Hepatic Stellate Cells Orchestrate Clearance of Necrotic Cells in a Hypoxia-Inducible Factor-1α–Dependent Manner by Modulating Macrophage Phenotype in Mice

Akie Mochizuki, Aaron Pace, Cheryl E. Rockwell, Katherine J. Roth, Aaron Chow, Kate M. O’Brien, Ryan Albee, Kara Kelly, Keara Towery, James P. Luyendyk and Bryan L. Copple
J Immunol April 15, 2014, 192 (8) 3847-3857; DOI: https://doi.org/10.4049/jimmunol.1303195

Abstract

Hypoxia-inducible factor-1α (HIF-1α) is activated in hepatic stellate cells (HSCs) by hypoxia and regulates genes important for tissue repair. Whether HIF-1α is activated in HSCs after acute injury and contributes to liver regeneration, however, is not known. To investigate this, mice were generated with reduced levels of HIF-1α in HSCs by crossing HIF-1α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i.e., HIF-1α-GFAP Cre+ mice). These mice and control mice (i.e., HIF-1α-GFAP Cre− mice) were treated with a single dose of carbon tetrachloride, and liver injury and repair were assessed. After carbon tetrachloride, HIF-1α was activated in HSCs. Although liver injury was not different between the two strains of mice, during resolution of injury, clearance of necrotic cells was decreased in HIF-1α-GFAP Cre+ mice. In these mice, the persistence of necrotic cells stimulated a fibrotic response characterized by extensive collagen deposition. Hepatic accumulation of macrophages, which clear necrotic cells from the liver after carbon tetrachloride, was not affected by HIF-1α deletion in HSCs. Conversion of macrophages to M1-like, proinflammatory macrophages, which have increased phagocytic activity, however, was reduced in HIF-1α-GFAP Cre+ mice as indicated by a decrease in proinflammatory cytokines and a decrease in the percentage of Gr1hi macrophages. Collectively, these studies have identified a novel function for HSCs and HIF-1α in orchestrating the clearance of necrotic cells from the liver and demonstrated a key role for HSCs in modulating macrophage phenotype during acute liver injury.

Introduction

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor activated in cells by hypoxia, as well as other mediators, including cytokines, growth factors, and oxidative stress (1, 2). In normoxic cells, a family of prolyl hydroxylases, called egg laying defective nine homologs, hydroxylate HIF-1α on proline residues within the oxygen degradation domain (3, 4). Prolyl hydroxylation immediately targets HIF-1α for proteasomal degradation. Under hypoxic conditions, prolyl hydroxylase activity is inhibited resulting in HIF-1α stabilization and translocation to the nucleus, where it regulates a wide array of genes involved in glycolysis, angiogenesis, immunomodulation, matrix dynamics, and cell proliferation (5, 6). Because of the diverse groups of genes regulated by HIF-1α, this protein has been implicated in a number of physiological and pathological processes, including tissue growth and repair.

Several studies have demonstrated that HIF-1α is activated in a variety of cell types in the liver after exposure to hepatotoxicants and other insults, including ethanol, acetaminophen, and bile duct ligation and in cultured liver cells exposed to hypoxia (7–15). What remains poorly understood is the cell-specific function of HIF-1α in the liver after injury and whether HIF-1α is indispensable for normal liver repair after acute injury. We recently demonstrated that HIF-1α is activated in hepatic stellate cells (HSCs) in diseased human livers and that hypoxia increases expression of numerous genes in HSCs in a HIF-1α–dependent manner (11, 12). Several of these genes are important for tissue repair processes including, angiogenesis, matrix remodeling, and immunomodulation, suggesting that activation of HIF-1α in HSCs may be important for liver regeneration. Whether stabilization of HIF-1α in HSCs is crucial for this process, however, is not known. Accordingly, the purpose of this study was to determine whether HIF-1α is activated in HSCs after acute liver injury and whether HIF-1α activation in these cells is critical for liver repair.

Materials and Methods

Animals

To reduce HIF-1α levels in HSCs, HIF-1αfl/fl mice (B6.129-Hif1atm3Rsjo/J; The Jackson Laboratory, Bar Harbor, ME), described in detail previously (16), were crossed with mice expressing Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (B6.Cg-Tg(Gfap-cre)73.12Mvs/J mice; The Jackson Laboratory) (17). Both strains of mice were backcrossed to C57BL/6J for at least 12 generations. HIF-1αfl/fl-GFAP Cre− (i.e., normal HIF-1α levels in GFAP+ cells) and HIF-1αfl/fl-GFAP Cre+ (i.e., decreased HIF-1α levels in GFAP+ cells) littermates were used for these studies.

To monitor prolyl hydroxylase (i.e., egg laying defective nine) activity as an indirect measure of HIF activation, ODD-luc mice were used (F,FVB.129S6-Gt(ROSA)26Sor < tm2(HIF1A/luc)Kael > /J; The Jackson Laboratory) (18).

All mice were maintained on a 12-h light/dark cycle under controlled temperature (18–21°C) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and tap water were allowed ad libitum. All procedures on animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health and approved by the Michigan State University Institutional Animal Care and Use Committee.

Carbon tetrachloride treatment

Acute injury.

Mice were treated with a single dose of 1 ml/kg carbon tetrachloride (Sigma-Aldrich) by i.p. injection. The carbon tetrachloride was diluted 1:10 in corn oil (Sigma-Aldrich) prior to injection.

Chronic injury

Mice were treated with 1 ml/kg carbon tetrachloride twice per week for a total of 4 wk.

Real-time PCR

Real-time PCR was performed as described previously (19). Primers used for PCR are shown in Table I.

Immunohistochemistry and immunofluorescence

Immunofluorescence was used to detect and quantify type I collagen, neutrophils, and macrophages (i.e., CD68 and F4/80) in 8-μm frozen sections of liver, as described previously (12).

To detect GFAP and firefly luciferase (Photinus pyralis) or HIF-1α in frozen sections of liver, the sections were fixed in 4% formalin and then incubated with goat anti-luciferase Ab diluted 1:50 (Promega, Madison, WI) or rabbit anti-HIF-1α Ab (1:100, NB100-479; Novus Biologicals, Littleton, CA) and chicken anti-GFAP Ab diluted 1:50 (Aves Laboratories, Tigard, OR). The sections were then incubated with secondary Abs conjugated to either Alexa Fluor 488 or Alexa Fluor 594 (Life Technologies, Grand Island, NY).

Proliferating cell nuclear Ag (PCNA) and α-smooth muscle actin (α-SMA) were detected in formalin-fixed, paraffin-embedded sections of a liver using the Vectastain Elite ABC Kit and Vector DAB (Vector Laboratories, Burlingame, CA). Anti-PCNA Ab (1:8000; Abcam, Cambridge, MA) and anti–α-SMA (1:100; Abcam) were added to the tissues and incubated overnight at 4°C.

Isolation of HSCs

The livers of anesthetized mice were perfused with calcium and magnesium-free HBSS containing 50 mM EGTA, 1 M glucose, and penicillin–streptomycin solution (all chemicals from Sigma-Aldrich). Next, the livers were perfused with HBSS containing 1 M calcium chloride (Sigma-Aldrich), 1 M glucose, penicillin–streptomycin solution, and 8 mg Pronase (Roche, Indianapolis, IN). Last, the livers were perfused with HBSS containing 1 M calcium chloride, 1 M glucose, penicillin–streptomycin solution, and 5 mg collagenase H (Sigma-Aldrich). The livers were removed and dissociated in DMEM containing 10% FBS, penicillin–streptomycin solution and 16 μg/ml DNase I (Sigma-Aldrich). The homogenate was centrifuged at 50 × g for 2 min to remove hepatocytes. The supernatant was centrifuged at 1000 × g for 6 min. The resulting pellet was resuspended in 12.6 ml HBSS. The HSCs were then separated from other nonparenchymal cells by using an 8% Histodenz gradient. Briefly, 4.9 ml 28.7% Histodenz (Sigma-Aldrich), made in sodium chloride-free HBSS, was added to the cell suspension. This solution was mixed and divided between two 15-ml tubes (Greiner Bio-One, Monroe, NC). These solutions were overlaid with 3 ml HBSS and centrifuged at 1500 × g for 15 min at 4°C without break. The HSCs, contained at the interface were removed and diluted to 50 ml with HBSS. This solution was centrifuged at 1000 × g for 8 min at 4°C. The HSCs were cultured in DMEM containing 10% FBS and penicillin–streptomycin solution.

Isolation of hepatocytes and Kupffer cells

Hepatocytes and Kupffer cells were isolated as described previously (13, 19).

Neutrophil depletion

Neutrophils were depleted from mice as described previously (20). Briefly, mice were treated with 1 mg anti–mLy-6G Ab (clone 1A8; BioXCell, West Lebanon, NH) or 1 mg isotype control Ab (rat IgG2a, clone 2A3; BioXCell) by i.p. injection. After 24 h, the mice were treated with carbon tetrachloride or vehicle. The mice then received a second dose of anti–mLy-6G Ab or isotype control 24 h after carbon tetrachloride treatment.

Flow cytometry

Nonparenchymal cells were isolated from the liver by digestion with collagenase H. The cells were washed and resuspended in FACS buffer (PBS, 1% FCS). The cells were then incubated with anti-CD11b/allophycocyanin, anti–F4-80/FITC, and anti-Gr1/PE for 30 min at 4°C, after which the cells were washed and resuspended in FACS buffer. The fluorescence was then detected and quantified with a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA). The data were analyzed using CFlow software (BD Accuri, San Jose, CA). The CD11b and F4/80 Abs were purchased from eBioscience (San Diego, CA) and the Gr1 Ab was purchased from BioLegend (San Diego, CA).

Macrophage depletion

Mice were treated with 200 μl liposome encapsulated clodronate (ClodronateLiposomes.com, Haarlem, The Netherlands) or PBS-containing liposomes by i.p. injection. After 48 h, the mice were treated with carbon tetrachloride.

Isolation of peritoneal macrophages

Mice were injected with 3 ml 3% Brewers thioglycollate medium (i.p.) (Sigma-Aldrich). After 3 d, the peritoneal cavity was lavaged with DMEM, and the cells were plated in DMEM containing 10% FBS.

Quantification of necrosis

The area of necrosis was quantified in 15 ×200 fields per H&E-stained liver section using ImageJ Software (National Institutes of Health). The analysis was performed in a blinded fashion.

Alanine aminotransferase activity

Hepatocyte injury was evaluated by measuring the serum activity of alanine aminotransferase (ALT) (Pointe Scientific, Canton, MI).

Statistics

Results are presented as the mean ± SEM. Each in vivo study used five to seven mice per group. Each in vitro study was performed at least three times, each experiment utilizing cells isolated from different mice. Data were analyzed by one- or two-way ANOVA, as appropriate. Data expressed as a fraction were transformed by arc sine square root prior to analysis. Comparisons among group means were made using the Student-Newman-Keuls test. The criterion for significance was p < 0.05 for all studies.

Results

Activation of HIF-1α in HSCs after carbon tetrachloride

HIF-1α was not detected in the livers of vehicle treated mice (data not shown). In the livers of mice treated with carbon tetrachloride, nuclear HIF-1α was detected in cells within centrilobular regions (Fig. 1A, 1B). Many of these cells were HSCs as demonstrated by colocalization of HIF-1α with GFAP (Fig. 1C). In addition, nuclear HIF-1α was detected in CD68+ macrophages (Fig. 1D). To confirm activation of HIF-1α, ODD-luc mice were treated with carbon tetrachloride. ODD-luc mice express firefly luciferase fused to the oxygen degradation domain (ODD) of HIF-1α (18). Similar to HIF-1α, in cells in normoxic conditions, luciferase is targeted for proteasomal degradation and not detected. In cells that become hypoxic, however, the luciferase protein is stabilized. Luciferase was not detected in the livers of mice treated with vehicle (Fig. 1E). In mice treated with carbon tetrachloride, however, numerous cells within centrilobular regions stained positive for luciferase (Fig. 1F). Many of these cells were HSCs as indicated by colocalization of luciferase with GFAP (Fig. 1G, 1H).

FIGURE 1.
FIGURE 1.

Activation of HIF-1α in the liver after carbon tetrachloride. C57BL/6 mice were treated with 1 ml/kg carbon tetrachloride. Eighteen hours later, the livers were removed, and immunofluorescence was used to detect HIF-1α [green staining in (A–D)], HSCs [GFAP, red staining in (C)], macrophages [CD68, red staining in (D)], and nuclei [DAPI, blue staining in (C) and (D) only]. Original magnifications, ×200 (A) and ×400 (B). Images in (C) and (D) show the same field (original magnification ×1000). Image in (C) shows colocalization of HIF-1α to nuclei within GFAP+ HSCs, indicated by solid white arrows. Dashed arrows indicate cells that are negative for GFAP but stain positive for HIF-1α. Solid arrows in (D) indicate CD68+ macrophages that stain positive for HIF-1α. ODD-luc mice were treated with vehicle (E) or carbon tetrachloride (F–H) for 24 h. Immunofluorescence was used to detect luciferase [green staining in (E–H)] and GFAP [red staining in (H)]. (G) and (H) show the same field. Block arrows indicate GFAP+ cells, which are also positive for luciferase. Insets show a higher power image of a GFAP+ HSC with nuclear HIF-1α (D) or luciferase (H) immunostaining. Bars, 50 μm. HIF-1α-GFAP Cre− (I, J) and HIF-1α-GFAP Cre+ (K, L) mice were treated with carbon tetrachloride. Eighteen hours later, HIF-1α (red staining) and HSCs (GFAP, green staining) were detected by immunofluorescence (original magnification ×1000). DAPI: blue staining in (J) and (L). (I) and (J) show the same field. (K) and (L) show the same field. Solid arrows indicate GFAP+ cells. Dashed arrows indicate GFAP− cells that stain positive for HIF-1α. CV, central vein. PP, periportal.

Characterization of HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice

Immunofluorescence was used to detect HIF-1α in sections of liver from HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice. HIF-1α was detected in GFAP+ HSCs in HIF-1α-GFAP Cre− mice treated with carbon tetrachloride (Fig. 1I, 1J). HIF-1α was not detected in GFAP+ HSCs in HIF-1α-GFAP Cre+ mice, although, HIF-1α was detected in neighboring, non-GFAP+ cells (Fig. 1K, 1L).

HSCs were isolated from HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice and exposed to room air or 1% oxygen. The HIF-1α target genes, glucose transporter-1 and vascular endothelial growth factor (VEGF), were upregulated in HSCs isolated from HIF-1α-GFAP Cre− mice but not HIF-1α-GFAP Cre+ mice, indicating functional inactivation of HIF-1α in HSCs from HIF-1α-GFAP Cre+ mice (Supplemental Fig. 1A, 1B). In contrast, upregulation of VEGF by hypoxia in hepatocytes and Kupffer cells was unaffected in HIF-1α-GFAP Cre+ mice (Supplemental Fig. 1C, 1D).

Effect of HIF-1α deletion in HSCs on liver injury after carbon tetrachloride

Serum ALT activity was not different between HIF-1α-GFAP Cre− mice and HIF-1α-GFAP Cre+ after carbon tetrachloride treatment (Fig. 2A). In liver sections from HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice, extensive areas of hepatocyte necrosis were observed in centrilobular regions of liver at 48 h after carbon tetrachloride (Fig. 2C, 2D). By 72 h, the necrotic hepatocytes were largely cleared from the livers of HIF-1α-GFAP Cre− mice (Fig. 2E). In striking contrast, large areas of necrosis remained in the livers of HIF-1α-GFAP Cre+ mice at 72 h after carbon tetrachloride (Fig. 2F). Quantification of the area of necrosis demonstrated no difference between HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice 48 h after carbon tetrachloride (Fig. 2B). At 72 h, however, the area of necrosis was decreased to a greater extent in HIF-1α-GFAP Cre− mice when compared with HIF-1α-GFAP Cre+ mice (Fig. 2B). These findings suggested that activation of HIF-1α in HSCs was critical for efficient clearance of necrotic hepatocytes from the liver after carbon tetrachloride.

FIGURE 2.
FIGURE 2.

Effect of deletion of HIF-1α in HSCs on necrosis in the liver after carbon tetrachloride. HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride. At the indicated times, serum ALT activity was measured (A), and the area of necrosis was quantified in liver sections (B). aSignificantly different from HIF-1α-GFAP Cre− mice at the same time point. Representative liver sections from HIF-1α-GFAP Cre− (C, E) and HIF-1α-GFAP Cre+ (D, F) mice at 48 h (C, D) and 72 h (E, F) after treatment with carbon tetrachloride. *Central vein. Bars, 50 μm.

Impact of HIF-1α deletion in HSCs on deposition of extracellular matrix after carbon tetrachloride

Forty-eight hours after carbon tetrachloride administration, mRNA levels of type I collagen were measured (Table I). Type I collagen levels were increased in the livers of HIF-1α-GFAP Cre− mice, but not in the livers of HIF-1α-GFAP Cre+ mice (Fig. 3A). In contrast, 72 h after carbon tetrachloride administration mRNA levels of type I collagen trended higher in HIF-1α-GFAP Cre+ mice when compared with HIF-1α-GFAP Cre− mice. Consistent with this, type I collagen protein deposition at 72 h was significantly higher in HIF-1α-GFAP Cre+ when compared with HIF-1α-GFAP Cre− mice (Fig. 3). Similarly, α-SMA mRNA and protein levels were significantly higher in HIF-1α-GFAP Cre+ at 72 h after carbon tetrachloride when compared with HIF-1α-GFAP Cre− mice (Fig. 3).

View this table:
Table I. Sequences of real-time PCR primers
FIGURE 3.
FIGURE 3.

Effect of deletion of HIF-1α in HSCs on type I collagen deposition and α-SMA in the liver. (A) Collagen type 1a1 mRNA levels, measured by real-time PCR (Table I), in the livers of HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice treated with carbon tetrachloride for the indicated time. aSignificantly different from vehicle-treated mice. (B) Quantification of the area of type I collagen protein immunofluorescence in the liver 72 h after carbon tetrachloride. aSignificantly different from HIF-1α-GFAP Cre− mice. Representative type I collagen immunostaining in liver sections from HIF-1α-GFAP Cre− mice (D) and HIF-1α-GFAP Cre+ mice (E) treated with carbon tetrachloride for 72 h. Positive staining appears black. Bar, 50 μm. (C) α-SMA mRNA levels in the livers of HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride for the indicated time. aSignificantly different from vehicle-treated mice. bSignificantly different from HIF-1α-GFAP Cre− mice. Representative α-SMA immunostaining in liver sections from HIF-1α-GFAP Cre− (F) and HIF-1α-GFAP Cre+ (G) mice 72 h after carbon tetrachloride treatment. Positive staining appears dark gray. Bar, 50 μm.

Effect of HIF-1α deletion in HSCs on hepatocyte proliferation after carbon tetrachloride

Considering the impact of HIF-1α deletion in HSCs on the clearance of necrotic hepatocytes and matrix deposition, we next evaluated whether hepatocyte proliferation was affected by quantifying PCNA protein by immunohistochemistry. Numerous PCNA+ hepatocytes were observed in HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice after carbon tetrachloride (Supplemental Fig. 2). The numbers of PCNA positive hepatocytes were not different between the two groups at either 48 or 72 h (Supplemental Fig. 2).

Impact of HIF-1α deletion in HSCs on macrophage and neutrophil accumulation in liver after carbon tetrachloride

A critical function of macrophages and neutrophils is the phagocytosis and clearance of dead cells and debris from damaged tissue. CD68+ and F4/80+ macrophages accumulated in the livers of HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice after carbon tetrachloride administration (Supplemental Fig. 3A–D). Quantification indicated no significant differences between the genotypes at any time after treatment (Fig. 4A). Consistent with this, monocyte chemotactic protein-1 mRNA levels were unaffected by deletion of HIF-1α in hepatic stellate cells (Supplemental Fig. 3E). Numerous neutrophils accumulated in centrilobular regions of the liver in HIF-1α-GFAP Cre− mice after carbon tetrachloride (Fig. 4B). In contrast, fewer neutrophils were observed in the livers of HIF-1α-GFAP Cre+ mice (Fig. 4B).

FIGURE 4.
FIGURE 4.

Role of neutrophils and macrophages in the clearance of dead cells from the liver after carbon tetrachloride. HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride. (A) At the indicated times, the area of CD68 and F4/80 immunofluorescence was quantified in sections of liver. (B) Representative photomicrographs of neutrophil immunofluorescence in liver sections from HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice treated with carbon tetrachloride for 24 h. Positive staining appears black. Bar, 50 μm. The area of neutrophil immunofluorescence was quantified. aSignificantly different from HIF-1α-GFAP Cre− mice. (C) C57BL/6 mice were treated with either isotype control IgG or anti–mLy-6G Ab, followed by treatment with carbon tetrachloride for 72 h. Quantification of neutrophil immunofluorescence. aSignificantly different from mice treated with isotype control IgG. Quantification of the area of necrosis. Representative H&E-stained liver sections from mice treated with carbon tetrachloride for 72 h and either isotype control IgG or anti–mLy-6G Ab. (D) C57BL/6 mice were treated with either PBS-containing liposomes or clodronate-containing liposomes. These mice were then treated with carbon tetrachloride for 72 h. Quantification of the area of F4/80 and CD68 immunofluorescence in sections of liver and quantification of the area of necrosis from mice treated with carbon tetrachloride and either PBS-containing liposomes or clodronate-containing liposomes. aSignificantly different from PBS liposome–treated mice. Representative H&E-stained liver sections from mice treated with carbon tetrachloride for 72 h and either PBS-containing liposomes or clodronate-containing liposomes. CV, central vein.

Effect of neutrophil depletion on clearance of necrotic hepatocytes from the liver after carbon tetrachloride

Because deletion of HIF-1α in HSCs reduced neutrophil accumulation after carbon tetrachloride, it is possible that this formed the basis for defective clearance of necrotic hepatocytes seen in these mice. To examine this possibility, mice were treated with anti–mLy-6G Ab to deplete neutrophils. Treatment of mice with anti–mLy-6G Ab reduced neutrophil accumulation in the liver after carbon tetrachloride (Fig. 4C, Supplemental Fig. 4A, 4B). Although neutrophil accumulation was reduced, the area of necrosis at 72 h after carbon tetrachloride was unaffected (Fig. 4C).

Effect of macrophage depletion on clearance of necrotic hepatocytes from the liver after carbon tetrachloride

Because neutrophil depletion did not affect clearance of necrotic cells from the liver, we next determined whether macrophages are critical for this process. To examine this, mice were treated with liposomal clodronate to deplete macrophages. Treatment of mice with liposomal clodronate substantially reduced hepatic numbers of both CD68+ and F4/80+ macrophages and reduced levels of proinflammatory cytokines (Fig. 4D, Supplemental Fig. 4C–G). Consistent with a role of macrophages in the clearance of necrotic hepatocytes, the area of necrosis was significantly higher in clodronate liposome–treated mice when compared with mice treated with PBS liposomes after carbon tetrachloride (Fig. 4D).

Impact of HIF-1α deletion in HSCs on macrophage function after carbon tetrachloride

Because deletion of HIF-1α in HSCs and macrophage depletion both prevented removal of necrotic cells from the liver suggests a functional link between these two cells types in this process. Although, deletion of HIF-1α in HSCs did not affect hepatic accumulation of macrophages, it is possible that macrophage activation was affected. To examine this, we first quantified the hepatic induction of markers of M1 (classical) macrophages. Treatment of HIF-1α-GFAP Cre− mice with carbon tetrachloride increased hepatic expression of several M1 macrophage markers, including inducible NO synthase (iNOS), TNF-α, MIP-2, keratinocyte-derived chemotactic factor (KC), Ccl3, and Ccl4 (Fig. 5A). In striking contrast, levels of all M1 macrophage markers investigated were substantially lower in HIF-1α-GFAP Cre+ mice (Fig. 5A). Activation of HIF-1α in cultured HSCs with hypoxia did not upregulate MIP-2, KC, or TNF-α, indicating that the decreased production of inflammatory mediators in HIF-1α-GFAP Cre+ mice was not the result of HIF-1α–dependent production of cytokines by HSCs and most likely represented reduced production of cytokines by macrophages (Supplemental Fig. 4H). To confirm that inflammatory macrophage phenotype was affected, flow cytometry was performed to quantify Gr1hi macrophages within the CD11bhiF4/80+ subset, which are recruited, M1-like, inflammatory macrophages (21, 22). Treatment of mice with carbon tetrachloride increased the percentage of Gr1hi macrophages in the liver, as reported previously (data not shown). Of interest, the percentage of Gr1hi macrophages in the livers of HIF-1α-GFAP Cre+ mice was significantly lower than that in HIF-1α-GFAP Cre− mice, indicating a reduction in macrophages exhibiting an inflammatory phenotype (Fig. 5B). In addition to M1 macrophage markers, we measured levels of M2 (alternative) macrophage markers. Levels of some M2 macrophage markers, including matrix metalloproteinase (MMP)2, MMP9, and MMP13 were lower in HIF-1α-GFAP Cre+ mice (Fig. 6). Collectively, these results indicate that the mechanism by which HIF-1α in HSCs promotes clearance of necrotic hepatocytes is by influencing macrophage phenotype.

FIGURE 5.
FIGURE 5.

Impact of HIF-1α deletion in HSCs on expression of M1 macrophage markers and on the Gr1hi macrophage population in the liver. HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride. (A) At various times, mRNA levels of the genes indicated in the graphs were measured. aSignificantly different from vehicle-treated mice of the same genotype. bSignificantly different from HIF-1α-GFAP Cre− mice at the same time point. (B) At 48 h after carbon tetrachloride, flow cytometry was used to quantify the percentage of the Gr1hi population (histogram on right) within the CD11bhiF4/80+ subset (dot plot on left). Average of four separate mice in each group. aSignificantly different from HIF-1α-GFAP Cre− mice.

FIGURE 6.
FIGURE 6.

M2 macrophage markers in the liver after carbon tetrachloride. HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride. (A–F) At various times, mRNA levels of the genes indicated in the graphs were measured. aSignificantly different from vehicle-treated mice of the same genotype. bSignificantly different from HIF-1α-GFAP Cre− mice at the same time point.

Impact of HIF-1α deletion in HSCs on upregulation of urokinase plasminogen activator in the liver after carbon tetrachloride

Studies have shown that urokinase plasminogen activator (uPA) is essential for removal of dead cells from the liver after carbon tetrachloride treatment (23). Accordingly, we determined whether uPA levels were affected in HIF-1α-GFAP Cre+ mice treated with carbon tetrachloride. uPA mRNA levels were upregulated in the livers of HIF-1α-GFAP Cre− mice at 48 h after carbon tetrachloride (Fig. 7A). Upregulation of uPA was completely prevented in HIF-1α-GFAP Cre+ mice (Fig. 7A). Exposure of HSCs to hypoxia increased expression of VEGF, but had no effect on uPA mRNA levels (Fig. 7B). Treatment of peritoneal macrophages with conditioned medium from hypoxic HSCs, however, upregulated uPA, suggesting that a mediator released from hypoxic HSCs upregulates uPA in macrophages (Fig. 7C).

FIGURE 7.
FIGURE 7.

Role of HIF-1α in upregulation uPA. (A) HIF-1α-GFAP Cre- and HIF-1α-GFAP Cre+ mice were treated with carbon tetrachloride. At various times, mRNA levels of uPA were measured. aSignificantly different from vehicle-treated mice of the same genotype. bSignificantly different from HIF-1α-GFAP Cre− mice at the same time point. (B) HSCs were isolated from mice and exposed to room air or 1% oxygen for 24 h. VEGF and uPA mRNA levels were measured. aSignificantly different from cells exposed to room air. (C) Conditioned medium from HSCs exposed to room air or 1% oxygen for 24 h was added to peritoneal macrophages for 24 h. uPA mRNA levels were measured in macrophages. aSignificantly different from cells exposed to conditioned medium from room air–exposed HSCs.

Impact of HIF-1α deletion in HSCs on the development of liver fibrosis

Because deletion of HIF-1α in HSCs increased collagen deposition in the liver after a single dose of carbon tetrachloride, we next evaluated whether this would affect development of liver fibrosis after chronic treatment with carbon tetrachloride. mRNA levels of α-SMA and type I collagen were not different between HIF-1α-GFAP Cre+ mice and HIF-1α-GFAP Cre− mice at 4 wk after biweekly injections of carbon tetrachloride (Fig. 8A, 8B). Collagen deposition in the liver, however, was significantly higher in HIF-1α-GFAP Cre+ mice (Fig. 8C–E).

FIGURE 8.
FIGURE 8.

Impact of HIF-1α deletion in HSCs on development of liver fibrosis. HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice received biweekly injections carbon tetrachloride for 4 wk. Type I collagen (A) and α-SMA mRNA (B) levels were measured. aSignificantly different from vehicle-treated mice of the same genotype. Representative type I collagen immunofluorescence in liver sections from HIF-1α-GFAP Cre− (C) and HIF-1α-GFAP Cre+ (D) mice. Positive staining appears black. (E) Quantification of the area of type I collagen protein immunofluorescence in the liver. aSignificantly different from HIF-1α-GFAP Cre− mice.

Discussion

These studies have identified a novel function for HSCs in orchestrating the clearance of necrotic hepatocytes from the liver. HSCs accomplish this function through modulation of macrophage phenotype. In these studies, ALT activity in the serum peaked in both HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice at 48 h after carbon tetrachloride treatment (Fig. 2). ALT activity was then substantially reduced by 72 h, with no difference in ALT activity detected at any time point between HIF-1α-GFAP Cre− and HIF-1α-GFAP Cre+ mice. This indicated that carbon tetrachloride–induced hepatocyte necrosis was not affected by deletion of HIF-1α in HSCs. Consistent with these findings, the area of necrosis was similar between these two strains at 48 h, the time where peak injury had occurred (Fig. 2). In HIF-1α-GFAP Cre− mice, necrotic hepatocytes were rapidly cleared from the liver as indicated by an 89% reduction in the area of necrosis between 48 and 72 h. In striking contrast, the area of necrosis was only reduced by 30% in HIF-1α-GFAP Cre+ mice, indicating that these mice had a defect in the clearance of necrotic hepatocytes. Because the initial hepatotoxic insult was unaffected, as measured by serum ALT activity, the greater area of necrosis observed in HIF-1α-GFAP Cre+ mice at 72 h could only be explained by a deficiency in the removal of dead cells from the liver.

An important function of macrophages and neutrophils is the clearance of dead cells and debris from damaged tissue by phagocytosis. Surprisingly, little is known about the contribution of these two cell types to the clearance of dead cells from the liver, even though this is an essential component of the liver repair process. In the current study, deletion of HIF-1α in HSCs attenuated the accumulation of neutrophils into the liver by ∼75%, suggesting that the reduced clearance of necrotic hepatocytes may have resulted from diminished accumulation of neutrophils (Fig. 4). Depletion of neutrophils to the same extent with a neutralizing Ab, however, did not affect the area of hepatic necrosis 72 h after carbon tetrachloride (Fig. 4). This indicates that neutrophils do not play a major role in the clearance of necrotic hepatocytes and that HIF-1α activation in HSCs does not promote clearance of dead cells by recruiting neutrophils. A possible reason for the lack of effect of neutrophil depletion on removal of necrotic cells is that neutrophil accumulation peaks in the liver at 24 h after carbon tetrachloride (B. Copple, unpublished observations), whereas liver injury does not peak until 48 h after carbon tetrachloride (Fig. 2). We found that by 48 h, neutrophil numbers were substantially decreased, a time where these cells would most likely contribute to the removal of dead cells from the liver. In contrast, depletion of macrophages with liposomal clodronate substantially reduced the clearance of necrotic hepatocytes after carbon tetrachloride treatment (Fig. 4). Unlike neutrophils, however, deletion of HIF-1α in HSCs did not affect accumulation of macrophages. Consistent with this, deletion of HIF-1α in HSCs did not prevent upregulation of monocyte chemotactic protein-1 (Supplemental Fig. 3), a chemokine that contributes to monocyte/macrophage recruitment to the liver after carbon tetrachloride treatment (24).

Although macrophage recruitment was unaffected, markers of inflammatory macrophage activation were substantially lower in mice deficient in HIF-1α in HSCs (Fig. 5). Macrophages have been broadly classified into two general categories, M1 or inflammatory macrophages and M2 or alternative macrophages. Although there is often not a clear-cut distinction between these two classes of macrophages, M1 macrophages are typically associated with production of inflammatory mediators and phagocytosis of dead cells and debris. In HSC-specific HIF-1α knockouts, several markers of M1 macrophage activation, including iNOS, TNF-α, MIP-2, KC, Ccl3, Ccl4, and macrophage migration inhibitory factor were lower, suggesting that HSCs influence macrophage phenotype during acute injury in a HIF-1α–dependent manner (Fig. 5). Consistent with this, deletion of HIF-1α in HSCs reduced the percentage of Gr1hi macrophages, which have been characterized as inflammatory-M1-like macrophages (Fig. 5) (21, 22). In addition to inflammatory mediators, uPA was not upregulated in the livers of HSC-specific HIF-1α knockouts after carbon tetrachloride treatment (Fig. 7A). As mentioned, uPA is required for the clearance of dead cells from the liver after carbon tetrachloride treatment, and the lack of uPA induction in HSC-specific HIF-1α knockouts may explain the mechanism by which there is reduced clearance of dead cells from the livers of HSC-specific HIF-1α knockout mice. Interestingly, activation of HIF-1α in HSCs did not upregulate uPA, but it stimulated release of a factor that upregulated uPA in macrophages (Fig. 7). Although our studies did not identify the specific factor(s) regulated by HIF-1α in HSCs that affect upregulation of uPA in macrophages, we showed previously that numerous genes are upregulated in hypoxic HSCs in a HIF-1α–dependent manner (11). It is possible that through upregulation of one or more of these genes that HSCs directly affect upregulation of uPA in macrophages through the release of a soluble mediator(s). Studies are under way to elucidate this mechanism.

After carbon tetrachloride treatment, α-SMA, and collagen type I were upregulated, indicating activation of HSCs. Of interest, upregulation of type I collagen was prevented in HSC-specific HIF-1α knockouts at 48 h, indicating a key role for HIF-1α in the initial upregulation of this collagen (Fig. 3). This is consistent with a previous study that demonstrated upregulation of type I collagen in hypoxic HSCs (25). In addition, we showed that global deletion of HIF-1α attenuated the increase in type I collagen after bile duct ligation (7). Surprisingly, however, at 72 h after carbon tetrachloride, type I collagen and α-SMA levels were higher in HSC-specific HIF-1α knockouts when compared with HIF-1α-GFAP Cre− mice, suggesting that the inadequate removal of necrotic hepatocytes stimulated collagen production (Fig. 3). This is similar to what was observed in plasminogen knockout mice, which also have a defect in the clearance of necrotic hepatocytes from the liver after carbon tetrachloride (26, 27). These mice also developed substantial fibrosis after a single dose of carbon tetrachloride. One possible explanation for the exacerbated fibrotic response may be the persistent exposure of HSCs to necrotic products from dead hepatocytes, such as HMGB1, which may enhance TGF-β–induced collagen production by HSCs in a TLR4-dependant manner (28, 29). Considering the impact of HIF-1α deletion in HSCs after a single dose of carbon tetrachloride, we also determined the effect on collagen deposition after chronic carbon tetrachloride treatment. Similar to the acute study, collagen protein in the liver was similarly increased in HIF-1α-GFAP Cre+ mice after chronic carbon tetrachloride treatment (Fig. 8).

A limitation of our study is the use of GFAP-Cre to selectively delete HIF-1α in HSCs. Although GFAP is only expressed in HSCs in the liver, it is expressed by other cells types in the intestines, kidneys, and brain, among other organs. Because of this, we cannot completely rule out the possibility that deletion of HIF-1α in one of these other cell types affected hepatic inflammation after carbon tetrachloride treatment. A recent study showed, however, that depletion of HSCs in the liver reduces hepatic inflammation after ischemia/reperfusion and LPS treatment (30). This result, along with our studies showing an interaction between HSCs and macrophages in vitro (Fig. 7), however, lends support for a role of HSCs in regulating hepatic inflammatory responses after liver injury. Development of a Cre-transgenic mouse that selectively expresses Cre recombinase in HSCs, would help to solidify these observations.

Collectively, these studies identified a novel function for HSCs in mediating clearance of necrotic cells from the liver by influencing macrophage phenotype. Furthermore, these studies identified macrophages as the primary cells that mediate clearance of necrotic cells from the liver. Overall, we propose a model whereby hepatocyte necrosis activates HIF-1α in HSCs either through the formation of hypoxic regions or by other mechanisms yet to be determined. HIF-1α then regulates production of a mediator in HSCs that stimulates recruited macrophages to produce inflammatory cytokines, uPA, and to remove necrotic hepatocytes by phagocytosis. If clearance of necrotic hepatocytes is impaired, liver fibrosis ensues. Accordingly, the current studies have potential implications for both drug-induced liver injury and liver fibrosis. Characterization of the cell types and mechanisms involved in the clearance of necrotic cells from the liver could provide insight into ways to facilitate this process and accelerate liver regeneration in patients suffering from acute liver failure. Similarly, the present studies may ultimately lead to the identification of therapeutic targets for the resolution of fibrosis. Specifically, further insight into this process could potentially identify ways to promote matrix degradation in patients with fibrosis.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grants DK073566 (to B.L.C.), ES018885 (to C.E.R.), and ES017537 (to J.P.L.). K.M.O. was supported by National Institutes of Health Training Grant T32ES007255.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ALT
    alanine aminotransferase
    GFAP
    glial fibrillary acidic protein
    HIF-1α
    hypoxia-inducible factor-1α
    HSC
    hepatic stellate cell
    iNOS
    inducible NO synthase
    KC
    keratinocyte-derived chemotactic factor
    MMP
    matrix metalloproteinase
    ODD
    oxygen degradation domain
    PCNA
    proliferating cell nuclear Ag
    α-SMA
    α-smooth muscle actin
    uPA
    urokinase plasminogen activator
    VEGF
    vascular endothelial growth factor.

  • Received November 27, 2013.
  • Accepted February 11, 2014.

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