Farnesoid X Receptor Activation Protects Liver From Ischemia/Reperfusion Injury by Up-Regulating Small Heterodimer Partner in Kupffer Cells

Abstract

Farnesoid X receptor (FXR) is the nuclear receptor of bile acids and is involved in innate immune regulation. FXR agonists have been shown to protect multiple organs from inflammatory tissue injuries. Because liver expresses high levels of FXR, we explored the potential therapeutic benefits and underlying mechanisms of pharmacologic FXR activation in a murine model of partial liver warm ischemia. Pretreatment of mice with FXR agonist 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064) attenuated liver ischemia/reperfusion injuries (IRIs) in wild-type but not FXR knockout mice. Posttreatment with GW4064 facilitated liver recovery from IRI. Mechanistically, Kupffer cells (KCs) expressed much higher levels of FXR than bone marrow-derived macrophages (BMMs). Pretreatment of KCs but not BMMs with GW4064 resulted in lower tumor necrosis factor α but higher interleukin-10 expressions following toll-like receptor stimulation. FXR-targeted gene small heterodimer partner (SHP) was critical for the regulation of KC response by GW4064. In vivo, the depletion of KCs but not cluster of differentiation (CD) 11b⁺ cells or knockdown of SHP diminished the immune regulatory effect of GW4064 in liver IRI. Thus, FXR activation protects liver from IRI by up-regulating SHP in KCs to inhibit the liver proinflammatory response.

Figure 1

FXR activation protects livers from IRI. B6 mice pretreated with or without (control) FXR agonist GW4064 were subjected to 90 minutes of partial liver warm ischemia followed by 6 hours of reperfusion, as described in Materials and Methods. (A) Plots of average FXR target gene/HPRT ratios in livers of different experimental groups. Gene expressions in liver tissues were measured by qRT‐PCR. (B) Western blotting of FXR and SHP in liver tissues. (C) Representative liver HE staining (magnification ×100), plots of average sALT levels, and liver Suzuki scores of different experimental groups. (D) Plots of average inflammatory gene/HPRT ratios in livers. Data represent Mean ± SD, n = 4‐6/group; *P < 0.05. Abbreviations: h, hour; NS, not significant.

Figure 2

FXR activation facilitates liver recovery from IRI. B6 mice were subjected to 90 minutes of partial liver warm ischemia and divided into two groups treated with daily injection of either the vehicle control or GW4064, as described in Materials and Methods. IR liver tissues were harvested at day 5 after reperfusion. (A) Representative liver HE sections (magnification ×100) and plots of average Suzuki scores of control and GW groups. (B,C) Plots of average target gene/HPRT ratios of different experimental groups. Inflammatory and FXR‐targeted gene expressions in sham and IR livers were measured by qRT‐PCR. Representative results of two independent experiments; Data represent Mean ± SD, n = 4‐6/group; *P < 0.05, **P < 0.01. Abbreviations: d, day; MerTK, Mer tyrosine kinase; TIM‐4, T‐cell immunoglobulin mucin protein 4.

Figure 3

FXR expressions and functions in macrophages. (A) Plots of average FXR/HPRT gene expression ratios in livers and macrophages. FXR expressions were determined by qRT‐PCR in sham or IR livers (6 hours after reperfusion), liver NPCs (isolated from sham or IR livers after 0 or 6 hours of reperfusion), or BMMs stimulated with LPS for 0 and 6 hours in vitro. (B) Western blots of FXR expressions in KCs and BMMs. (C, D) Plots of target gene/HPRT ratios in KCs and BMMs. KCs were isolated from sham livers, and BMMs were derived from 7‐day cultures of bone marrow cells, as described in Materials and Methods. These macrophages were pretreated with either vehicle controls or GW4064 followed by 3 hours of stimulation of LPS. Gene expressions were determined by qRT‐PCR. Representative results of at least two independent experiments; Data represent Mean ± SD, n = 3‐6/group; *P < 0.05. Abbreviations: DMSO, dimethyl sulfoxide; PBS, phosphate‐buffered saline.

Figure 4

KCs mediate the immune regulatory effect of FXR activation in liver IRI. Groups of WT B6 mice were treated with either blank or clodronate liposomes 48 hours before, followed by the treatment of either vehicle controls or GW4064 2 hours before the onset of liver ischemia. Liver injuries and inflammatory immune responses were evaluated at 6 hours after reperfusion. (A) Plots of average sALT levels, representative liver histologic analysis (HE staining, magnification ×100), and plots of average Suzuki's scores in different experimental groups. (B,C) Plots of average target gene/HPRT expression ratios in different experimental groups. Liver inflammatory and FXR‐targeted gene expressions were determined by qRT‐PCR. Representative results of at least two independent experiments; Data represent Mean ± SD, n = 3‐6/group; *P < 0.05. Abbreviation: NS, not significant.

Figure 5

CD11b+ BMMs are dispensable for FXR‐mediated liver protection. CD11b‐DTR mice were treated with either phosphate‐buffered saline (Ctl) or DT 48 hours before, followed by the treatment of either vehicle controls or GW4064 2 hours before the onset of liver ischemia, as described in Materials and Methods. Liver injuries and inflammatory immune responses were evaluated at 6 hours after reperfusion. (A) Plots of average sALT levels and Suzuki scores in different experimental groups. Representative HE staining (magnification ×100) of IR livers. (B, C) Plots of average target gene/HPRT expression ratios in different experimental groups. Expressions of liver inflammatory and FXR‐targeted genes were determined by qRT‐PCR. Representative results of at least two independent experiments; Data represent Mean ± SD, n = 3‐6/group; *P < 0.05. Abbreviation: h, hour.

Figure 6

SHP is critical for FXR‐mediated liver protection. Scrambled or SHP‐specific siRNA was injected into different groups of B6 mice, followed by the administration of vehicle control or GW4064, as described in Materials and Methods. Liver IRI was evaluated at 6 hours after reperfusion. Expressions of FXR‐target genes and inflammatory genes in IR livers were measured by qRT‐PCR. (A) Plots of average ratios of target gene/HPRT expressions in different experimental groups. (B) Plots of average sALT levels and Suzuki's scores in different experimental groups. Representative liver histologic images (HE staining, magnification ×100). (C) Plots of average target gene/HPRT rations in different experimental groups. Gene expressions were determined by qRT‐PCR. Representative results of at least two independent experiments; Data represent Mean ± SD, n = 3‐6/group; *P < 0.05. Abbreviations: h, hour; ssiRNA, scrambled small interfering RNA.

Figure 7

SHP is critical for FXR‐mediated immune regulation in KCs. KCs were transfected with either scrambled or SHP‐specific siRNA. Transfected cells were pretreated with either PBS or GW4064, followed by 3 hours of stimulation of LPS. Gene expressions were determined by qRT‐PCR. Average ratios of target gene/HPRT ratios in different experimental groups were plotted. Representative results of at least two independent experiments; Data represent Mean ± SD, n = 3/group; *P < 0.05. Abbreviations: PBS, phosphate‐buffered saline; SC, scrambled small interfering RNA.