Liver X receptor signaling is a determinant of stellate cell activation and susceptibility to fibrotic liver disease

Abstract

Background & aims: Liver X receptors (LXRs) are lipid-activated nuclear receptors with important roles in cholesterol transport, lipogenesis, and anti-inflammatory signaling. Hepatic stellate cells activate during chronic liver injury and mediate the fibrotic response. These cells are also major repositories for lipids, but the role of lipid metabolism during stellate cell activation remains unclear. We investigated the role of LXR signaling stellate cell activation and susceptibility to fibrotic liver disease.

Methods: Immortalized and primary stellate cells purified from mice were treated with highly specific LXR ligands. Carbon tetrachloride and methionine/choline deficiency were used as chronic liver injury models. Reciprocal bone marrow transplants were performed to test the importance of hematopoietically derived cells to the fibrotic response.

Results: LXR ligands suppressed markers of fibrosis and stellate cell activation in primary mouse stellate cells. Lxrαβ(-/-) stellate cells produce increased levels of inflammatory mediators, and conditioned media from Lxrαβ(-/-) cells increases the fibrogenic program of wild-type cells. Furthermore, Lxrαβ(-/-) stellate cells exhibit altered lipid morphology and increased expression of fibrogenic genes, suggesting they are primed for activation. In vivo, Lxrαβ(-/-) mice have marked susceptibility to fibrosis in 2 injury models. Bone marrow transplants point to altered stellate cell function, rather than hematopoietic cell inflammation, as the primary basis for the Lxrαβ(-/-) phenotype.

Conclusions: These results reveal an unexpected role for LXR signaling and lipid metabolism in the modulation of hepatic stellate cell function.

Figure 1. Reciprocal lipogenic / anti-inflammatory action of LXRs in immortalized stellate cells

Gene expression in human LX-2 stellate cells by qPCR. (A–B) LX-2 cells were cultured to near confluence and treated with increasing amounts of ligand for 12–18 hours: GW3965 (0 → 1 µM), 22R-hydroxycholesterol (0 → 5 µM). (C) LX-2 stellate cells were pretreated for 12 hours with 1 µM GW3965 or vehicle (DMSO), then exposed to LPS (0 → 5 ng/mL) for another 6 hours.

Figure 2. LXR agonism suppresses myofibroblastic gene expression and promotes neutral lipid accumulation in stellate cells

(A) Gene expression from hepatic non-parenchymal fractions of WT and Lxrαβ −/− mice treated for 7 days with 1 µM GW3965 or vehicle (DMSO). (B) Freshly isolated, highly purified primary stellate cells from WT mice are shown in phase contrast overlaid with autofluorescence (blue DAPI excitation) of the retinoid content in each cell, 200×. (C) WT stellate cells (day 5 of culture activation) stained with a FITC-conjugated, α-smooth muscle actin monoclonal antibody (green), 50× and 100×. (D) Neutral lipid staining by BODIPY (green) in WT primary stellate cells (day 5), incubated with 1 µM GW3965 or DMSO for 36 hours, 400×. Nuclei are stained with DAPI (C & D).

Figure 3. LXR null stellate cells have marked alterations in lipid / retinoid distribution and increased fibrotic and inflammatory gene expression

(A) High-power (630×) micrographs of BODIPY stained (green), purified stellate cells from WT and Lxrαβ −/− mice one day after isolation. Stellate cells from wild-type mice have characteristic multiple, small droplets while Lxrαβ −/− cells typically have a particularly large droplet, almost the same size as the nucleus (blue DAPI). Autofluorescence of retinoid overlaps entirely with the lipid droplets (not shown). (B) Gene expression in primary WT and Lxrαβ −/− stellate cells early in culture activation (day 3). (C) Gene expression in primary WT stellate cells treated continuously with either 1 µM T0901317 (abbreviated T1317) or vehicle (DMSO) throughout culture activation.

Figure 4. Conditioned media from LXR null stellate cells increases the fibrotic and inflammatory program in WT cells

Purified, primary stellate cells from each genotype were isolated in parallel (cf. Methods) and plated on plastic for spontaneous culture activation. (A) Experimental design. (B,C) Gene expression by qPCR from day 5 culture activated cells showing myofibroblastic (B) and inflammatory (C) genes.

Figure 5. LXR null mice are susceptible to liver injury from CCl₄

(A) α-smooth muscle actin expression (dark brown) by immunohistochemistry, 72 hours after a single dose of CCl₄. N=10 per genotype, 100×. (B) WT and Lxrαβ−/− mice were challenged biweekly for one month with intraperitoneal CCl₄ or vehicle (cf Methods). Liver sections of intraparenchymal fibrotic septae (blue Masson’s trichrome) after chronic CCl₄ injury are shown, 100×. (C) ALT levels from CCl₄-treated mice. (D) Collagen α1(I) gene expression by qPCR from CCl₄-treated mice. (E) Hepatic fibrosis as quantified by digital scanning of entire liver sections (cf. Methods). N = 6–8 mice per group, repeated on 2 separate occasions.

Figure 6. LXR null mice develop more fibrosis on MCD diet

(A) Masson’s trichrome staining of liver sections from WT and Lxrαβ−/− mice after a month of MCD or control diets, 100×. (B) ALT levels from mice fed an MCD diet. (C) Collagen α1(I) gene expression by qPCR from mice fed the MCD diet. (D) Quantified hepatic fibrosis after MCD diet (cf. Methods). N = 6–10 per group.

Figure 7. Reciprocal bone marrow transplants between WT and LXR null mice

Lethally irradiated WT or Lxrαβ−/− (KO) recipient mice were reconstituted with bone marrow from WT or KO donors (cf. Methods). Eight weeks after transplantation the mice were then challenged with intraperitoneal CCl₄ injections as before. (A) Masson’s trichrome staining of liver sections from vehicle and CCl₄-treated chimeric animals. N = 8–10 per group, magnification 100×. (B) As in (A), except all recipient mice were Lxrαβ−/−, N = 4–7 per group, magnification 100×. (C) Col1αI gene expression by qPCR and quantified fibrosis in WT recipients after chronic CCl₄ administration. (D) Col1αI gene expression by qPCR and quantified fibrosis in Lxrαβ−/− recipients after chronic CCl₄ administration.