Introduction

Liver diseases, despite tremendous efforts, remain a global health burden accounting for more than two million deaths annually1. Till date, there is no clinically approved treatment for liver diseases. Regardless of etiology (alcohol, metabolic dysfunction, viral hepatitis), the progression of liver diseases encompasses liver injury, inflammation and fibrogenesis that culminates into liver cirrhosis, end-stage liver failure and liver cancer when untreated2,3.

During liver injury, damaged hepatocytes secrete pro-inflammatory factors that activates resident liver macrophages (Kupffer cells, KCs)4. Damaged hepatocytes and KCs secrete chemokines such as C–C motif chemokine ligand-2 (CCL2) that instigate the recruitment of circulating bone-marrow derived monocytes (Fig. 1). The recruited monocytes differentiate into inflammatory macrophages referred to as monocytes-derived macrophages (MoMFs) causing liver inflammation, and activation of hepatic stellate cells (HSCs) via transforming growth factor beta (TGF-β)5,6. HSCs following activation transdifferentiate into highly proliferative myofibroblasts that secretes excessive amounts of collagen-rich extracellular matrix (ECM) that disrupts the architecture and function of the liver7,8. Activated HSCs also stimulate monocyte recruitment by secreting CCL2 (Fig. 1). The involvement of the CCL2/CCR2 axis in the pathogenesis of liver disease has prompted extensive research into its potential as a therapeutic target. Several studies via genetic or pharmacological inactivation of CCR2 have demonstrated a significant reduction in monocyte infiltration and disease progression in different liver disease etiologies9,10,11,12,13,14,15,16,17. In addition to its extensively studied role in hepatic inflammation and monocyte recruitment, CCR2 is also expressed on HSCs and plays a pivotal role in hepatic fibrosis18. Owing to the crucial role of CCL2-CCR2 pathway in different liver diseases, Cenicriviroc (CVC), a CCR2/CCR5 antagonist have been investigated greatly in several preclinical with promising results14,15,19,20, and in clinical trials21,22. Regrettably, CVC encountered challenges during phase III clinical trials, primarily due to a lack of efficacy23.

Figure 1
figure 1

Schematic showing the pathogenesis of liver fibrosis, and the combination treatment strategy (Cenicriviroc and Matrix Metalloproteinase 1, MMP1) to promote the resolution of liver fibrosis. During liver injury, injured hepatocytes secrete pro-inflammatory factors that activate liver resident Kupffer cells (KCs). Damaged hepatocytes and KCs secrete chemokines especially CCL2 that induces recruitment of CCR2-expressing monocytes via CCL2-CCR2 axis. Following recruitment, monocytes differentiate into macrophages referred to as monocytes-derived macrophages (MoMFs). KCs and MoMFs secrete pro-fibrogenic factors such as transforming growth factor beta (TGF-β) that activates hepatic stellate cells (HSCs). Activated HSCs stimulate monocyte recruitment by secreting CCL2 and produce excessive amounts of extracellular matrix proteins primarily collagen that leads to fibrosis development. Cenicriviroc (CVC, a CCR2/CCR5 antagonist) inhibits monocyte recruitment and thereby blunts TGF-β secretion and HSCs activation which further inhibit monocyte recruitment. MMP1 degrades collagen-rich ECM thereby inhibits HSCs activation and monocyte recruitment. The combination of CVC and MMP1 can potentially contribute to fibrosis resolution.

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Monocytes/macrophages contribute to liver fibrosis by inducing liver inflammation, but they are also involved in disease resolution by secreting matrix metalloproteinases (MMPs) to degrade ECM, thereby inhibiting HSCs activation, and monocyte recruitment (driven by ECM and activated HSCs)24,25,26. During the fibrotic process, the equilibrium between collagen deposition and degradation is disrupted by the downregulation of MMPs and the upregulation of tissue inhibitors of MMPs (TIMPs)27,28,29. We and others have demonstrated that the MMP1 delivery by cell/gene therapy or drug delivery carriers e.g., polymersomes, ameliorates liver fibrogenesis in vivo29,30. In this study, we propose a combination therapeutic approach by combining CVC and MMP1 to inhibit intrahepatic monocyte recruitment and increasing collagen degradation respectively to ameliorate liver inflammation and promote fibrosis resolution (Fig. 1).

Materials and methods

Materials

Carbon tetrachloride (CCl4) (Cat. no. 02671, Sigma Aldrich, St. Louis, MO, USA); olive oil (Cat. no. O1514, Sigma Aldrich); (2-Hydroxypropyl)-β-cyclodextrin (Cat. no. 778966, Sigma Aldrich); Kolliphor HS15 (Cat. no. 42966, Sigma Aldrich); collagenase from Clostridium histolyticum (MMP1) (Cat. no. C0130, Sigma Aldrich); Cenicriviroc (CVC) (HY-14882, MedChemExpress, South Brunswick Township, NJ, USA); Roswell Park Memorial Institute (RPMI) 1640 with L-Glutamine (Capricorn Scientific, Ebsdorfergrund, Germany); Penicillin/Streptomycin, (Capricorn Scientific); Bovine Serum Albumin (BSA) (Cat. no. A7906, Sigma Aldrich); Fetal Bovine Serum (FBS) (Cat. no. F7524, Sigma Aldrich); Phosphate Buffered Saline (PBS) tablets (Cat. no. 524650, Millipore, Watford, UK); Formaldehyde (Cat. no. F8775, Sigma Aldrich); Hoechst 33342, Trihydrochloride, Trihydrate (Cat. no. H1399, Thermo Fisher Scientific, Wesel, Germany); Epredia Cryomatrix™ embedding resin (Cat. no. 6769006, Epredia, Runcorn, UK); 2-methylbutane (Cat. no. M32631, Sigma Aldrich); 3-Amino-9-ethylcarbazole (AEC) (Cat. no. A6926, Sigma Aldrich); Dimethylformamide (DMF) (Cat. no. 227056, Sigma Aldrich); Hematoxylin solution according to Mayer (Cat. no. 51275, Sigma Aldrich); Aqueous Mounting Medium Aquatex (Cat. no. 1085620050, Sigma Aldrich); SV Total RNA Isolation System (Cat. no. Z3105, Promega, Leiden, Netherlands); iScript cDNA synthesis kit (Cat. no. 1708891, BioRad, Veenendaal, Netherlands); SensiMix Plus SYBR & Fluorescein (Cat. no. QT615-20, GC biotech, Waddinxveen, Netherlands).

In vitro studies

Cell lines

Mouse RAW 264.7 macrophages and NIH/3T3 mouse fibroblasts used in this study were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). RAW 264.7 macrophages were cultured in RPMI medium supplemented with 10% FBS, 50 U/mL penicillin and 50 μg/mL streptomycin. 3T3 cells were cultured in DMEM medium supplemented with 10% FBS, 50 U/mL penicillin and 50 μg/mL streptomycin. The cells were passaged twice a week as per established experimental protocols.

In vitro transwell assays

For the transwell migration assay, mouse RAW macrophages (1 × 105 cells/100 µL) were seeded into the 24-well inserts with 5 µm polycarbonate membranes in RPMI medium supplemented with 100 ng/mL LPS (Sigma) and 10 ng/mL IFNγ (Peprotech, Thermo Fisher Scientific) to activate macrophages. 3T3 fibroblasts (3 × 105 cells/300 µL) were seeded in the lower chamber, where applicable. In the lower well containing 600 µL medium, 10 ng/mL CCL2 was added to induce transwell migration and 5 ng/mL TGF-β (Peprotech) was added, where applicable, to activate 3T3 fibroblasts and to induce transwell migration. CVC (10 µM) was added in the upper chamber while MMP1 (2 µg/mL) was added in the lower chamber. After 24 h of incubation, the upper wells were cleaned with a cotton swap to remove non-migrated cells. The migrated cells on the bottom side were washed 3 times with PBS and fixed for 20 min at RT with 4% formaldehyde in PBS. After fixation, cells were washed 3 times in PBS and permeabilized for 5 min in ice-cold methanol. Cells were again washed 3 times in PBS and membranes were cut out, put on a glass slide, mounted in DAPI containing mounting medium and representative images were captured at 10 × under a microscope. Nuclei were counted using ImageJ. 3T3 fibroblasts in the lower chamber were lysed with RNA lysis buffer and stored at − 80 °C for further analysis.

CCl4-induced acute liver injury mouse model

These animal experiments were carried out in strict accordance with the guidelines and regulations for the Care and Use of Laboratory Animals, Utrecht University, The Netherlands. All the experimental protocols were approved by the Institutional Animal Ethics Committee of the University of Twente, The Netherlands. All the methods are reported in accordance with the ARRIVE guidelines. CCl4 was prepared in olive oil (0.2 mL/kg), MMP1 was dissolved in PBS, and CVC was formulated in (2-Hydroxypropyl)-β-cyclodextrin (10%) + Kolliphor HS15 (5%) in MilliQ (85%) (hereafter called vehicle). Male C57BL/6 mice (10–12 weeks old) received a single intraperitoneal (IP) injection of olive oil (healthy n = 7) or 0.2 mL/kg CCl4 (n = 26) on day 1. On day 2 and 3, the CCl4-treated mice received vehicle (IP, n = 7), 500 µg/kg MMP1 (intravenously (IV), n = 7), 1 µmol/kg CVC (IP, n = 6), or both 500 µg/kg MMP1 (IV) and 1 µmol/kg CVC (IP) (n = 6), once daily. On day 4, all animals were euthanized, and their blood, liver, lungs, kidneys, spleen, and heart were collected. Heparinized blood samples were centrifuged at 2300 × g for 10 min at 4 °C. Plasma was transferred in new Eppendorf’s and stored at − 80 °C until further analysis. Total alanine transaminase (ALT) and aspartate transaminase (AST) levels were measured in the plasma as per standard biochemical assays. Organs were weighed and liver tissues were processed for further analysis.

Immunohistochemistry staining

Liver tissues were harvested and transferred to Cryomatrix™ embedding resin, and snap-frozen in 2-methylbutane chilled on dry ice. Cryosections (6 µm) were cut using a Leica CM 1860 cryostat. The sections were air-dried until dry and fixed in acetone for 20 min at RT. Thereafter, tissue cryosections were rehydrated in PBS and incubated with the primary antibody i.e., Rat anti-mouse F4/80 (clone CI: A3-1, Bio-Rad); Rat anti-mouse CD11b (clone M1/70, BioLegend, Amsterdam, Netherlands); goat anti-mouse collagen-I antibody (cat. no. 1310-01, Southern Biotech, Birmingham, USA) at 4 °C overnight. Next day, sections were washed 3 times with PBS. Endogenous peroxidase activity was blocked by 0.3% H2O2 prepared in methanol for 30 min. Sections were washed 3 times with PBS and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies i.e., rabbit anti-rat (Southern Biotech) or rabbit anti-goat (Thermo Fisher Scientific) for 1 h at RT. Thereafter, sections were washed 3 times with PBS and incubated with HRP-conjugated tertiary antibody (goat anti-rabbit, Dako, Agilent, CA, USA) for 1 h at RT and washed again 3 times with PBS. 3-Amino-9-ethylcarbazole (AEC) solution was freshly prepared by combining 4.5 mL MilliQ, 500 µL 1 M sodium acetate pH 5.5 and 250 µL AEC in dimethylformamide (DMF) (1 tablet per 2.5 mL DMF). This was filtered using a 4.5 µm nylon filter and lastly 5.2 µL 30% H2O2 was added to the AEC solution. When prepared, peroxidase activity was developed using AEC solution for 20 min at RT, and nuclei were counterstained with hematoxylin for 5 min. After 5 min of washing under tap water sections were mounted in Aquatex mounting medium. Slides were digitized using a NanoZoomer 2.0 HT whole slide scanner (Hamamatsu, Shizuoka, Japan) and resulting digital images were visualized at 1680 × 1050-pixel resolution with NDP software and analyzed using FIJI/ImageJ software.

Hematoxylin and eosin staining

Sections were fixed with 4% formalin for 20 min and then rinsed with distilled water. The sections were incubated with hematoxylin for 15 min followed by washings with tap water. Thereafter, sections were incubated with eosin solution for 1.5 min followed by washing in 96% ethanol, dehydrating with ethanol, and mounted with VectaMount mounting medium (Vector Laboratories, Burlingame, CA).

CHP staining

A PBS solution containing 5 μM of 5 FAM-labeled collagen hybridization peptide (F-CHP, 3Helix Inc., Salt Lake City, USA) was heated for 5 min at 80 °C in water bath, followed by immediate incubation in an ice/water bath (for 90 s) to quench the hot solution to room temperature. The quenched solution was immediately added onto the paraformaldehyde-fixed liver sections and incubated for overnight. The sections were washed with PBS and mounted using aqueous DAPI-mounting medium (Fluoroshield with DAPI, Sigma) and stored at 4 °C in the dark until imaging. Fluorescent images were made using a Nikon E400 microscope (Nikon), and ImageJ was used to make the overlay images.

Quantitative real-time PCR

Total RNA was extracted using GenElute Total RNA Miniprep Kit (Sigma) for 3T3 cells or SV total RNA isolation system (Promega Corporation, Fitchburg, WI, USA) for mouse liver tissues according to the manufacturer’s instructions. The RNA concentration was quantified using NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Waltham, USA). Total RNA (1 µg) was reverse transcribed using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. For quantitative real-time PCR, 20 ng cDNA was used for each PCR reaction and was performed with 2 × SensiMix SYBR and Fluorescein Kit (Bioline GmbH, QT615-05, Luckenwalde, Germany) and pre-tested and gene-specific primers (see Table 1), using a BioRad CXF-384 Real-Time PCR detection system (BioRad). Finally, cycle threshold (Ct) values were normalized to reference 18S ribosomal RNA (18S rRNA), and relative gene expression were calculated using the 2−ΔΔCt-method.

Table 1 Sequence of the primers used for quantitative real-time PCR.
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Data and graphs

All graphs were made, and statistical analysis was performed using GraphPad Prism version 10.0.1 (GraphPad Prism, La Jolla, CA, USA). The results are expressed as the mean + standard error of the mean (SEM). Comparisons between two groups were performed using the unpaired students’ t-test and multiple comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, respectively.

Results

CCL2 and fibroblasts with or without TGF-β potentiates macrophage migration

Previous studies have shown that CCL2 secreted by damaged hepatocytes, KCs and activated HSCs promote monocyte recruitment (Fig. 1). We therefore first evaluated if CCL2 and/or other chemokines secreted by fibroblasts without and with TGF-β activation potentiates macrophage recruitment using transwell migration assays. As can be seen in the schematic in Fig. 2A, we cultured macrophages in the upper transwell insert without CCL2, with CCL2, with 3T3 fibroblasts cultured in the lower chamber, with 3T3 fibroblasts and CCL2, with 3T3 fibroblasts and TGF-β, and with 3T3 fibroblasts, CCL2 and TGF-β. We observed that the CCL2 showed 4.7-fold increase in macrophage migration similar to 3T3 fibroblasts (about 4.9-fold) suggesting that 3T3 fibroblasts secrete chemokines that favor macrophage recruitment (Fig. 2B). Moreover, we observed that macrophage migration is strongly increased upon co-addition of CCL2 and 3T3 suggesting synergistic effect of CCL2 and 3T3 (Fig. 2B). Macrophage migration is further potentiated by 3T3 fibroblasts and TGF-β, while addition of CCL2 to 3T3 fibroblasts and TGF-β did not promote further migration suggesting that maximum macrophage recruitment is achieved by 3T3 fibroblasts and TGF-β (Fig. 2B). These results suggest that macrophage recruitment is indeed driven by CCL2 while TGF-β activated fibroblasts strongly potentiate macrophage recruitment via secretion of chemokines including CCL2 indicating the role of CCL2 and TGF-β activated fibroblasts in intrahepatic macrophage recruitment and in liver inflammation.

Figure 2
figure 2

CCL2 and 3T3 fibroblasts with or without TGF-β potentiates migration of RAW 264.7 macrophages. (A) Schematic showing the experimental design of the transwell migration assay. RAW 264.7 macrophages were cultured in the upper transwell insert without CCL2 (control), with CCL2, with 3T3 fibroblasts cultured in the lower chamber, with 3T3 fibroblasts and CCL2, with 3T3 fibroblasts and TGF-β, or with 3T3 fibroblasts, CCL2 and TGF-β. (B) Graph shows the relative RAW 264.7 macrophage migration (relative to control) expressed in percentage (%) in response to the conditions (indicated in the table below the graph). The data are presented as truncated violin plots showing all the individual experimental points. All experiments were conducted at least four times to be able to make statistical comparisons. Multiple comparisons between different groups were performed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001 (versus control); ##p < 0.01; ###p < 0.001 (versus CCL2); †p < 0.05; †††p < 0.001 (versus 3T3 fibroblasts).

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CVC and CVC + MMP1 inhibit TGF-β activated fibroblasts driven macrophage migration, and MMP1 and CVC + MMP1 inhibit TGF-β induced collagen-I expression in fibroblasts

Next, we evaluated the following: (a) if Cenicriviroc (CVC), a CCR2/CCR5 antagonist, can inhibit TGF-β activated fibroblasts driven macrophage migration by antagonizing CCL-CCR axis; (b) if MMP1 can inhibit macrophage migration; or (c) if the combination of CVC and MMP1 show a synergistic effect inhibiting macrophage migration by antagonizing CCL-CCR axis and inhibiting fibroblast activation respectively. We used the transwell assays where we cultured RAW 264.7 macrophages in the transwell inserts and fibroblasts in the lower chamber with and without TGF-β (Fig. 3A). We observed threefold increase in macrophage migration upon TGF-β activation (Fig. 3B) as seen previously (Fig. 2). This TGF-β activated fibroblasts driven macrophage migration was inhibited by CVC while MMP1 alone showed no inhibition. The combination of CVC and MMP1 completely blunted TGF-β-activated fibroblasts driven macrophage migration (Fig. 3B). These results indicate that antagonizing CCL-CCR axis in combination with inhibiting the fibroblasts activation is an effective approach in attenuating intrahepatic macrophage recruitment and liver inflammation.

Figure 3
figure 3

CVC and CVC + MMP1 inhibit TGF-β activated fibroblasts driven macrophage migration, and MMP1 and CVC + MMP1 inhibit TGF-β induced collagen-I expression in fibroblasts. (A) Schematic showing the experimental design of the transwell migration assay where we cultured RAW 264.7 macrophages with and without CVC in the transwell inserts and fibroblasts in the lower chamber without and with TGF-β without or with MMP1. (B) Graph shows the relative RAW 264.7 macrophage migration (relative to TGF-β activated 3T3 fibroblasts induced macrophage migration) expressed in percentage (%) in response to the conditions (indicated in the table below the graph). (C) Graph shows the relative collagen-I mRNA expression (relative to TGF-β activated 3T3 fibroblasts), normalized to 18S rRNA, as analysed in 3T3 fibroblasts in response to the conditions indicated in the table below the graph. The data are presented as truncated violin plots showing all the individual experimental points. All experiments were conducted at least four times to be able to make statistical comparisons. Multiple comparisons between different groups were performed by one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001.

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We also evaluated the collagen-I mRNA expression in the fibroblasts cultured in the lower chamber. It can be clearly seen that TGF-β induced collagen-I gene expression in 3T3 fibroblasts (Fig. 3B). We further observed that TGF-β induced collagen-I expression in 3T3 fibroblasts remain unchanged following CVC treatment while MMP1 and more significantly CVC + MMP1 inhibited TGF-β induced collagen gene expression in 3T3 fibroblasts.

Altogether, these results suggest CVC strongly inhibits macrophage migration while MMP1 inhibits fibroblasts activation, and the combination of CVC and MMP1 show synergistic effects by inhibiting both macrophage migration driven by TGF-β activated fibroblasts and collagen-I expression in TGF-β activated fibroblasts.

CVC, MMP1 and CVC + MMP1 treatment decreased normalized liver weights and improved liver function in vivo in CCl4-induced acute liver injury mouse model

Following the promising in vitro results, we next evaluated if CVC, MMP1 and CVC + MMP1 treatment have an effect in vivo in an acute CCl4-induced liver injury-early liver fibrosis mouse model. The schematic overview of the in vivo study can be seen in Fig. 4A. Following CCl4 administration, mice were treated with CVC, or MMP1 or CVC + MMP1. Here, we first investigated the effect of CVC + MMP1 treatment on liver weights normalized to body weight (Fig. 4 and Supplementary Fig. S1). Acute CCl4 administration resulted in significantly increased liver weight respective to body weight (p < 0.05) (Fig. 4B). CVC, MMP1 or CVC + MMP1 treatments showed a significant decrease (p < 0.05) in the normalized liver weights. Furthermore, we analyzed total plasma transaminase (AST and ALT) levels as indicators of liver function. We found that both MMP1 and CVC + MMP1 significantly improved the CCl4-induced AST and ALT levels while CVC alone showed modest inhibition in the AST and ALT levels (Fig. 4C,D).

Figure 4
figure 4

CVC, MMP1 and CVC + MMP1 treatment decreased normalized liver weights and improved liver function in vivo in CCl4-induced acute liver injury mouse model. (A) Schematic overview of the in vivo study. (B) Liver weights (respective to body weight) expressed in percentage. (C) Total alanine transaminase (ALT) plasma levels expressed in units (U)/L. (D) Total aspartate transaminase (AST) plasma levels expressed in units (U)/L. The data are represented as mean + SEM. Multiple comparisons between different groups (n = 6–7 mice per group) were performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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We also analyzed the effect of CVC, MMP1 and CVC + MMP1 treatments on other organs by analyzing the normalized organ weights. No significant differences were observed in the normalized organ weights in the treatment groups compared to the CCl4-vehicle group (Supplementary Fig. S1). As shown previously, the spleen weight in mice is increased upon CCl4 administration reflecting the development of splenomegaly. CVC treatment reduced the normalized spleen weights while other treatments i.e., MMP1 or CVC + MMP1 showed no major changes in the spleen weights.

We further analyzed the effect of CVC, MMP1 and CVC + MMP1 treatments on body weights of the mice by normalizing the body weights from the starting body weights. We found that CVC significantly reduced the body weights when compared with healthy, CCl4-vehicle, CCl4-MMP1, CCl4-CVC + MMP1 mice (Supplementary Fig. S1). These results suggest that combining MMP1, we could prevent the adverse effects of CVC and indicating the safety profile of CVC + MMP1 combined treatment.

CVC + MMP1 treatment attenuated monocyte recruitment and liver inflammation in vivo in CCl4-induced acute liver injury mouse model

We next evaluated the effect of CVC, MMP1 and CVC + MMP1 on monocyte-macrophage recruitment and subsequent liver inflammation in vivo in an acute CCl4-induced liver injury-early liver fibrosis mouse model. We performed immunostaining for F4/80 (pan-macrophage marker). Moreover, we analyzed mRNA expression for F4/80 and tumor necrosis factor alpha (TNFα, an inflammatory cytokine) in the liver tissues of mice. The results show increased F4/80 expression in CCl4-mice (2.1-fold, p < 0.01) compared to control mice suggesting an increased expansion of hepatic macrophage pool (Fig. 5A,B). F4/80 expression (staining) was significantly decreased with CVC (3.1-fold, p < 0.001), MMP1 (1.9-fold, p < 0.05), and CVC + MMP1 (eightfold, p < 0.0001) treatments versus vehicle-treatment group. Moreover, CVC + MMP1 treatment reduced F4/80 mRNA expression which was reflected in reduction in TNFα mRNA expression (Fig. 5C).

Figure 5
figure 5

CVC + MMP1 treatment attenuated macrophage infiltration and liver inflammation in vivo in CCl4-induced acute liver injury mouse model. (A) Representative images (scalebar = 250 µm) of F4/80-stained liver sections. (B) Quantified positive stained area of F4/80 staining. (C) Relative mRNA expression (normalized to 18S rRNA) of Adgre1 (F4/80) and Tnfα (relative to vehicle control). The data are represented as mean + SEM. Multiple comparisons between different groups (n = 6–7 mice per group) were performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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To assess if the reduction in F4/80 expression is related to the monocyte recruitment, we performed staining for CD11b (a marker for liver infiltrating MoMFs). We evidenced that marked increase in the CD11b expression upon CCl4-induced liver injury (26-fold, p < 0.001) versus control mice (Fig. 6A,B). CD11b expression was significantly attenuated in CVC (3.9-fold, p < 0.01), MMP1 (2.7-fold, p < 0.05) and CVC + MMP1 (5.9-fold, p < 0.01) treatment groups (Fig. 6A,B).

Figure 6
figure 6

CVC + MMP1 treatment attenuated monocyte recruitment in vivo in CCl4-induced acute liver injury mouse model. (A) Representative images (scalebar = 100 µm) of CD11b-stained liver sections. (B) Quantified positive stained area of CD11b staining. The data are represented as mean + SEM. Multiple comparisons between different groups (n = 6–7 mice per group) were performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001.

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Altogether, these results indicate that CVC, MMP1 and CVC + MMP1 treatments can inhibit monocyte recruitment either by antagonizing CCL2-CCR2 antagonism and/or ECM degradation and HSCs inactivation. The effects are however more pronounced in the combined CVC + MMP1 treatment group indicating the synergistic effect of CVC and MMP1 treatment.

CVC + MMP1 treatment ameliorated liver fibrogenesis in vivo in CCl4-induced acute liver injury mouse model

We next evaluated the effect of CVC, MMP1 and CVC + MMP1 on HSCs activation and fibrogenesis in vivo in an acute CCl4-induced liver injury-early liver fibrosis mouse model. Hematoxylin and eosin (H&E) staining indicated that CCl4 injection caused acute liver injury which was improved following MMP1 treatment and more strongly with CVC + MMP1 treatment, while CVC alone showed mild improvement (Supplementary Fig. S2). Moreover, we observed that a single CCl4 injection in mice led to a significant increase in collagen-I (1.5-fold increase, p < 0.001) staining compared to healthy control mice (Fig. 7A,B). Increased collagen-I expression was significantly decreased following CVC (1.2-fold, p < 0.05), MMP1 (1.6-fold, p < 0.0001) and CVC + MMP1 (twofold, p < 0.0001) treatment compared to CCl4 mice (Fig. 7A,B). We further investigated the effects of treatments on gene expression of major fibrotic parameter (Procollagen1a1) and HSC activation marker (Acta2) in acute CCl4-induced liver fibrosis (Fig. 7C). Acute CCl4 administration caused upregulation of mRNA expression of Procollagen1a1 and Acta2, which was highly significantly inhibited following CVC + MMP1 treatment (Fig. 7C). Notably, MMP1 showed reduction in collagen-I protein levels and not significantly Procollagen1a1 and Acta2 gene expression suggesting that MMP1 has a direct role in collagen-I protein by degrading collagen however, combined CVC and MMP1 treatment show increased anti-fibrotic effects. Moreover, we evaluated the collagen degradability by MMP1 and CVC + MMP1 treatment groups using 5-Carboxyfluorescein labelled collagen hybridization probe (5FAM-CHP) that binds to degraded collagen (Supplementary Fig. S3). We observed that indeed MMP1 and CVC + MMP1 show increased collagen degradation in vivo as stained by 5FAM-CHP while 5FAM staining was very weak/undetectable in control group and mild 5FAM-CHP staining was observed in CCl4-vehicle and CCl4 + CVC groups (Supplementary Fig. S3).

Figure 7
figure 7

CVC + MMP1 treatment ameliorated liver fibrogenesis in vivo in CCl4-induced acute liver injury mouse model. (A) Representative images (scalebar = 250 µm) of collagen I-stained liver sections. (B) Quantified positive stained area of collagen-I staining. (C) Relative mRNA expression (normalized to 18S rRNA) of Procollagen 1α1 (collagen I) and Acta 2 (α-SMA) (relative to vehicle control). The data are represented as mean + SEM. Multiple comparisons between different groups (n = 6–7 mice per group) were performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. The differences were considered significant when *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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These results indicate that MMP1 and CVC + MMP1 treatment is effective in inhibiting collagen deposition, and the superiority of the dual therapy (CVC + MMP1) over monotherapy (CVC or MMP1) in inhibiting liver fibrogenesis.

Discussion

Beside the efforts, there is still no therapy approved for liver disease31,32,33. Despite promising preclinical results, the highly regarded small molecule CCR2/5 antagonist, known as CVC, sadly proved ineffective in the Phase III AURORA clinical trial23. While CVC was safe and well tolerated in patients, the primary end point of fibrosis regression (22.3% in CVC versus 25.5% in placebo) without worsening of NASH (23.0% in CVC versus 27.2% in placebo) was not achieved23,34. Since macrophages not only contribute to liver fibrosis by inducing liver inflammation; they are also involved in disease resolution by secreting MMPs to degrade ECM, thereby inhibiting HSCs activation, and monocyte recruitment (driven by ECM and activated HSCs)24,25,26. Hence, in this study, we explored if combining CVC with MMP1 would be an efficient therapeutic approach for the treatment of liver fibrosis. We observed indeed inhibiting monocyte recruitment by CVC and increased ECM degradation by MMP1 improved liver function, decreased liver inflammation and fibrogenesis without causing adverse effects.

CCL2-driven recruitment of CCR2-expressing monocytes and their subsequent differentiation into inflammatory macrophages strongly contribute to liver inflammation upon acute and chronic liver injury9,10,11,12,13,14,15,16,17. Consequently, CCL2-CCR2 axis gained tremendous attention in last years15,25,35,36,37. Using genetic models and pharmacological inhibition studies, it has been shown that targeting this axis is an attractive approach for the treatment of liver inflammation and liver fibrosis14,15,19,20. Studies have also shown that depleting macrophages or inhibiting monocyte recruitment delayed or hindered fibrosis regression since monocytes-macrophage secrete MMPs to degrade extracellular matrix24,25,26,38. In our study, we first investigated the effect of CVC, MMP1, and CVC + MMP1 on monocyte-macrophage infiltration in vitro and in vivo, and liver inflammation in vivo. Aligned with previous studies, we observed that CVC and CVC + MMP1 inhibited monocyte-macrophage recruitment as evidenced by F4/80 and CD11b staining while MMP1 showed no-modest effect. The effects were more pronounced in CVC + MMP1 treatment group which is attributed to the direct effect of CVC as shown previously, combined with ECM-degradation and reduced HSCs activation by MMP1 as ECM-induced stiffness, ECM-induced HSCs activation and CCL2 secretion by activated HSCs are shown to promote monocyte/macrophage infiltration38,39,40. Moreover, it has been reported that MMP1 can cleave CCL2 thereby reducing the availability of CCL2 and the activation of CCR241. Additionally, while it is hypothesized MMP1 may contribute to the inflammatory response by cleaving pro-inflammatory cytokines such as IL1β and TNFα42, treatment with MMP1 has not been associated with aggravating inflammation43,44. Collagen-I degradation by MMP1 has shown to induce hepatocyte-ECM interaction leading to improved hepatocyte proliferation44. In turn, hepatocyte proliferation can reduce inflammation by decreasing IL8 and CCL2 expression45. In our study, we did not observe an effect of MMP1 on TNFα mRNA expression however we observed that combined CVC + MMP1 treatment reduced TNFα mRNA expression by the underlying mechanisms mentioned above.

Beside inflammation, fibrosis is a hallmark of liver diseases2. Since in fibrosis, the accumulation of excessive ECM is initiated by a disbalanced expression of MMPs and their inhibitors, TIMPs, it is hypothesized that MMPs themselves could have therapeutic effects28,29,30. CCR2 also contributes to fibrosis indirectly via infiltration of inflammatory monocytes and by activation of quiescent HSC18. Therefore, combining CVC and MMP1 would be expected to have synergistic anti-fibrotic effects as clearly evidenced in our study. To confirm that the effect observed in fibrosis is partly due to collagen-degradation by MMP1, we evaluated collagen degradation using CHP, a collagen hybridizing peptide and found higher collagen degradation in MMP1 treatment groups suggesting that MMP1-degraded collagen-I resulting in reduced collagen staining in MMP1 and CVC + MMP1 treatment groups. While the combined effect is more clearly evident also in mRNA expression of collagen-I and α-SMA which is attributed to both direct and/or indirect and/or combined effects of MMP1 and CVC.

Liver function tests further support our histology and PCR results. We observed that CVC + MMP1 combined treatment highly significantly improved liver function especially when compared with CVC-alone treatment as also reported earlier14,19,46. Krenkel et al., argued that CVC attenuates pro-inflammatory and pro-fibrotic pathways, but does not influence restorative pathways46. Degradation of fibrous ECM induces a regenerative process, therefore MMP1 is associated with improved liver regeneration and liver function28,43. Finally, CVC-treatment significantly reduced body weights while this reduction in body weights were blunted in the CVC + MMP1 treatment group suggesting that MMP1 overcomes the adverse effects of CVC indicating that higher doses of CVC could be used when combined with MMP1 for improved therapeutic efficacy without compromising safe and tolerability of the treatment.

One of the limitations of our study is the acute livery injury mouse model that we acknowledge does not reflect the clinical situation. Patients normally present to the clinic when liver damage progresses to chronic-to-advanced liver disease. More studies are hence warranted in different (etiological) and more advanced liver disease models to examine the therapeutic potential of CVC + MMP1-dual therapy.

In conclusion, in this study, we have successfully analyzed the therapeutic potential of CVC + MMP1 dual therapy in acute liver injury. We have shown that combining MMP1 with CVC, we could regress liver inflammation and fibrogenesis in vivo with improved liver function and reduced adverse effects. This study warrants the further exploration of CVC in combination with drugs that target fibrosis by inhibiting HSCs activation and/or promoting ECM degradation for future clinical applications.