FXR protects lung from lipopolysaccharide-induced acute injury

Abstract

Acute lung injury and its more severe form, acute respiratory distress syndrome, are characterized by an acute inflammatory response in the airspaces and lung parenchyma. The nuclear receptor farnesoid X receptor (FXR) is expressed in pulmonary artery endothelial cells. Here, we report a protective role of FXR in a lipopolysaccharide-induced mouse model of acute lung injury. Upon intratracheal injection of lipopolysaccharide, FXR-/- mice showed higher lung endothelial permeability, released more bronchoalveolar lavage cells to the alveoli, and developed acute pneumonia. Cell adhesion molecules were expressed at higher levels in FXR-/- mice as compared with control mice. Furthermore, lung regeneration was much slower in FXR-/- mice. In vitro experiments showed that FXR activation blocked TNFα-induced expression of P-selectin but stimulated proliferation of lung microvascular endothelial cells through up-regulation of Foxm1b. In addition, expression of a constitutively active FXR repressed the expression of proinflammatory genes and improved lung permeability and lung regeneration in FXR-/- mice. This study demonstrates a critical role of FXR in suppressing the inflammatory response in lung and promoting lung repair after injury.

Fig. 1.

FXR is expressed in the mouse lung. A, RT-QPCR analysis of expression levels of FXR transcript in WT mouse ileum, WT lung, and FXR−/− lung. B, Immunoblot analysis of FXR protein levels in WT and FXR−/− lung tissues. C, QRT-PCR analysis of expression levels of FXR transcript from isolated lung EC. D, Immunofluorescence staining of FXR and Pecam-1 on frozen lung tissues sections (green, FXR; red, Pecam-1; blue, DAPI). **, P < 0.01.

Fig. 2.

FXR−/− mice are more susceptible to LPS-induced ALI. A–D, Histologic analysis revealed elevated neutrophilic alveolar and interstitial infiltration by d 1 and 2 after LPS inhalation in FXR−/− mice, with increased interstitial thickening and mixed cellular infiltration on d 2 in the lungs of FXR−/− mice compared with the control WT. Arrow indicates increased interstitial thickening and mixed cellular infiltration. E and F, Representative micrographs of terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end labeling (TUNEL) staining of WT and FXR−/− lung tissue after LPS challenge. Lung sections of WT (E) and FXR−/− (F) mice collected 24 h after LPS challenge were stained with FITC-conjugated TUNEL to identify apoptotic cells. G, Number of the TUNEL-positive cells on D1 after LPS treatment. H, Total BAL cell counts of WT and FXR−/− mice after treatment with LPS (cellular infiltration in the airways after exposure to LPS). *, P < 0.05. TUNEL staining revealed there were approximately twice as many positive cells in lung tissue from FXR−/− mice (n = 4) compared with WT control mice (n = 4). The number of cells is the average of at least three different fields from each mouse (E–G). In FXR−/− animals, LPS treatment also triggered an altered profile of leukocyte influx into the airspace, with a marked increased of BAL cells at d 1 (D1) (n = 6) and 2 (D2) (n = 6), as compared with the WT controls (H). H&E, Hematoxylin and eosin.

Fig. 3.

FXR displays antiinflammatory activities in the lung after LPS treatment. Quantitative real-time PCR analysis of the expression of proinflammatory genes in lungs from WT and FXR−/− mice that were treated with a single dose of LPS (4 μg/10 g of BW) or PBS [as controls (Con) (Veh)]. RNA expression levels of TNFα (A), IFNγ (B), IL-6 (C), iNOS (D), and MCP1 (E) were analyzed on d 1 (D1) after LPS or PBS treatments. F, RNA levels of P-selectin expression were analyzed on d 1 (D1) and d 2 (D2) after LPS treatment. *, P < 0.05; **, P < 0.01. Con, Control; Veh, vehicle.

Fig. 4.

Defective lung regeneration in FXR−/− mice after LPS-induced ALI. A, Representative micrographs of BrdU immunostaining of WT and FXR−/− lungs after LPS-induced lung microvascular injury. Sections (5 μm) of lungs collected 48 h after LPS challenge were stained with FITC-conjugated anti-BrdU antibody to identify proliferating cells; nuclei were counterstained with propidium iodide. B, Quantification of BrdU-positive nuclei of the lung sections described in A at indicated time points after LPS challenge [d 1 (D1), n = 4; and d 2 (D2), n =4]. C, QRT-PCR analysis of Foxm1b mRNA levels in lungs collected from WT or FXR−/− mice at the indicated time points after LPS exposure. D, Fold change in Foxm1b gene transcription induction (D2/D0). E, Quantitative analysis of Cyclin D1 mRNA levels after treatment of WT or FXR−/− mice with LPS. F, Pulmonary permeability of WT or FXR−/− mice as a function of Evans blue dye extravasation at the indicated time points after treatment with LPS [d 1 (D1), n = 4 and d 2 (D2), n = 4]. *, P < 0.05. Con, Control; PI, propidium iodide.

Fig. 5.

Expression of a constitutively active FXR rescues promotes lung repair in FXR−/− mice. FXR−/− mice were infected by iv injection with either a control VP-16 adenovirus or an adenovirus that expresses FXR-VP16. After 4 d, the mice were treated with vehicle control (Con) or LPS (4 μg/10 g of BW). A, Lung vascular permeability was measured at the indicated time points [d 1 (D1), and d 2 (D2)] after LPS treatment. B, Quantification of BrdU-positive nuclei. C–H, Relative mRNA levels of Foxm1b (C), TNFα (D), IFNγ (E), iNOS (F), MCP1 (G), and P-selectin (H) as measured by RT-QPCR using total RNA prepared from the whole lung. *, P < 0.05. Con, Control.

Fig. 6.

FXR ligands suppress TNFα-induced P-selectin gene expression and stimulate DNA synthesis in primary lung EC. A, Primary lung EC from WT and FXR−/− mice were treated with TNFα or vehicle control for 5 h after pretreatment with or without 6eCDCA for 19 h. The mRNA levels of P-selectin were measured by RT-QPCR. B, Representative micrographs of BrdU immunostaining of 6eCDCA-treated primary lung EC. Nuclei were counterstained with DAPI. Cells were isolated from WT and FXR−/− lungs with an CD31 antibody and cultured for 5 d in M199 plus 20% FBS. Then the medium was changed to 5% resin-charcoal-superstripped serum for 2 d. Cells were then treated with 2 μm 6eCDCA or vehicle (dimethylsulfoxide) for 24 h before being subjected to immunostaining. C, Quantification of the BrdU-positive nuclei in WT and FXR−/− EC. The number of nuclei is the average of at least three different fields. D, CDCA and 6eCDCA-mediated up-regulation of Foxm1b mRNA expression in WT EC compared with FXR−/− cells. *, P < 0.05. Veh, Vehicle.