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Cited by 128Liver physiology and pathologyCardiac Fibrosis and RemodelingLiver Disease Diagnosis and Treatment

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

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Weng, Shih-Yen, Wang, Xiaoyu, Vijayan, Santosh, Tang, Yilang, Kim, Yong Ook, Padberg, Kornelius, Regen, Tommy, Molokanova, Olena, Chen, Tao, Bopp, Tobias, Schild, Hansjörg, Brombacher, Frank, Crosby, Jeff R., McCaleb, Michael L., Waisman, Ari, Bockamp, Ernesto, Schuppan, Detlef·2018·EBioMedicine
DOI 10.1016/j.ebiom.2018.01.028PMC PMC5925448
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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.

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Figures

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. 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.CC BY-NC-ND · PubMed Central
Fig. 2 IL-4Rα is required for infiltration by monocyte-derived inflammatory macrophages (Ly-6Chi CD11bhi F4/80int). 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 (CD11bint F4/80hi) and monocytes/macrophages (CD11bhi F4/80lo) are shown. CD11bhi F4/80lo cells were further subdivided into pro-inflammatory (Ly-6Chi) and anti-inflammatory (Ly-6Clo) 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. 2 IL-4Rα is required for infiltration by monocyte-derived inflammatory macrophages (Ly-6Chi CD11bhi F4/80int). 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 (CD11bint F4/80hi) and monocytes/macrophages (CD11bhi F4/80lo) are shown. CD11bhi F4/80lo cells were further subdivided into pro-inflammatory (Ly-6Chi) and anti-inflammatory (Ly-6Clo) 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.CC BY-NC-ND · PubMed Central
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. 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.CC BY-NC-ND · PubMed Central
Fig. 4 Myeloid IL-4Rα deficiency attenuates CCl4-induced liver fibrosis. Il4raf/− (control) and myeloid cell IL-4Rα-deleted (Il4raΔLysM) 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. 4 Myeloid IL-4Rα deficiency attenuates CCl4-induced liver fibrosis. Il4raf/− (control) and myeloid cell IL-4Rα-deleted (Il4raΔLysM) 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.CC BY-NC-ND · PubMed Central
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. 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.CC BY-NC-ND · PubMed Central
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. 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.CC BY-NC-ND · PubMed Central
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 Il4raΔLysM 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 Il4rafl/− 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.
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 Il4raΔLysM 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 Il4rafl/− 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.CC BY-NC-ND · PubMed Central

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Authors
Weng, S.-Y., Wang, X., Vijayan, S., Tang, Y., Kim, Y. O., Padberg, K., Regen, T., Molokanova, O., Chen, T., Bopp, T., Schild, H., Brombacher, F., Crosby, J. R., McCaleb, M. L., Waisman, A., Bockamp, E., & Schuppan, D.
Affiliations
  • Johannes Gutenberg University Mainz (DE)
  • South African Medical Research Council (ZA)
  • Ionis Pharmaceuticals (United States) (US)
  • Beth Israel Deaconess Medical Center (US)
Corresponding author
Schuppan, Detlef
ORCID
Venue
EBioMedicine
Publisher
Elsevier BV
Published
2018-03
Volume
29
Pages
92-103
ISSN
2352-3964 (ISSN-L)
DOI
10.1016/j.ebiom.2018.01.028
PMC
PMC5925448
OpenAlex
W2790513179
Cited by
128
References
60
Citations by year
2026: 14 · 2025: 18 · 2024: 11 · 2023: 18 · 2022: 17 · 2021: 21 · 2020: 9 · 2019: 15 · 2018: 4
FWCI
7.68
Type
journal article
Language
en
Open access
Licence
Locations
  • EBioMedicine (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Gutenberg Open Science (submittedVersion, cc-by-nc-nd)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Gutenberg Open Science
Funders
  • National Science Council
  • European Commission
  • European Research Council
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiac Fibrosis and Remodeling
  • Health Sciences › Medicine › Hepatology › Liver Disease Diagnosis and Treatment
Keywords
IL-4 receptor alpha, liver fibrosis, macrophage polarization, M2 macrophages, fibrosis reversal, chronic hepatitis, STAT6 signaling, MMP-12, IL-13, IL-4, inflammatory monocytes, antisense oligonucleotides, liver cirrhosis
MeSH terms
RAW 264.7 Cells, Animals, Disease Models, Animal, Liver Cirrhosis, Macrophage Activation, Macrophages, Spleen, Signal Transduction, Gene Expression, T-Lymphocyte Subsets, Cytokines, Mice, Knockout, Disease Progression, Matrix Metalloproteinases, Myeloid Cells, STAT6 Transcription Factor, Mice, Interleukin-4 Receptor alpha Subunit, Non-alcoholic Fatty Liver Disease

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