LOXL2 Inhibition Paves the Way for Macrophage-Mediated Collagen Degradation in Liver Fibrosis

Abstract

Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins and enzymes, especially fibrillary collagens, and represents a major cause of morbidity and mortality worldwide. Lysyl oxidases (LOXs) drive covalent crosslinking of collagen fibers, thereby promoting stabilization and accumulation of liver fibrosis while limiting its resolution. Here we show in a carbon tetrachloride (CCl₄)-induced liver fibrosis murine model that treatment with a novel anti-lysyl oxidase like 2 (LOXL2) neutralizing antibody, which targets extracellular LOXL2, significantly improves fibrosis resolution. LOXL2 inhibition following the onset of fibrosis accelerated and augmented collagen degradation. This was accompanied by increased localization of reparative monocyte-derived macrophages (MoMFs) in the proximity of fibrotic fibers and their representation in the liver. These cells secreted collagenolytic matrix metalloproteinases (MMPs) and, in particular, the membrane-bound MT1-MMP (MMP-14) collagenase. Inducible and selective ablation of infiltrating MoMFs negated the increased "on-fiber" accumulation of MMP-14-expressing MoMFs and the accelerated collagenolytic activity observed in the anti-LOXL2-treated mice. Many studies of liver fibrosis focus on preventing the progression of the fibrotic process. In contrast, the therapeutic mechanism of LOXL2 inhibition presented herein aims at reversing existing fibrosis and facilitating endogenous liver regeneration by paving the way for collagenolytic macrophages.

Keywords: liver fibrosis; liver macrophages; lysyl oxidase like 2 (LOXL2); matrix metalloproteinase-14 (MMP-14); matrix metalloproteinases (MMPs); monocyte-derived macrophages.

Figure 1

Matrix metalloproteinase (MMP)-governed collagenolytic activity naturally appears at the resolution phase of carbon tetrachloride (CCl₄)-induced fibrosis. (A) Schematic representation of the liver fibrosis model. inj, injection. (B) Representative images of Sirius red stain (top) and in situ zymography (bottom) of samples collected from mice sacrificed at different time points after the last (ninth) injection of CCl₄ or oil control (Sirius red scale−250 μm; zymography scale−50 μm). (C) Sirius red quantification by calculating the fraction of collagen-covered area. Analysis was done using ImageJ software (n = 4). (D) Representative images of livers excised from CCl₄-injected mice at the peak of fibrosis (24 h) and recovery phase (120 h). Zymography signal (green) and collagen signal by second harmonic generation (SHG, red) were obtained using two-photon microscopy (scale−50 μm) (n ≥ 2). The co-localization of the zymography and collagen signals were quantified by measuring the intensity of the zymography signal that overlapped with the main collagen fiber in the image. Data were analyzed by one-way ANOVA with Dunnett post hoc comparison. Results are presented as mean ± SEM with significance: *p < 0.05, ***p < 0.001. Data in (A–D) represent a single experiment.

Figure 2

GS341 antibody targets specifically lysyl oxidase like 2 (LOXL2) and affects collagen morphology. (A) Upper panel: Representative second harmonic generation (SHG) images of an in vitro native fibroblast-derived three-dimensional (3D) matrix treated with GS341 or phosphate buffered saline (PBS) (scale−50 μm). Lower panel: Representative fiber directionality analysis plots depicting the frequency of fibers in a specific orientation. Analysis was done using ImageJ software. (B) The thickness of the in vitro native fibroblast-derived 3D matrix treated with GS341 or PBS. (C) Immunoprecipitation (IP) of fibrotic liver lysate with GS341. Beads conjugated with GS341 were incubated with a fibrotic liver lysate, and the eluted fraction was tested in Western blot against commercial anti-LOX and LOXL2 antibodies (lane 1 in each membrane). LOX and LOXL2 recombinant proteins (lane 2) served as positive controls. IP, immunoprecipitation; r, recombinant. Data were analyzed via an unpaired, two-tailed t-test. Results are presented as mean ± SEM with significance: *p < 0.05. Data in (A–C) represent one single experiment.

Figure 3

Inhibition by GS341 demonstrates improved recovery in chronic carbon tetrachloride (CCl₄)-induced liver fibrosis and morphological change to fibrotic fibers. (A) Schematic representation of the experimental setting. inj, injection. (B) Representative images of Sirius red staining performed on paraffin-embedded slides of livers from GS341-treated and control Ab-treated mice. Livers were excised at 48 h after the last (ninth) injection of CCl₄ (scale−200 μm). (C) Quantification of the percentage area covered by collagen, performed by ImageJ. (D) Graph showing histopathological scoring in GS341-treated mice compared to control Ab-treated mice (C,D–n ≥ 6). (E) qRT-PCR analysis of fibrosis marker genes at 24 h after CCl₄-induced liver fibrosis (n = 6). (F) Representative SEM images of de-cellularized three-dimensional (3D) extracellular matrix (ECM) liver scaffolds excised 48 h after the last CCl₄ injection (scale−200 nm). (G) Quantification of fiber thickness in the scanning electron microscope (SEM) images was performed using ImageJ software. Comparison of fibril diameters in control vs. GS341-treated samples shows significant reduction of the fibril diameters of the latter. Statistics are obtained from the four biologic replicates for each case, 12 images per treatment (n = 4). (H) Western blotting (WB) with commercial anti-lysyl oxidase like 2 (LOXL2) and anti-LOX antibodies (Abs) of liver samples treated with control Ab or GS341, excised 48 h after the last CCl₄ injection. LOXL2/LOX expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Quantification of the WB was done using ImageJ software. Data were analyzed by unpaired, two-tailed t-test. Results are presented as mean ± SEM with significance: *p < 0.05, **p < 0.01. Data in (A–D) represent three independent experiments. Data in (E–G) represent two independent experiments. Data in (H) represent one single experiment.

Figure 4

Inhibition of lysyl oxidase like 2 (LOXL2)-governed extracellular matrix (ECM) crosslinking paves the way for macrophages to the fibrotic fibers. CX₃CR1^gfp/+ transgenic reporter mice subjected to carbon tetrachloride (CCl₄)-induced liver fibrosis and, after 2 weeks, to treatment with GS341 or a control antibody (Ab). Livers were excised 48 h following the last CCl₄ injection, stained with anti-green fluorescent protein (GFP) antibody, and then two-photon microscopy imaging was used for the mutual detection of CX₃CR1-GFP expression and the second harmonic generation (SHG) signal at the portal tract fibrotic area. (A) Upper panel: representative images of the distribution of CX₃CR1-GFP⁺ monocyte-derived macrophages (MoMFs) (green) near collagen fibers (red) in GS341- and control Ab-treated mice; yellow arrows indicate CX₃CR1-GFP⁺ MoMFs adjacent to the fibers; scale−50 μm. Lower panel: representative images of Imaris analysis for quantification of the distance of infiltrating CX₃CR1-GFP⁺ MoMFs from the fiber. CX₃CR1-GFP⁺ MoMFs are color coded according to their distance from the fiber; yellow arrows indicate CX₃CR1-GFP⁺ macrophages adjacent to the fibers; scale−50 μm. (B) Quantification of the CX₃CR1-GFP⁺ signal's volume within 15 μm from the fiber normalized to the total collagen fiber volume; scale−50 μm (n ≥ 5). (C) Imaris images showing that macrophages adjacent to the fiber are wrapping the fiber and embracing its shape; scale−10 μm. (D) Representative flow cytometry images showing the gating strategy used to identify MoMFs (CD45⁺CD11b⁺Ly6G⁻CX₃CR1⁺Ly6C^loF4/80⁺CD64⁺ MHCII⁺) and a summarizing graph showing their frequency normalized to tissue mass (g) at 48 h following CCl₄-induced liver fibrosis. KC, kupffer cells; Neut, neutrophils; MoMFs, monocyte-derived macrophages. Data were analyzed by an unpaired, two-tailed t-test. Results are presented as mean ± SEM with significance: *p < 0.05, **p < 0.01. Data in (A,B) represent a single experiment, and data in (D) represent three independent experiments.

Figure 5

Fibrotic macrophages express different collagenolytic matrix metalloproteinases (MMPs) in injured liver. (A) Mmp8, Mmp12, Mmp13, Mmp14, and Cstk gene expression in sorted Ly6C^hi monocytes and monocyte-derived macrophages (MoMFs) 24 and 72 h following carbon tetrachloride (CCl₄)-induced fibrosis, respectively, as extracted from the ArrayExpress database (www.ebi.ac.uk/arrayexpress, accession no. E-MEXP-3177). Their expression was normalized to the expression of the adipocyte marker Adipoq, which served as a background expression level (n = 3). (B) Mmp8, Mmp12, Mmp13, Mmp14, and Cstk gene expression in sorted Ly6C^hi monocytes 24 h following acetaminophen-induced liver injury (AILI) and in sorted Kupffer cells (KCs) and MoMFs 72 h post AILI. Data were extracted from our existing database (GSE55606). Their expression was normalized to the expression of the adipocyte marker Adipoq, which served as a background expression level. Black asterisks indicate a significant difference from KCs at 72 h. Gray asterisks indicate a significant difference between Ly6C^hi monocytes at 24 h and MoMFs at 72 h. (C) cyTOF analysis of livers following CCl₄-induced liver injury. Mice were injected three times with CCl₄ without any treatment, after which livers were excised 48 h following the last CCl₄ injection. Presented are plots depicting the gating strategy and analysis of neutrophils and macrophages percentage from total MMP-14⁺ or MMP-9⁺ cells (n = 3). (D) Representative flow cytometry images showing the expression of MMP-14 in liver infiltrating neutrophils (CD11b⁺Ly6G⁺CX₃CR1⁻CD64⁻), Ly6C^hi monocytes (CD11b⁺Ly6G⁻ CX₃CR1⁺Ly6C^hiCD64^lo), and MoMFs (CD11b⁺Ly6G⁻CX₃CR1⁺Ly6C^loCD64⁺) 48 h following CCl₄-induced liver fibrosis. (E) Mean fluorescent intensity (MFI) expression of MMP-14 in MoMFs as depicted by flow cytometry analysis. (F) Representative images displaying the localization of CX3CR1-GFP⁺ MoMFs (green) and MMP-14 (red) in GS341- and control Ab-treated mice. White arrows indicate co-localization of CX3CR1-GFP⁺ and MMP-14 signals. Mono, monocyte; Neut, neutrophils; MoMFs, monocyte-derived macrophages. Data were analyzed using an unpaired, two-tailed t-test (A,C,E) or one-way ANOVA with Tukey's Honestly Significant Difference post hoc comparison (B). Results are presented as mean ± SEM with significance: *p < 0.05, **p < 0.01, and ***p < 0.001. Data in (A–F) represent a single experiment.

Figure 6

Ly6C^hi monocytes and monocyte-derived macrophages (MoMFs) facilitate collagen degradation following lysyl oxidase like 2 (LOXL2) inhibition. (A) Representative images of in situ zymography of fragment antigen binding (Fab)-treated and control livers 48 h after the last carbon tetrachloride (CCl₄) injection. Spatial localization differences are demonstrated in collagen type I-degrading enzymes in Fab-treated livers compared to control [n = 5, green–zymography; red–second harmonic generation (SHG); scale−50 μm]. (B) Representative images of in situ zymography and tissue inhibitor of metalloproteinase (TIMP)1 staining of Fab-treated and control livers 48 h after the last CCl₄ injection. (C) Representative images of in situ zymography of GS341– and GS341+ MC-21-treated mice 48 h after the last CCl₄ injection (green–zymography; red–SHG; scale−50 μm). (D) Representative SHG images of 48 h liver samples treated with control antibody (Ab)/GS341/GS341+MC-21 Abs stained with MMP-14 antibody (scale−50 μm). (E) Quantification of the MMP-14 signal volume within 15 μm from the fiber, normalized to the total collagen fiber volume (n = 3). Data were analyzed by one-way ANOVA with Dunnett post hoc comparison. Results are presented as mean ± SEM with significance: *p < 0.05, **p < 0.01. Data in (A–E) represent a single experiment.