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. 2015;11(8):1280-92.
doi: 10.1080/15548627.2015.1058473.

Macrophage autophagy protects against liver fibrosis in mice

Jasper Lodder  1 , Timothé Denaës, Marie-Noële Chobert, JingHong Wan, Jamel El-Benna, Jean-Michel Pawlotsky, Sophie Lotersztajn, Fatima Teixeira-Clerc
Affiliations

Macrophage autophagy protects against liver fibrosis in mice

Jasper Lodder et al. Autophagy. 2015.

Abstract

Autophagy is a lysosomal degradation pathway of cellular components that displays antiinflammatory properties in macrophages. Macrophages are critically involved in chronic liver injury by releasing mediators that promote hepatocyte apoptosis, contribute to inflammatory cell recruitment and activation of hepatic fibrogenic cells. Here, we investigated whether macrophage autophagy may protect against chronic liver injury. Experiments were performed in mice with mutations in the autophagy gene Atg5 in the myeloid lineage (Atg5(fl/fl) LysM-Cre mice, referred to as atg5(-/-)) and their wild-type (Atg5(fl/fl), referred to as WT) littermates. Liver fibrosis was induced by repeated intraperitoneal injection of carbon tetrachloride. In vitro studies were performed in cultures or co-cultures of peritoneal macrophages with hepatic myofibroblasts. As compared to WT littermates, atg5(-/-) mice exposed to chronic carbon tetrachloride administration displayed higher hepatic levels of IL1A and IL1B and enhanced inflammatory cell recruitment associated with exacerbated liver injury. In addition, atg5(-/-) mice were more susceptible to liver fibrosis, as shown by enhanced matrix and fibrogenic cell accumulation. Macrophages from atg5(-/-) mice secreted higher levels of reactive oxygen species (ROS)-induced IL1A and IL1B. Moreover, hepatic myofibroblasts exposed to the conditioned medium of macrophages from atg5(-/-) mice showed increased profibrogenic gene expression; this effect was blunted when neutralizing IL1A and IL1B in the conditioned medium of atg5(-/-) macrophages. Finally, administration of recombinant IL1RN (interleukin 1 receptor antagonist) to carbon tetrachloride-exposed atg5(-/-) mice blunted liver injury and fibrosis, identifying IL1A/B as central mediators in the deleterious effects of macrophage autophagy invalidation. These results uncover macrophage autophagy as a novel antiinflammatory pathway regulating liver fibrosis.

Keywords: Kupffer cell; inflammation; interleukin-1; liver injury; myofibroblast.

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Figures

Figure 1 (See previous page).
Figure 1 (See previous page).
Macrophage from atg5−/− mice show impaired autophagy and proinflammatory properties. (A) LC3 and SQSTM1 protein expression and quantification by immunocytochemistry in Kuppfer cells exposed for 6 h to 100 nM rapamycin or its vehicle. Data are shown as mean ± SEM of 4 independent experiments. *, p < 0.05 for rapamycin vs vehicle, &, p < 0.05 for WT vs atg5−/−. (B) ELISA analysis of IL1A and IL1B in supernatant fractions from Kupffer cells and neutrophils isolated from atg5−/− or WT mice and exposed to 10 ng/ml LPS for 6 h. Data are shown as mean ± SEM and are representative of 2 independent experiments. &, p < 0.05 for WT vs atg5−/−. (C) Left, Il1a and pro-il1b mRNA expression in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 ng/ml LPS for 6 h. Right, ELISA analysis of IL1A and IL1B in supernatant fractions from peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 ng/ml LPS for 24 h. Data are shown as mean ± SEM and are representative of 2 independent experiments. &, p < 0.05 for WT vs atg5−/−. (D) RT-PCR analysis of Il1a and pro-il1b mRNA in RAW264.7 cells exposed for 1 h to 100 nM rapamycin or vehicle, and further incubated with 10 ng/ml LPS for 6 h. Data are the mean ± SEM from sextuplate repeats. *, p < 0.05 for vehicle vs rapamycin.
Figure 2.
Figure 2.
ROS generation and activation of MAPK14 is associated with enhanced IL1A/B production by macrophages from atg5−/− mice. (A) ROS production in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 ng/ml LPS. Data are the mean ± SEM (n = 4). &, p < 0.05 for WT vs atg5−/− (B) Il1a and pro-il1b mRNA expression and production in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 mM NAC or its vehicle for 1 h and further stimulated with 10 ng/ml LPS for 6 h. Data are the mean ± SEM from sextuplate repeats. *, p < 0.05 for NAC vs vehicle and &, p < 0.05 for WT vs atg5−/−. (C) Representative P-MAPK14 protein expression by immunocytochemistry in peritoneal macrophages exposed for 15, 30 or 60 min to 10 ng/ml LPS or its vehicle. (D) P-MAPK14 and MAPK14 expression by western blotting in peritoneal macrophages exposed for 15 or 30 min to 10 ng/ml LPS or its vehicle. *, p < 0.05 for WT vs atg5−/− (E) Il1a and pro-il1b mRNA expression in peritoneal macrophages isolated from atg5−/− or WT mice and exposed to 10 μM SB203580 for 1 h and further stimulated with 10 ng/ml LPS for 6 h. Data are the mean ± SEM from sextuplate repeats. *, p < 0.05 for vehicle vs SB203580, &, p < 0.05 for WT vs atg5−/−.
Figure 3.
Figure 3.
atg5−/− mice show enhanced liver inflammation. (A) ELISA quantification of cytokine levels in liver homogenates in WT or atg5−/− mice exposed to CCl4 for 2.5 weeks or its vehicle (mineral oil, MO). Representative ADGRE1 (B) and MPO (C) staining (original magnification x400) and quantification in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or its vehicle (MO). Arrows indicate positive cells. (D) Hepatic mRNA expression of Adgre1, Ly6c1, Ccr2, Ly6g, and Mpo in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or its vehicle (MO). (E) Hepatic mRNA expression of Ccl2, Ccl3, Ccl4 and Cxcl2 in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or its vehicle (MO). Data are shown as mean ± SEM; *, p < 0.05 for MO vs CCl4 and &, p < 0.05 for WT vs atg5−/−. n = 3 for WT mice MO; n = 4 for atg5−/− mice MO; n = 14 for WT mice CCl4; n = 12 for atg5−/− mice CCl4.
Figure 4.
Figure 4.
atg5−/− mice display enhanced liver injury. (A) Left, representative staining of liver tissue sections stained with hematoxylin and eosin (original magnification x200) in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or its vehicle (MO). Arrows indicate necrotic areas. Right, quantification of necrosis area by morphometry. (B) Left, representative TUNEL staining (original magnification x200) in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or its vehicle (MO). Arrows indicate TUNEL-positive cells. Right, quantification of TUNEL staining expressed as number of positive cells per field. (C) Serum levels of GOT and GPT in WT or atg5−/− mice exposed to CCl4 for 2.5 wk or MO. Data are shown as mean ±SEM ; *, p < 0.05 for MO vs CCl4 and &, p<0.05 for WT vs atg5−/−. n = 3 for WT mice MO; n = 4 for atg5−/− mice MO; n = 14 for WT mice CCl4; n = 12 for atg5−/− mice CCl4.
Figure 5.
Figure 5.
atg5−/− mice display exacerbated liver fibrosis. (A) Left, representative liver tissue sections stained with Sirius Red (original magnification x200) in mice exposed to CCl4 for 2.5 or 4 wk. Right, quantification of fibrosis area by morphometry. (B) Left, representative hepatic ACTA2 immunostaining (original magnification x200) in mice exposed to CCl4 for 2.5 or 4 wk. Right, quantification of ACTA2 immunostaining by morphometry. (C) RT-PCR analysis of hepatic Tgfb1, Mmp9, and Serpine1 mRNA expression in mice exposed to CCl4 for 2.5 wk. Data are shown as mean ± SEM; *, p < 0.05 for MO vs CCl4 and, & p < 0.05 for WT vs atg5−/−. n = 3 for WT mice MO 2.5 wk; n = 5 for WT mice MO 4 wk; n = 4 for atg5−/− mice MO 2.5 wk; n = 5 for atg5−/− mice MO 4 wk; n = 14 for WT mice CCl4 2.5 wk; n = 14 for WT mice CCl4 4 wk; n = 12 for atg5−/− mice CCl4 2.5 wk; n = 11 for atg5−/− mice CCl4 4 wk.
Figure 6.
Figure 6.
Atg5-deficient macrophages enhance the fibrogenic properties of hepatic myofibroblasts, via an IL1-dependent pathway. (A) Conditioned media (CM) were obtained from atg5-/− or WT peritoneal macrophages exposed for 24 h to LPS, and were further incubated for 1 h with IL1A/B neutralizing antibodies or control IgG. RT-PCR analysis of Timp1, Serpine1 and Mmp9 mRNA was performed in myofibroblasts exposed for 6 h to CM. *, p<0.05 for CM atg5−/− + control IgG vs CM WT + control IgG and &, p<0.05 for CM atg5−/− + control IgG vs CM atg5−/− + anti-IL1A/B. Data are shown as mean ± SEM and are representative of 2 independent experiments. (B) RT-PCR analysis of Timp1, Serpine1 and Mmp9 mRNA in hepatic myofibroblasts exposed to recombinant IL1A or IL1B for 6 h. *, p<0.05 for vehicle vs IL1. Data are the mean ± SEM of 2 independent experiments.
Figure 7.
Figure 7.
Treatment with recombinant IL1RN rescues atg5−/− mice from CCl4-induced liver fibrosis and injury. Mice were exposed to CCl4 and treated with recombinant IL1RN or its vehicle for 2.5 wk (A) Left, representative liver tissue sections stained with Sirius Red (original magnification x200). Right, quantification of fibrosis area by morphometry. (B) Left, representative staining of liver tissue sections stained with hematoxylin and eosin (original magnification x200). Arrows indicate necrotic areas. Right, quantification of necrosis area by morphometry. *, p < 0.05 for IL1RN vs vehicle and &, p < 0.05 for atg5−/− vs WT. n = 7 for WT vehicle, n = 6 for atg5−/− vehicle, n = 4 for WT IL1RN and n = 5 for atg5−/− IL1RN.
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