IL-4 Receptor Alpha Signaling through Macrophages Differentially Regulates Liver Fibrosis Progression and Reversal

Abstract

Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα^-/- as well as in macrophage-specific IL-4Rα^-/- (IL-4Rα^ΔLysM) mice. However, with deletion of IL-4Rα^ΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies.

Research in context: Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.

Keywords: Fibrosis; IL-4 receptor alpha; Liver; MMP12; Macrophage; Progression; Reversal.

Fig. 1

Loss of IL-4Rα signaling retards liver fibrosis progression. (A–D) Il4ra^−/− and WT mice received CCl₄ or vehicle (mineral oil) for 6 weeks by oral gavage. (A–B) Representative (A) Sirius Red and (B) α-smooth muscle actin (α-SMA) staining of WT and Il4ra^−/− liver sections; bar: 200 μm. The diagrams depict mean percentages of stained areas from 10 microscopic fields of each mouse (n = 6 per group). (C) Total liver collagen (hydroxyproline, Hyp). (D) Serum alanine aminotransferase (ALT) levels in WT (white bars) and Il4ra^−/− (black bars) mice with or without CCl₄ treatment (n = 6 per group). (E-G) Il4/Il13 DKO and WT type mice received CCl₄ or vehicle (mineral oil) for 6 weeks by oral gavage. (E) Representative Sirius Red staining (n = 5) of WT and Il4/Il13 DKO liver sections; bar: 200 μm. The diagrams depict mean percentages of stained areas from 10 microscopic fields of each mouse. (F) Col1a1 mRNA (normalized to Gapdh mRNA, n = 5). (G) Hepatic transcript levels of Mmp8, 12 and 13 and Timp1 in mice received vehicle or CCl₄ (n = 5). *p < 0.05, **p < 0.01, ***p < 0.005 (unpaired Student's t-test). All data are expressed as means ± SEM. Results are representative of two independent experiments.

Fig. 2

IL-4Rα is required for infiltration by monocyte-derived inflammatory macrophages (Ly-6C^hi CD11b^hi F4/80^int). Bar diagrams indicating absolute (A) splenic CD45⁺, and (B) hepatic CD45⁺, (C) CD19⁺ (B cell), CD4 and CD8 T cell numbers of WT and Il4ra^−/− mice that had received either mineral oil or CCl₄ for 6 weeks. Analysis was performed one day, one and two weeks after CCl₄ withdrawal (n = 4 per group). (D) Representative FACS plots showing liver monocytes/macrophages (see also Fig. S2) of WT and Il4ra^−/− mice that were analyzed at the end of 6 weeks of treatment with CCl₄. In the two plots on the left resident macrophages (CD11b^int F4/80^hi) and monocytes/macrophages (CD11b^hi F4/80^lo) are shown. CD11b^hi F4/80^lo cells were further subdivided into pro-inflammatory (Ly-6C^hi) and anti-inflammatory (Ly-6C^lo) monocytes/macrophages. Bar diagrams show the mean numbers of monocytes/macrophages per liver (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.005 (unpaired Student's t-test). All data are expressed as means ± SEM. Results are representative of ≥2 independent experiments.

Fig. 3

Inhibition of IL-4Rα expression by specific antisense oligonucleotides decreases fibrosis progression. (A) Mice received 9 doses of either control oligonucleotide (C) or IL-4Rα-ASO2 during three weeks of CCl₄ treatment. (B) Sirius Red and (C) CD68 (macrophage) staining in livers of mice with fibrosis progression. Bar: 200 and 50 μm, respectively (n = 4–5). (D) Relative hepatic transcript levels of key fibrosis-related genes in CCl₄-treated mice receiving either the control oligonucleotide or the IL-4Rα-specific ASO2. (E) Transcript levels for macrophage marker genes (Cd68, Mrc1 and iNOS) for control oligonucleotide- and IL-4Rα-specific ASO2-treated livers during fibrosis progression (n = 5). *p < 0.05, **p < 0.01 (unpaired Student's t-test). All data are expressed as means ± SEM. Data are representative of three independent experiments.

Fig. 4

Myeloid IL-4Rα deficiency attenuates CCl₄-induced liver fibrosis. Il4ra^f/− (control) and myeloid cell IL-4Rα-deleted (Il4ra^ΔLysM) mice were treated with CCl₄ for 6 weeks. Mice were analyzed 3 days after the last dose of CCl₄. (A) Representative Sirius Red (upper) and α-SMA (lower) stained sections of CCl₄ treated mice. Bar: 200 μm. The diagrams on the right depict mean percentages of stained areas from 10 microscopic fields per liver (n = 5). (B) Hepatic hydroxyproline (Hyp) levels (n = 5). (C) Serum ALT levels of control and Il4ra^∆LysM mice (n = 5). (D) Hepatic Col1a1 and α-SMA transcript levels (n = 5). Transcript levels were normalized to Gapdh mRNA. *p < 0.05, **p < 0.01 (unpaired Student's t-test). All data are expressed as means ± SEM. Results are representative of two independent experiments.

Fig. 5

Reduction of both M1- and M2-type macrophage markers in Il4ra^∆LysM mice during CCl₄-induce liver fibrosis. (A) Transcript levels of Cd68, iNOS, Il1b, Tgfb1. Arg1 and Mrc1 in control and Il4ra^∆LysM mice treated with mineral oil or CCl₄ for 6 weeks (n = 5). (B) Representative YM1 immunohistochemistry in control and Il4ra^∆LysM mice treated with mineral oil or CCl₄ for 6 weeks (n = 5). Bar: 50 μm. *p < 0.05, **p < 0.01 (unpaired Student's t-test). All data are expressed as means ± SEM.

Fig. 6

IL-4Rα antisense oligonucleotides attenuate resolution and inhibit M2-type polarization during spontaneous fibrosis reversal. (A) Liver Hyp level of control and Il4ra^∆LysM mice after 2 weeks of CCl₄ withdrawal (n = 5). (B) Transcript levels of Col1a1 and α-SMA of control and Il4ra^∆LysM mice after 2 weeks of CCl₄ withdrawal (n = 6). (C–G) ASO2-mediated IL-4Rα reduction caused the retardation of fibrosis reversal. (C) Mice received 3 doses of control oligonucleotide or ASO2 during the first week of reversal, off CCl₄. Mice were analyzed one day after the last dose of ASO2. (D) Representative Sirius Red staining in livers of mice during the reversal phase (n = 6). Bar: 200 μm. Bar diagrams represent results from 10 microscopic fields per liver. (E) Transcript levels for Col1a1. (F) Representative CD206 (M2-type macrophage) staining in livers of mice during the reversal phase (n = 6). Bar: 50 μm. Bar diagrams represent results from 10 microscopic fields per liver. (G) Transcript levels for macrophage specific genes (Cd68, Mrc1, Arg1 and iNOS, n = 4). *p < 0.05 (unpaired Student's t-test). All data are expressed as means ± SEM. Results are representative of ≥2 independent experiments.

Fig. 7

IL-4 and IL-13 mediated STAT6 activation induces macrophage MMP-12 expression. (A) Relative Mmp transcripts (normalized to Gapdh RNA) in livers of control and Il4ra^ΔLysM mice that were analyzed at 24 h and 72 h after 6 weeks of CCl₄-treatment (n = 5). *p < 0.05, **p < 0.01; n, not significant (one-way ANOVA). (B,C) Peritoneal macrophages (pMφ) or bone marrow-derived macrophages (BMDM) were either untreated or treated with IL-4 (20 ng/mL) and/or IL-13 (20 ng/mL) for 24 h. Mmp12 transcript were measured (n = 4 per group). (D) Immune cells from Il4ra^fl/− control (C) and Il4ra^∆LysM (ΔLysM) livers were harvested at day three following CCl₄ treatment and stimulated with IL-4 or IL-13 for 30 min for the detection of phospho-STAT6 in macrophages (n = 4 per group). (E) Mmp12 transcript levels in RAW264.7 macrophages transfected with the indicated oligonucleotide. (F) Representative Sirius Red staining of liver sections from mice three days after 6 weeks of fibrosis induction with CCl₄ or from mice after 2 weeks of recovery after the last CCl₄ dose. Mice in the reversal group were injected with empty pcDNA3 vector (C) or pcDNA3-MMP12 (Mmp12). Bar diagrams show the quantification results. Bar: 200 μm. *p < 0.05, **p < 0.01, ***p < 0.005 (unpaired Student's t-test). All data are expressed as means ± SEM. See also the supplemental table of Mmp transcript levels.