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. 2021 Apr 29;12(5):421.
doi: 10.1038/s41419-021-03704-w.

Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment

Sara Flamini  1   2 , Philipp Sergeev  3   4 , Zenobio Viana de Barros  1 , Tommaso Mello  5 , Michele Biagioli  1 , Musetta Paglialunga  1 , Chiara Fiorucci  1 , Tatiana Prikazchikova  3 , Stefano Pagano  1 , Andrea Gagliardi  1 , Carlo Riccardi  1 , Timofei Zatsepin  3   6 , Graziella Migliorati  1 , Oxana Bereshchenko #  7 , Stefano Bruscoli #  1
Affiliations

Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment

Sara Flamini et al. Cell Death Dis. .

Abstract

Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Lack of GILZ results in increased CCl4-induced liver fibrosis development.
A Sirius Red staining of liver sections from 4-month-old WT and GILZ KO mice that had been treated with oil (control; upper panels) or CCl4 (lower panels) for 6–7 weeks. Arrows indicate the areas of collagen deposition. B Graph shows the quantitative measurement of collagen deposition in all experimental groups. C, D AST (C) and ALT (D) levels in blood from 4-month-old WT and GILZ KO mice that had been treated with CCl4 for 6–7 weeks. E LI of 4-month-old WT and GILZ KO mice that had been treated with oil (control) or CCl4 for 6–7 weeks. F qPCR analysis of Asma mRNA expression in livers from 4-month-old WT and GILZ KO mice that had been treated for 6–7 weeks with oil (control) or CCl4. All data are presented relative to Actb mRNA expression. Each dot represents an individual mouse; bars indicate the mean. Results are presented as the means ± SEM of three (A, B, F) or two (C–E) independent experiments (*p < 0.05, ***p < 0.001, ns not significant).
Fig. 2
Fig. 2. GILZ deficiency is associated with elevated HSC activation.
A, B Immunohistochemical analysis of α-SMA expression in the liver sections of WT and GILZ KO mice that had been treated with oil or CCl4 for 72 h (A) or 7 weeks (B). Original magnification, 5× or 10×, as indicated. Arrows indicate the areas of α-SMA positivity. Scale bar, 100 μm (5× magnification images) or 200 μm (10× magnification images).
Fig. 3
Fig. 3. Lack of GILZ results in increased leukocyte infiltration in the liver upon CCl4 treatment.
A, B Number of infiltrated leukocytes in livers from 4-month-old WT and GILZ KO mice that had been treated with oil (control) or CCl4 for 72 h (A) and 6–7 weeks (B). Results are presented as the means ± SEM. Each dot represents an individual mouse; bars indicate the mean. Data were pooled from five (A) or three (B) independent experiments (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001, ns not significant).
Fig. 4
Fig. 4. Lack of GILZ results in increased monocyte, CD4+ T cell, and NK cell recruitment to the liver.
A–C Number of CD4+ (A), Mac1+Gr1− (B), and NK (C) cells infiltrated in the livers of 4-month-old WT and GILZ KO mice that had been treated with oil (control) or CCl4 for 72 h. Data were pooled from three independent experiments. Results are presented as the means ± SEM; each dot represents an individual mouse; scale bars indicate the mean (*p < 0.05, **p < 0.005 ***p < 0.001, ****p < 0.0001, ns not significant).
Fig. 5
Fig. 5. Lack of GILZ results in increased Ccl2 mRNA expression in the liver following CCl4 treatment.
A, B qPCR analysis of Ccl2 mRNA expression in the livers of 4-month-old WT and GILZ KO mice that had been treated for 72 h (A) or 6–7 weeks (B) with oil (control) or CCl4. C, D qPCR analysis of Ccr2 mRNA expression in the livers of 4-month-old WT and GILZ KO mice that had been treated for 72 h (C) or 6–7 weeks (D) with oil (control) or CCl4. All data are presented relative to Actb mRNA expression. Each dot represents an individual mouse; scale bars indicate the mean. Results are presented as the means ± SEM. Data were pooled from three (A–C) or two (B–D) independent experiments (*p < 0.05, **p < 0.005, ns not significant).
Fig. 6
Fig. 6. Silencing CCR2 reverts enhanced leukocyte infiltration in GILZ KO mouse livers.
A The number of leukocytes isolated from the livers of 4-month-old WT and GILZ KO mice that had been pretreated with siCCR2 or siLUC (LUC) for 1 week and then treated with oil (control) or CCl4 for 72 h. B Immunohistochemical analysis of α-SMA expression in the liver sections of WT and GILZ KO mice that had been pretreated with siLUC or siCCR2, and then treated with oil or CCl4 for 72 h. Arrows indicate the areas of α-SMA positivity. Original magnification, 5×; scale bar, 200 µM. Results are presented as the means ± SEM. Each dot represents an individual mouse; scale bars indicate the mean. Data were pooled from two independent experiments (**p < 0.005, ns not significant).
Fig. 7
Fig. 7. GILZ is downregulated in patients with LF.
TSC22D3 and CCL2 mRNA expression was analyzed in liver samples from NAFLD patients, according to GSE48452 (NASH patients) and GSE49541 (NAFLD patients) cohorts accessed in Gene Expression Omnibus. A, B TSC22D3 mRNA expression in NASH patients. Samples were classified as A high fat score when presenting a score of ≥70 (n = 9) and B fibrosis stages 1–4, representing the samples in this score range (n = 14). C, D TSC22D3 (C) and CCL2 (D) gene expression in NAFLD patients with mild (stages 0–1; n = 40) and advanced (stages 3–4; n = 34) fibrosis. E, F Pearson correlation between TSC22D3 and CCL2 mRNA expression in NAFLD patients. E Pearson correlation coefficient in NAFLD patients with mild (stages 0–1) and (F) advanced (stages 3–4) fibrosis. Data are presented as a box and whiskers plot. *p < 0.05, **p < 0.01.
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