected with S. pneumoniae. A and B: lung homogenates were assayed for IL-6, IL-10, IL-17, MIP-2, TNF-α, and IL-12 p40 by ELISA. Bars represent means ± SE of n = 5–10 mice per group from 2 separate experiments. C: alveolar macrophages (AMs) were stimulated with lipoteichoic acid (LTA, 10 μg/ml) for 24 h, and concentrations of IL-6, IL-10, and TNF-α were measured in supernatants by ELISA. Bars represent means ± SE of n = 3. *P <0.05 compared with WT by Student's t-test.

Fig. 6.

Effects of mPGES-1 deficiency on alveolar macrophage (AM) bactericidal function: phagocytosis (A), killing (B), reactive oxygen intermediate (ROI; C), nitrite production (D), and inducible nitric oxide synthase (iNOS) expression (E) in AMs from WT and mPGES-1 KO mice. A: phagocytosis of S. pneumoniae was assessed in AMs from WT and mPGES-1 KO mice. B: intracellular killing of S. pneumoniae was assessed in AMs from WT and mPGES-1 KO, mPGES-1 KO with PGE₂ (1 μM), or mPGES-1 KO with indomethacin (10 μM) where indicated. C: ROI production was assessed in AMs stimulated with heat-killed S. pneumoniae. D: nitrite production was assessed in AMs stimulated with lipoteichoic acid (LTA, 10 μg/ml) and IFN-γ (100 ng/ml) for 24 h. E: quantitative real-time PCR was used to quantify mRNA levels of inducible nitric oxide synthase (iNOS); data were normalized to GAPDH and expressed relative to WT. Bars represent means ± SE of n = 3–7 experiments with at least 5 replicates per experiment. *P <0.05 compared with WT by ANOVA. ND, none detected.

Fig. 7.

The NO donor SNAP restores bacterial killing in AMs from mPGES-1 KO mice. Intracellular killing of S. pneumoniae serotype 3 (A) or the D39 strain of S. pneumoniae (B) was assessed in AMs from WT mice, mPGES-1 KO mice, and mPGES-1 KO mice treated with SNAP (1 μM). Bars represent means ± SE of n = 3–4 experiments with at least 5 replicates per experiment. *P <0.05 compared with WT and mPGES-1 KO + SNAP, ^#P < 0.05 vs. WT by ANOVA.