Early use of dexamethasone increases Nr4a1 in Kupffer cells ameliorating acute liver failure in mice in a glucocorticoid receptor-dependent manner

Abstract

Background and objective: Acute liver failure (ALF) is a type of disease with high mortality and rapid progression with no specific treatment methods currently available. Glucocorticoids exert beneficial clinical effects on therapy for ALF. However, the mechanism of this effect remains unclear and when to use glucocorticoids in patients with ALF is difficult to determine. The purpose of this study was to investigate the specific immunological mechanism of dexamethasone (Dex) on treatment of ALF induced by lipopolysaccharide (LPS)/D-galactosamine (D-GaIN) in mice.

Methods: Male C57BL/6 mice were given LPS and D-GaIN by intraperitoneal injection to establish an animal model of ALF. Dex was administrated to these mice and its therapeutic effect was observed. Hematoxylin and eosin (H&E) staining was used to determine liver pathology. Multicolor flow cytometry, cytometric bead array (CBA) method, and next-generation sequencing were performed to detect changes of messenger RNA (mRNA) in immune cells, cytokines, and Kupffer cells, respectively.

Results: A mouse model of ALF can be constructed successfully using LPS/D-GaIN, which causes a cytokine storm in early disease progression. Innate immune cells change markedly with progression of liver failure. Earlier use of Dex, at 0 h rather than 1 h, could significantly improve the progression of ALF induced by LPS/D-GaIN in mice. Numbers of innate immune cells, especially Kupffer cells and neutrophils, increased significantly in the Dex-treated group. In vivo experiments indicated that the therapeutic effect of Dex is exerted mainly via the glucocorticoid receptor (Gr). Sequencing of Kupffer cells revealed that Dex could increase mRNA transcription level of nuclear receptor subfamily 4 group A member 1 (Nr4a1), and that this effect disappeared after Gr inhibition.

Conclusions: In LPS/D-GaIN-induced ALF mice, early administration of Dex improved ALF by increasing the numbers of innate immune cells, especially Kupffer cells and neutrophils. Gr-dependent Nr4a1 upregulation in Kupffer cells may be an important ALF effect regulated by Dex in this process.

Keywords: Glucocorticoid; Dexamethasone; Kupffer cells; Acute liver failure; Nuclear receptor subfamily 4 group A member 1 (Nr4a1).

Fig. 1

LPS/D-GaIN-induced mouse ALF model (a) Schematic illustration of ALF mouse sample processing. (b) Changes of serum ALT and AST levels during mice ALF progression. All groups are compared with a PBS control group (n=5 or 9). (c) Representative images of mouse liver samples and hepatic hematoxylin and eosin (H&E) staining (n=5; scan bar=50 μm (×200) or 25 μm (×400)). (d) Serum levels of IL-10, IL-6, TNF-α, and CCL-2 after different time durations of LPS/D-GaIN stimulation (n=5 or 4). All groups are compared to a PBS control group. Data are expressed as mean±standard error of the mean (SEM). The number and value of each sample are shown by various marks. ^* P<0.05; ^** P<0.01. LPS, lipopolysaccharide; D-GaIN, D-galactosamine; ALF, acute liver failure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IL-10, interleukin-10; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; CCL-2, chemokine (C-C motif) ligand 2

Fig. 2

Changes of liver and blood immune cell numbers during LPS/D-GaIN-induced ALF in mice (a) Gating strategy for surface maker analysis within the total liver nonparenchymal cells. (b, c) t-SNE map of CD45⁺ cells analyzed by expression of CD45, B220, CD3, CD4, CD8, CD49b, CD11b, Ly6G, and F4/80 in liver based on 15 000 CD45⁺ cells pooled from 15 mice and stacked bar plots of cell numbers within different subsets. (d) Gating strategy for makers within the blood immune cells. (e, f) t-SNE map distribution of CD45⁺ blood cells analyzed by intensities of CD45, B220, CD3, CD4, CD8, CD49b, CD11b, and Ly6G markers from 15 000 CD45⁺ cells and stacked bar plots of cell numbers within different subsets. The results are expressed as mean±standard error of the mean (SEM; n=3 per group per time point). LPS, lipopolysaccharide; D-GaIN, D-galactosamine; t-SNE, t-distributed stochastic neighbor embedding; SSC-A, side scatter area; FSC-A, forward scatter area; FSC-H, forward scatter height; MDM, monocyte derived macrophage; PBS, phosphate-buffered saline

Fig. 3

Effects of early administration of Dex on ALF mice survival and liver injury Male C57 mice were treated with LPS/D-GaIN and Dex at early (0 h) and later (1 h) time. (a) Schematic illustration of mice processing. (b) Kaplan-Meier survival of ALF mice receiving Dex 1 mg/kg at 0 and 1 h (n=15). Log-rank test P<0.001 between LPS/D-GaIN group and LPS/D-GaIN+Dex 0 h group. Log-rank test P>0.05 between LPS/D-GaIN group and LPS/D-GaIN+Dex 1 h group. (c) Representative sample images and hematoxylin and eosin (H&E) staining of livers harvested at 4 h after the administrations of LPS/D-GaIN (n=5; scan bar=50 μm (×200) or 25 μm (×400)). (d) Changes in serum ALT and AST levels (n=8). (e) CBA methods detect serum levels of IL-6, TNF-α, CCL-2, and IL-10 (n=5). All groups are compared with the LPS/D-GaIN control group. The data are shown as mean±standard error of the mean (SEM). The number and value of each sample are shown by various marks. ^** P<0.01. LPS, lipopolysaccharide; D-GaIN, D-galactosamine; Dex, dexamethasone; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CBA, cytometric bead array; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; CCL-2, chemokine (C-C motif) ligand 2; IL-10, interleukin-10

Fig. 4

Effects of early use of Dex on immune cells in LPS/D-GaIN-induced ALF mice Samples of LPS/D-GaIN-induced WT mice treated with or without Dex were collected at 4 h. (a, b) The proportion of different immune cells in CD45⁺ cells in liver (a) and blood (b) with or without Dex treatment. (c) Distribution of Ly6G⁻ CD11b⁺F4/80⁺ Kupffer cells between ALF mice without and with Dex treatment by flow cytometry. (d) F4/80 protein expression in ALF mice without or with Dex treatment by immunohistochemical (IHC) techniques. Scan bar=50 μm. (e) Differential mRNA expression levels of Cd86 (left) and Cd163 (right) in Kupffer cells of ALF mice without or with Dex treatment. The data are shown as mean±standard error of the mean (SEM), n=5 per group. ^* P<0.05; ^** P<0.01. n.s., not significant; LPS, lipopolysaccharide; D-GaIN, D-galactosamine; Dex, dexamethasone; WT, wild-type; Mon, monocyte; Neu, neutrophil

Fig. 5

Effects of Dex on Nr4a1 expression in the liver and Kupffer cells in LPS/D-GaIN-induced ALF mice LPS/D-GaIN-induced WT mice were treated with or without Dex and liver samples were collected 4 h later. (a) RNA sequencing of Kupffer cells sorted from the liver in two groups (mentioned above). Gene expression heatmap showed the top differentially expressed genes between two samples. Yellow indicates high expression; purple and black indicate low expression. (b) Volcano plot of the Kupffer cells sequencing data. (c, d) mRNA expression levels of Nr4a1 in mice liver samples (c) and Kupffer cells (d) between four groups (n=5 per group). All groups are compared with the LPS/D-GaIN group. The data are shown as mean±standard error of the mean (SEM). ^* P<0.05; ^** P<0.01. LPS, lipopolysaccharide; D-GaIN, D-galactosamine; Dex, dexamethasone; WT, wild-type

Fig. 6

Effects of Gr inhibition on LPS/D-GaIN-induced ALF in mice and Nr4a1 levels in Kupffer cells (a) Gr mRNA expression of the Kupffer cells with or without LPS/D-GaIN and Dex administration (n=5). (b) Kaplan-Meier survival estimation of LPS/D-GaIN-challenged WT mice receiving no treatment, Dex treatment, or Dex and Gr antagonist RU486 (n=15). Log-rank test P<0.01 between Dex group and Dex-RU486 group. (c) Liver hematoxylin and eosin (H&E) staining after Dex or RU486 stimulation in LPS/D-GaIN-challenged WT mice (n=5; scan bar=100 μm). (d) Serum ALT and AST level changes among all groups (n=8). (e) Gr inhibition downregulates Nr4a1 mRNA levels in Kupffer cells in LPS/D-GaIN-induced WT mice (n=5). The results are shown as mean±standard error of the mean (SEM). ^** P<0.01. LPS, lipopolysaccharide; D-GaIN, D-galactosamine; Dex, dexamethasone; Gr, glucocorticoid receptor; WT, wild-type; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PBS, phosphate-buffered saline; Nr4a1, nuclear receptor subfamily 4 group A member 1