Effects of liver X receptor agonist treatment on pulmonary inflammation and host defense - PubMed (original) (raw)

Effects of liver X receptor agonist treatment on pulmonary inflammation and host defense

Kathleen Smoak et al. J Immunol. 2008.

Abstract

Liver X receptor (LXR) alpha and beta are members of the nuclear receptor superfamily of ligand-activated transcription factors. Best known for triggering "reverse cholesterol transport" gene programs upon their activation by endogenous oxysterols, LXRs have recently also been implicated in regulation of innate immunity. In this study, we define a role for LXRs in regulation of pulmonary inflammation and host defense and identify the lung and neutrophil as novel in vivo targets for pharmacologic LXR activation. LXR is expressed in murine alveolar macrophages, alveolar epithelial type II cells, and neutrophils. Treatment of mice with TO-901317, a synthetic LXR agonist, reduces influx of neutrophils to the lung triggered by inhaled LPS, intratracheal KC chemokine, and intratracheal Klebsiella pneumoniae and impairs pulmonary host defense against this bacterium. Pharmacologic LXR activation selectively modulates airspace cytokine expression induced by both LPS and K. pneumoniae. Moreover, we report for the first time that LXR activation impairs neutrophil motility and identify inhibition of chemokine-induced RhoA activation as a putative underlying mechanism. Taken together, these data define a novel role for LXR in lung pathophysiology and neutrophil biology and identify pharmacologic activation of LXR as a potential tool for modulation of innate immunity in the lung.

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Figures

FIGURE 1

Functional LXR is expressed in alveolar macrophages and alveolar epithelial cells. A, Fifty micrograms (Bradford assay) of lung, liver, and spleen homogenate protein from C57BL/6 mice was immunoblotted with goat anti-LXRα, rabbit anti-LXRβ, and mouse anti-α-tubulin (loading control) Abs. Results from three representative animals are shown for each organ. ns, Nonspecific band. B, Immunohistochemistry was performed for LXRα (murine IgG2A as control) and for pro-SPC (normal rabbit serum as control) on 5-μm serial sections of paraformaldehyde-fixed normal C57BL/6 mouse lung. (upper left) Arrowheads indicate two alveolar macrophages with positive nuclear staining for LXRα. Lower left, Arrow indicates an alveolar epithelial type II cell with positive nuclear staining for LXRα. In the images at the right, arrows indicate alveolar type II cells with positive cytoplasmic staining for pro-SPC and arrowheads indicate alveolar macrophages with absent pro-SPC staining. Images are representative of findings in four independent animals. C, Lung homogenates (10 μg of protein) from C57BL/6 mice treated orally with vehicle or TO-901317 (50 mg/kg per day for 5 days) were immunoblotted with rabbit anti-ABCA1 (an LXR target gene) and also for LXRαβ using a rabbit dual recognition Ab. Representative blots are shown from two animals per treatment. D, BALF from C57BL/6 mice treated orally with vehicle or TO-901317 (50 mg/kg per day for 5 days) was screened for LXR-modifying activity by incubation upon HEK293 cells transiently transfected with LXRE luciferase and TK-Renilla luciferase and with LXRα, LXRβ, and RXRα expression constructs. Normalized luciferase activity was measured in triplicate from three to five animals per treatment in two independent experiments (*, p < 0.0001).

FIGURE 2

LXR agonist treatment reduces influx of PMNs to the LPS-exposed lung. A, C57BL/6 mice were treated orally with vehicle or TO-901317 (50 mg/kg per day for 5 days) and then exposed to aerosolized LPS. BAL total white blood cells were counted 4, 6, and 24 h after exposure (*, p < 0.001). MPO activity, a quantitative measure of PMNs, was assayed in lung homogenates of vehicle- and TO-901317-treated animals 8 h (B) and 24 (C) h after LPS exposure (*, p < 0.05). Data shown are representative of three independent experiments.

FIGURE 3

LXR agonist treatment selectively inhibits LPS-induced airspace cytokine expression. BALF MIP-2 (A), TNFα (B), and KC (C) were measured by ELISA 2 and 6 h after LPS exposure, and LIX (D) was measured 2 h after LPS exposure in vehicle- and TO-901317-treated C57BL/6 mice (*, p < 0.05; all other comparisons were NS). E, NF-κB activation was measured in lung nuclear isolates of vehicle- and TO-901317-treated animals 0 and 2 h following LPS inhalation using an ELISA that measures DNA binding of the p65 component of NF-κB, as previously described (32) (p = NS for vehicle/LPS vs TO-901317/LPS). Data shown represent four independent experiments involving n = 16 mice/experiment.

FIGURE 4

LXR agonist treatment inhibits PMN migration in vivo and ex vivo and attenuates chemokine-induced RhoA activation. A, Vehicle- and TO-901317-treated C57BL/6 mice underwent i.t. instillation of 0.5 μg of KC. BAL PMNs were quantified 6 h later (*, p < 0.05). Murine bone marrow-derived PMNs were lysed and immunoblotted using a dual anti-LXRαβ Ab. B, Human PMNs were incubated with 0.1% DMSO vehicle or 10 μM TO-901317 (4 h, 37°C). Chemotaxis to IL-8 and nondirectional migration to buffer were then assayed in a modified Boyden chamber as previously described (34) (p < 0.0001 × two-way ANOVA for effect of TO-901317 upon both chemotaxis and nondirectional migration). Data shown are representative of three independent experiments. C, Human PMNs pretreated with DMSO vehicle or TO-901317 as in B were tested for PMA- and fMLP-induced superoxide anion (O2-) generation as previously described (35). D, Bone marrow PMNs were harvested from vehicle- and TO-901317-treated animals as previously described (29). Cells were then exposed to 25 ng/ml KC for the indicated times, and RhoA activation was assayed by Rhotekin-binding domain pull-down, as described previously (32), from three independent experiments. Lysates were immunoblotted for total RhoA as a loading control. Normalized RhoA activation was quantified by densitometry (p < 0.05 for TO-901317 treatment by two-way ANOVA).

FIGURE 5

LXR agonist treatment modifies lung inflammation triggered by a host defense against Klebsiella pneumoniae. Vehicle- and TO-901317-treated C57BL/6 mice underwent i.t. inoculation with 2000 CFU of K. pneumoniae. A, BAL PMNs were quantified 6, 24, and 48 h postinoculation (*, p < 0.05). B, Lung histopathology was quantified by a pathologist blinded to treatment conditions in the left lung of vehicle- and TO-901317-treated mice 48 h following K. pneumoniae inoculation using a 0 -4 composite scoring system that grades inflammatory cell infiltration of alveoli and bronchioles, bronchial wall changes, and alveolar macrophage accumulation (38, 39) (p = NS, n = 20 animals/treatment pooled from four independent experiments). C, BALF TNFα and MIP-2 were quantified 6 h postinoculation (*, p < 0.05 for both). Data shown represent three to four independent experiments. D and E, Vehicle- and TO-901317-treated C57BL/6 mice underwent i.t. inoculation with 2000 CFU of K. pneumoniae. At the indicated times following inoculation, lung (D) and splenic (E) homogenate CFUs were quantified as previously described (*, p < 0.05) (29). Data shown represent four independent experiments.

FIGURE 6

LXR agonist treatment enhances mortality induced by intratracheal K. pneumoniae. C57BL/6 mice were pretreated with vehicle or TO-901317 (50 mg/kg per day for 3 days) and then inoculated i.t. with 2000 CFU of K. pneumoniae. Daily treatment was continued postinoculation and mortality was monitored (*, p = 0.002 by log rank test). Data shown represent n = 10 mice/treatment group from one of two representative independent experiments.

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Effects of liver X receptor agonist treatment on pulmonary inflammation and host defense - PubMed (original) (raw)