Abstract

   The accumulation of extracellular matrix (ECM) proteins in the liver
   leads to liver fibrosis and end-stage liver cirrhosis. C-C motif
   chemokine receptor 2 (CCR2) is an attractive target for treating liver
   fibrosis. However, limited investigations have been conducted to
   explore the mechanism by which CCR2 inhibition reduces ECM accumulation
   and liver fibrosis, which is the focus of this study. Liver injury and
   liver fibrosis were induced by carbon tetrachloride (CCl[4]) in
   wild-type mice and Ccr2 knockout (Ccr2^-/-) mice. CCR2 was upregulated
   in murine and human fibrotic livers. Pharmacological CCR2 inhibition
   with cenicriviroc (CVC) reduced ECM accumulation and liver fibrosis in
   prevention and treatment administration. In single-cell RNA sequencing
   (scRNA-seq), CVC was demonstrated to alleviate liver fibrosis by
   restoring the macrophage and neutrophil landscape. CVC administration
   and CCR2 deletion can also inhibit the hepatic accumulation of
   inflammatory FSCN1^+ macrophages and HERC6^+ neutrophils. Pathway
   analysis indicated that the STAT1, NFκB, and ERK signaling pathways
   might be involved in the antifibrotic effects of CVC. Consistently,
   Ccr2 knockout decreased phosphorylated STAT1, NFκB, and ERK in the
   liver. In vitro, CVC could transcriptionally suppress crucial
   profibrotic genes (Xaf1, Slfn4, Slfn8, Ifi213, and Il1β) in macrophages
   by inactivating the STAT1/NFκB/ERK signaling pathways. In conclusion,
   this study depicts a novel mechanism by which CVC alleviates ECM
   accumulation in liver fibrosis by restoring the immune cell landscape.
   CVC can inhibit profibrotic gene transcription via inactivating the
   CCR2-STAT1/NFκB/ERK signaling pathways.

   Keywords: single-cell RNA sequencing, macrophage, neutrophil, STAT1,
   NFκB, ERK

Introduction

   In the context of chronic liver injuries, the accumulation of
   extracellular matrix (ECM) proteins in the liver leads to liver
   fibrosis and its end-stage liver cirrhosis.[41]^1 Liver cirrhosis is
   currently the 11^th leading cause of death worldwide due to its poor
   prognosis.[42]^2^-[43]^4 Although many investigations have been
   conducted to clarify the mechanism and treatment of liver cirrhosis, no
   drugs with satisfactory anti-fibrosis effects have been approved.[44]^5
   Immune cells have been demonstrated to be crucially involved in
   proinflammatory signaling pathways and ECM accumulation in liver
   fibrosis.[45]^6^-[46]^11 In the injured liver, liver sinusoidal
   endothelial cells (LSECs) rapidly respond to injuries by recruiting
   other immune cells to the liver, eventually constructing a
   proinflammatory and profibrotic ECM deposition
   microenvironment.[47]^8^, [48]^11^-[49]^13 Of these recruited immune
   cells, macrophages and neutrophils are the predominant cells that are
   distributed in the fibrotic septa and promote liver fibrosis by
   secreting proinflammatory mediators.[50]^9^-[51]^12 Thus, therapies
   that target blocking immune cell infiltration have served as a new
   strategy for the clinical treatment of liver cirrhosis. However,
   limited therapeutic targets have been explored.

   Monocyte-derived macrophages (MDMs), the major immune cell responses to
   liver injury, can influence ECM accumulation in liver fibrogenesis and
   its regression[52]^14^-[53]^17. C-C motif chemokine receptor 2 (CCR2)
   is a classic G-protein-coupled transmembrane receptor distributed on
   immune cells, especially on MDMs.[54]^18 When binding to its ligand C-C
   motif chemokine ligand 2 (CCL2), CCR2 controls monocyte migration from
   the bone marrow into the liver and initiates the development of many
   liver diseases.[55]^19^, [56]^20 We have previously verified that
   CCR2^+ monocytes extensively accumulate in the fibrotic liver, and
   suppressing CCR2^+ monocyte infiltration by blocking CCL2 could
   effectively ameliorate ECM accumulation and liver fibrosis.[57]^12
   Consistently, CCR2^+ MDMs have been verified to participate in liver
   fibrosis and angiogenesis.[58]^12^, [59]^21^-[60]^23 Thus, CCR2 is
   supposed to be an attractive target for the clinical treatment of liver
   cirrhosis.

   Cenicriviroc (CVC) is an oral, dual antagonist of CCR2/CCR5 that was
   first developed to treat human immunodeficiency virus (HIV)
   infection.[61]^24^, [62]^25 Experimentally, CVC reduces hepatic
   steatosis and fibrosis by inhibiting the recruitment of Ly-6C^high
   monocytes and macrophages in murine nonalcoholic steatohepatitis (NASH)
   and alcohol-related liver disease (ALD).[63]^15^, [64]^25^, [65]^26 In
   a randomized, placebo-controlled trial and a phase IIb clinical trial,
   CVC has been successfully used to treat NASH and NASH-associated liver
   fibrosis.[66]^27^, [67]^28 However, CVC showed no significant
   improvement in liver fibrosis in a recent phase III clinical trial,
   which questioned the beneficial effects of CVC on human
   fibrosis.[68]^29 These controversial clinical and experimental data
   inspire us to explore the in-depth cellular and molecular mechanisms of
   CVC in liver fibrosis.

   CVC was previously reported to attenuate liver fibrosis by inhibiting
   hepatic immune cell infiltration and angiogenesis.[69]^23^, [70]^25
   Unlike previous reports in which CVC could attenuate liver fibrosis in
   NASH and ALD models, this study aimed to explore the antifibrotic role
   and mechanism of CVC in carbon tetrachloride (CCl[4])-induced murine
   liver fibrosis. Indeed, both prevention and treatment administration of
   CVC can attenuate ECM accumulation in liver fibrosis. Single-cell RNA
   sequencing (scRNA-seq) and bulk RNA sequencing indicate that CVC
   reduces liver fibrosis by inhibiting hepatic accumulation of
   inflammatory fascin actin-bundling protein 1 (FSCN1)^+ macrophages and
   HECT and RLD domain containing E3 ubiquitin protein ligase family
   member 6 (HERC6)^+ neutrophils. CVC exerts antifibrotic and
   anti-inflammatory effects by transcriptionally inhibiting profibrotic
   genes (Xaf1, Slfn4, Slfn8, Ifi213, and Il1β) through inactivating the
   signal transducer and activator of transcription 1 (STAT1)/nuclear
   factor-κB (NFκB)/extracellular regulated protein kinase (ERK) signaling
   pathways.

Materials and Methods

Mice and induction of liver fibrosis

   All animal experiments were approved by the Animal Care and Use
   Committee of West China Hospital, Sichuan University. Wild-type C57BL/6
   mice were purchased from GemPharmatech (Nanjing, China). Ccr2 knockout
   (Ccr2^-/-) mice were a gift from Jing Wang (Shanghai Jiaotong
   University). Liver fibrosis was induced by CCl[4] (1 μL/g body weight,
   Sigma‒Aldrich #319961) intraperitoneally (i.p.) injection. Mice in the
   control group (olive oil group) were i.p. injected with olive oil for 6
   weeks. For the murine preventive model, wild-type mice were i.p.
   administered 4 dosages of CVC (15 mg/kg body weight, Selleck #S8512)
   and 2 dosages of CCl[4] per week for 6 consecutive weeks. For the
   murine treatment model, mice were i.p. injected with CCl[4] for 3 weeks
   to induce liver fibrosis, and then 4 dosages of CVC and 2 dosages of
   CCl[4] per week were administered for another 3 weeks. For the acute
   liver injury experiment in Ccr2^-/- mice, 2 doses of CCl[4] were i.p.
   injected. All mice were sacrificed 48 hours by an overdose of
   pentobarbital sodium after the final injection, and livers were taken
   for subsequent experiments.

Human liver tissues

   Seven normal control and 11 cirrhotic livers were collected during
   hepatectomy at West China Hospital. All research was conducted
   following both the Declaration of Helsinki and Istanbul. The study was
   approved by the Ethical Committee of West China Hospital and registered
   in the Chinese Clinical Trial Registry (ChiCTR2200063108). Written
   informed consent forms were received from all patients. The detailed
   clinical information is listed in Supporting [71]Table S1.

Single-cell RNA sequencing (scRNA-seq) and bioinformatics analysis

   scRNA-seq and bioinformatics analysis were performed by Shanghai OE
   Biotech Inc. (Shanghai, China). Three murine livers from 3 groups of
   the murine preventive model were mixed and assigned to scRNA. The raw
   scRNA-seq data have been deposited in the NCBI Gene Expression Omnibus
   ([72]GSE218496). The detailed procedure is listed in the Supporting
   Materials.

Statistical analysis

   All data are presented as the mean ± standard deviation and were
   analyzed using GraphPad software (version 8, GraphPad Software Inc., La
   Jolla, CA, USA). One-way ANOVA for multiple-group comparisons followed
   by Bonferroni's post hoc test and t-test were used to analyze the data.
   A p-value <0.05 was considered statistically significant.

   The Supporting Materials include scRNA-seq, RNA sequencing,
   bioinformatic analysis of the public GEO dataset ([73]GSE136103 and
   [74]GSE157088),[75]^30^, [76]^31 hematoxylin and eosin (H&E) staining,
   Sirius red staining, immunohistochemistry (IHC), immunofluorescence
   (IF), Western blot (WB), cell culture and treatments, quantitative
   RT‒PCR (qPCR), primary murine hepatic stellate cell (HSC) isolation,
   primary murine HSC and macrophage Raw264.7 co-culture, and flow
   cytometry (FCM).

Results

Reduction of liver fibrosis by preventive pharmacological CCR2 inhibition
with CVC

   RNA sequencing was performed in control and CCl[4]-induced fibrotic
   murine livers (Supporting [77]Figure S1A). The top DEGs and KEGG
   pathway analysis indicated that Ccr2 and the PI3K-Akt, JAK-STAT, and
   NFκB signaling pathways were significantly activated in fibrotic livers
   (Supporting [78]Figure S2A-B). The upregulation of CCR2 was also
   confirmed in murine and human fibrotic livers by WB (Supporting
   [79]Figure S2C-D).

   As CCR2 is upregulated in fibrotic livers, CVC preventive treatment was
   applied to explore the antifibrotic effects of pharmacological CCR2
   inhibition. CVC significantly inhibited the CCR2^+ immune cells and
   CCR2 protein levels compared to the CCl[4] group, as determined by IF
   and WB (Figure [80]1A-Β). The increased number of CD45^+/CCR2^+ immune
   cells in the peripheral blood induced by CCl[4] was also significantly
   abrogated by CVC (Figure [81]1C). Consistently, liver fibrosis and
   hepatic stellate cell (HSC) activation induced by CCl[4] were also
   significantly attenuated when preventively treated with CVC, as
   quantified by decreased fibrotic areas and protein levels of collagen I
   and αSMA (Figure [82]1A-B). Meanwhile, the H&E staining of tissues
   taken from the three groups showed that CVC did not cause damage to
   other organs, including the heart, kidney, lung, stomach, intestine,
   and spleen (Supporting [83]Figure S3).

Figure 1.

   [84]Figure 1
   [85]Open in a new tab

   Reduction of liver fibrosis by preventive pharmacological CCR2
   inhibition with CVC. (A-C) In the prevention experiment, wild-type mice
   were administered 2 dosages of CCl[4] in addition to either 4 dosages
   of saline (CCl[4] group) or CVC (CVC group) per week for 6 weeks. Mice
   in the olive oil group were i.p. injected with olive oil for 6 weeks.
   Liver fibrosis was analyzed by Sirius red staining and IF of collagen I
   and αSMA (A). The protein levels of CCR2, collagen I, and αSMA were
   determined by WB (B). The number of CCR2^+ immune cells in the liver
   and peripheral blood was quantified by IF and FCM (A, C), n=6/group.
   (D-E) RNA sequencing was performed in murine livers from the CCl[4] and
   CVC groups. A heatmap of the top 30 DEGs in the two groups (D) and KEGG
   pathway analysis of the targeted DEGs (E) are shown. n=3/group.
   **p<0.01, ***p<0.001, ****p<0.0001.

   After that, RNA sequencing was applied in the CCl[4] group and CCl[4]
   plus CVC group (CVC group) to explore the disturbed DEGs and signaling
   pathways with CVC preventive treatment. In total, 757 downregulated and
   447 upregulated DEGs were displayed in the CVC group compared to the
   CCl[4] group (Supporting [86]Figure S1B). Compared to the CCl[4] group,
   proinflammatory and profibrotic genes (Ccr2, Tlr4, Il18, Ly6c1, Herc6,
   Gbp2, Slfn8, Xaf1, Ifi213) were significantly decreased in the CVC
   group (Figure [87]1D). Furthermore, the KEGG signaling pathway analysis
   of the 757 downregulated DEGs also indicated that associated
   profibrotic signaling pathways, including the cytokine‒cytokine
   receptor interaction, PI3K-Akt, JAK-STAT, and NFκB signaling pathways,
   were inhibited (Figure [88]1E). In summary, CVC attenuates liver
   fibrosis by inhibiting CCR2 and CCR2-associated proinflammatory and
   profibrotic signaling pathways.

Attenuation of liver fibrosis by therapeutic pharmacological CCR2 inhibition
with CVC

   To further verify the effects of CVC on existing liver fibrosis, CVC
   treatment was administered 3 weeks after CCl[4] injection (Figure
   [89]2A). The enhanced protein levels of CCR2 and CCR2^+ immune cells in
   the CCl[4] group were significantly decreased after treatment with CVC
   (Figure [90]2A-C). Similar to preventive administration, the ECM
   accumulation and HSC activation induced by CCl[4] injection, as
   quantified by increased fibrotic areas and protein levels of collagen I
   and αSMA, were also significantly ameliorated by 3 weeks of CVC
   treatment (Figure [91]2A-B).

Figure 2.

   [92]Figure 2
   [93]Open in a new tab

   Attenuation of liver fibrosis by therapeutic pharmacological CCR2
   inhibition with CVC. (A-C) For the treatment experiment, wild-type mice
   were i.p. injected with CCl[4] for 3 weeks, in addition to either 4
   dosages of saline (CCl[4] group) or CVC (CVC group) per week for
   another 3 weeks. Mice in the olive oil group were i.p. injected with
   olive oil for 6 weeks. Liver fibrosis was analyzed by Sirius red
   staining and IF of collagen I and αSMA (A). The protein levels of CCR2,
   collagen I, and αSMA were determined by WB (B). The number of CCR2^+
   immune cells in the liver and peripheral blood was quantified by IF and
   FCM (A, C), n=6/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Restoration of the immune cell landscape by CVC in murine fibrotic liver

   Immune cells play a central role in regulating proinflammatory
   signaling pathways and liver fibrosis.[94]^7^-[95]^11 The
   abovementioned results identified that the proinflammatory and
   profibrotic signaling pathways participate in the antifibrotic effects
   of CVC. Next, scRNA-seq was applied to nonparenchymal cells (NPCs) to
   clarify how CVC preventive treatment ameliorates liver fibrosis at the
   single-cell level (Figure [96]3A). In total, 22,672 NPCs from murine
   livers of the 3 groups were assigned to 14 clusters according to the
   reference marker genes (Figure [97]3B, Supporting [98]Figure S4). NPCs
   in the 3 groups showed a differential distribution of cell clusters,
   including T cells, NK cells, B cells, neutrophils, and macrophages
   (Figure [99]3C-D). Cell-to-cell communication analysis was then
   performed to reveal the interactions among 14 cell clusters. NKT cells
   (Ccr5/Ccl4), B cells (Cd74/Mif), neutrophils (Ccr1/Ccl2), and
   macrophages (Cd74/Mif) mainly interacted with HSCs (Figure [100]3E-F).
   Thus, NKT cells, B cells, neutrophils, and macrophages were further
   divided into subclusters according to the reference marker genes.

Figure 3.

   [101]Figure 3
   [102]Open in a new tab

   Restoration of the immune cell landscape by CVC in murine fibrotic
   liver. Murine livers from the prevention experiment were subjected to
   scRNA-seq to determine the immune cell landscape. (A) The flow of
   scRNA-seq analysis. (B-D) The relative abundance of 14 cell clusters in
   the liver (B), the differential distribution of NPCs in the three
   groups (C), and the changes in the abundance of NPCs in the three
   individual groups (D) are shown by UMAP plots. (E-F) The interactions
   of 14 cell clusters in the liver (E) and the number of interacting
   ligand-receptor pairs of 14 cell types (F) are shown. (G-H) The ratio
   changes of 6 macrophage clusters in three groups (G) and pseudotime
   analysis for 3 pseudotime states of macrophages (H) are displayed.
   (I-J) The ratio changes of 4 neutrophil clusters in three groups (I)
   and 5 pseudotime states of neutrophils (J) are shown.

   Of the 6 clusters of macrophages, increased ratios of clusters 1 and 6
   and decreased ratios of clusters 2-5 were observed in the CCl[4] group
   compared to the olive oil group (Figure [103]3G). However, the
   increased ratio of proinflammatory cluster 6 (Fscn1) and decreased
   ratio of anti-inflammatory cluster 2 (Xcr1) were abolished by CVC
   treatment (Figure [104]3G, Supporting [105]Figure S5A-B). Additionally,
   the populations of macrophages in diverse differentiation states were
   obtained through pseudotime analysis (Figure [106]3H). An enhanced
   percentage of pseudotime state 1 and decreased percentages of
   pseudotime states 2 and 3 were observed in the CCl[4] group compared to
   the olive oil group. However, CVC treatment did not restore these
   changes (Figure [107]3H).

   For the 4 clusters of neutrophils, the decreased ratio of cluster 1
   (Itga4) and increased ratio of cluster 4 (Herc6) induced by CCl[4]
   injection were abrogated by CVC treatment (Figure [108]3I, Supporting
   [109]Figure S6A-B). Similarly, the increased percentage of pseudotime
   state 4 induced by CCl[4] injection was reversed by CVC treatment
   (Figure [110]3J). Although some clusters of B cells and T cells were
   altered between the olive oil group and the CCl[4] group, there were no
   significant changes in the 6 clusters of T cells and 10 clusters of B
   cells between the CCl[4] group and CVC group (Supporting [111]Figure
   S7A-D). The increased ratio of macrophages and neutrophils was also
   validated in the human cirrhotic GEO scRNA-seq dataset [112]GSE136103
   (Supporting [113]Figure S8A). Consistently, FSCN1 and HERC6 were
   confirmed as cell markers for macrophages and neutrophils, respectively
   (Supporting [114]Figure S8B-C). In summary, CVC restores immune cell
   homeostasis in the murine fibrotic liver by regulating the neutrophil
   and macrophage landscape. Thus, neutrophils and macrophages were
   selected as the target cells for further analysis.

Inhibition of inflammatory macrophage and neutrophil accumulation by CVC

   The distributions of macrophages and neutrophils were determined to
   investigate the disturbed homeostasis of macrophages and neutrophils.
   In the murine model of CVC prevention, more extensive inflammatory cell
   infiltration, as determined by H&E staining, was observed in the CCl[4]
   group than in the olive oil group (Figure [115]4A). Consistently,
   enhanced F4/80^+ macrophages and myeloperoxidase (MPO)^+ neutrophils
   were observed in fibrotic areas of the CCl[4] group compared to the
   olive oil group (Figure [116]4A). FSCN1 and HERC6 are new cell markers
   for murine proinflammatory macrophage cluster 6 and neutrophil cluster
   4 as determined by scRNA-seq (Supporting [117]Figures S5-S6) and human
   macrophages and neutrophils (Supporting [118]Figures S8B-C). CCl[4]
   administration insult increased FSCN1^+ inflammatory macrophages and
   HERC6^+ inflammatory neutrophils compared with the olive oil group.
   Consistently, the protein levels of F4/80, FSCN1, MPO, and HERC6 were
   also significantly enhanced in the CCl[4] group compared to the olive
   oil group (Figure [119]4B). However, the abovementioned increases in
   inflammatory macrophages (F4/80^+, FSCN1^+) and inflammatory
   neutrophils (MPO^+, HERC6^+) were remarkably abolished by preventive
   CVC administration (Figure [120]4A-B). Similar results were obtained in
   the CVC treatment murine model. The enhanced hepatic inflammatory
   macrophage and inflammatory neutrophil accumulation determined by H&E,
   IF/IHC and WB of F4/80, FSCN1, MPO, and HERC6 in the CCl[4] group was
   also significantly abrogated by CVC treatment (Figure [121]4C-D). In
   summary, CVC inhibits inflammatory macrophage and neutrophil
   accumulation in prevention and treatment murine models.

Figure 4.

   [122]Figure 4
   [123]Open in a new tab

   Inhibition of inflammatory macrophage and neutrophil accumulation by
   CVC. (A-B) Inflammatory infiltration was determined in the murine
   livers of the prevention experiment. H&E staining, IHC of macrophage
   markers (F4/80, FSCN1), and neutrophil markers (MPO, HERC6, A), and WB
   for these markers (B) were performed. (C-D) Inflammatory infiltration
   was determined in the murine livers of the treatment experiment. H&E
   staining, IHC of macrophage markers (F4/80, FSCN1), neutrophil markers
   (MPO, HERC6, C), and WB for these markers (D) were performed.
   n=6/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Attenuation of macrophage and neutrophil recruitment by CCR2 knockout
following liver injury

   In liver fibrosis, macrophage and neutrophil recruitment occur early
   following liver injury.[124]^18 Therefore, an acute liver injury mouse
   model was established in Ccr2^-/- mice to confirm the protective role
   of CCR2 blockade on macrophage and neutrophil recruitment. CCR2
   deletion was confirmed by IF and WB in both olive oil- and
   CCl[4]-treated mice (Figure [125]5A-B). The enhanced protein levels of
   CCR2 and CCR2^+ immune cells in CCl[4]-treated Ccr2^+/+ mice were
   significantly attenuated after Ccr2 knockout. (Figure [126]5A-B). As
   shown by H&E staining, acute liver injury insult led to immune cells
   gathering around the portal and central vein areas compared to the
   olive oil group (Figure [127]5A). These accumulated immune cells in
   vascular areas comprised F4/80^+ macrophages, FSCN1^+ inflammatory
   macrophages, MPO^+ neutrophils, and HERC6^+ inflammatory neutrophils
   (Figure [128]5A). The distribution of these inflammatory macrophages
   and neutrophils in acute liver injury paralleled the fibrotic septa in
   liver fibrosis. The accumulation of inflammatory macrophages and
   neutrophils in the CCl[4] group compared to the olive oil group was
   also confirmed by increased protein levels of F4/80, FSCN1, MPO, and
   HERC6 (Figure [129]5B). However, the increased inflammatory macrophages
   and neutrophils following acute liver injury were significantly
   restored by Ccr2 knockout (Figure [130]5A-B). However, comparable
   levels of the abovementioned immune cells were observed in Ccr2^+/+ and
   Ccr2^-/- mice treated with olive oil (Figure [131]5A-B). In summary,
   CCR2 inhibition attenuates inflammatory macrophage and neutrophil
   recruitment following liver injury.

Figure 5.

   [132]Figure 5
   [133]Open in a new tab

   Attenuation of macrophage and neutrophil recruitment by CCR2 knockout
   following liver injury. (A-B) Liver injuries were induced by 2 doses of
   CCl[4] in Ccr2^+/+ and Ccr2^-/- mice, and mice injected with olive oil
   were used as controls. H&E staining, IHC and IF of macrophage markers
   (F4/80, FSCN1), neutrophil markers (MPO, HERC6), and immune cell
   markers (CCR2, A), and WB for these markers (B) were quantified.
   n=6/group, *p<0.05, **p<0.01, ****p<0.0001.

The potential mechanism by which CVC restores the immune cell landscape

   To further explore the potential mechanism by which CVC improves
   hepatic inflammatory macrophage and neutrophil accumulation, DEGs, GO
   enrichment, and KEGG pathway analysis of scRNA-seq were performed. In
   macrophages, 222 DEGs were found in the CVC group compared to the
   CCl[4]group, with the represented downregulated DEGs being Fscn1,
   Stat1, Xaf1, Slfn4, Slfn8, Ifi213, and Il1β (Figure [134]6A). Moreover,
   the JAK-STAT, NOD-like receptor, TNF, and MAPK signaling pathways were
   significantly enriched in the KEGG pathway analysis (Figure [135]6B).
   The GO enrichment also showed that the cellular response to
   lipopolysaccharide (LPS) and interferon γ, the immune system process,
   and DNA-binding transcription factors were crucial in the functions of
   CVC in restoring the macrophage landscape (Supporting [136]Figure S9A).
   To understand the deep molecular mechanisms, the gene regulatory
   networks between transcription factors and DEGs were analyzed with
   SCENIC software. As shown in the heatmap of the regulon activity score
   (RAS) for individual macrophages and the integrated three groups, and
   regulon specificity score (RSS) ranking plots, the activities of
   transcription factors in the JAK-STAT pathway (Stat1, Stat2), TNF-NFκB
   pathway (Irf1, Irf7), and MAPK-ERK pathway (Gata4, Ets2) were increased
   in the CCl[4] group compared with the olive oil group (Figure [137]6C,
   Supporting [138]Fig. S10A-C). However, increased activities of these
   transcription factors were partly abrogated by CVC treatment, with
   STAT1 being the most remarkable (Figure [139]6C, Supporting [140]Figure
   S10A-C). Presumably, CVC may improve disturbed macrophage homeostasis
   by regulating Xaf1, Slfn4, Slfn8, Ifi213, and Il1β via the JAK-STAT1,
   TNF-NFκB, and MAPK signaling pathways. The increased IL1β in
   macrophages of the human cirrhotic liver ([141]GSE136103) and enhanced
   Xaf1, Ifi213, Il1β in macrophages of murine fibrotic livers
   ([142]GSE157088) were also validated by bioinformatic analysis
   (Supporting [143]Figure S8D-E).

Figure 6.

   [144]Figure 6
   [145]Open in a new tab

   The potential mechanism by which CVC restores the immune cell
   landscape. Murine livers from the prevention experiment were subjected
   to scRNA-seq to determine the immune cell landscape. (A-C) A heatmap of
   the top 20 downregulated and upregulated DEGs in macrophages of the
   CCl[4] and CVC groups (A), KEGG pathway enrichment analysis for all
   DEGs (B), and a heatmap of RSS in macrophages of the three groups (C)
   were determined. (D-F) A heatmap of the top 20 downregulated and
   upregulated DEGs in neutrophils of the CCl[4] and CVC groups (D), KEGG
   pathway enrichment analysis for all DEGs (E), and a heatmap of RSS in
   neutrophils of the three groups (F) were displayed. (G) The heatmap of
   RAS for the whole liver in the integrated three groups was shown. (H)
   The transcription factor network analysis in the integrated three
   groups was revealed.

   Similar to macrophages, CVC can also abrogate disturbed neutrophil
   homeostasis via DEGs and related signaling pathways. Compared to the
   CCl[4] group, 156 DEGs were found in the CVC group, with the
   represented downregulated DEGs being Gbp2, Gbp5, Gbp7, and Herc6
   (Figure [146]6D). In the KEGG pathway analysis, the tumor necrosis
   factor (TNFα), cytosolic DNA sensing, NOD-like receptor, NFκB, and
   cellular senescence signaling pathways were significantly enriched
   (Figure [147]6E). Meanwhile, the GO enrichment indicated that antigen
   processing and presentation, cellular response to interferon γ, MHC I
   protein complex, and TAP binding process were involved in CVC restoring
   the neutrophil landscape (Supporting [148]Figure S9B). Gene regulatory
   network analysis was then performed. The enhanced activities of
   transcription factors in the JAK-STAT pathway (Stat1, Stat2, Stat3) and
   TNF-NFκB pathway (Irf1, Irf2, Irf7) induced by CCl[4] were restored by
   CVC treatment (Figure [149]6F, Supporting [150]Figure S10D-F).
   Similarly, the gene regulatory network analysis in the whole liver also
   indicated that these pathways induced by CCl[4] were partly abrogated
   by CVC treatment (Figure [151]6G, Supporting [152]Figure S10G-I).
   Moreover, general transcription factor network analysis also indicated
   that STAT1, NFκB1, ETS1, and FOS can regulate groups of proinflammatory
   genes (Figure [153]6H).

   In summary, CVC may ameliorate inflammatory macrophage and neutrophil
   accumulation and liver fibrosis via the JAK-STAT1, TNF-NFκB, and MAPK
   signaling pathways.

Suppression of STAT1/NFκB/ERK signaling pathway by CVC treatment in murine
liver and macrophages

   The profibrotic signaling pathways, including the JAK-STAT, TNF-NFκB,
   and MAPK-ERK signaling pathways, were inhibited by CVC in both
   scRNA-seq and bulk RNA sequencing. Therefore, we examined the
   interconnections between CCR2 and these signaling pathways in the
   murine liver. Compared to those in the olive oil group, the protein
   levels of phosphorylated STAT1 (p-STAT1), phosphorylated NFκB-p65
   (p-NFκB), and phosphorylated ERK (p-ERK) were significantly increased
   in the CCl[4]-induced liver injury group (Figure [154]7A). However, the
   increased p-STAT1, p-NFκB, and p-ERK levels in injured livers were
   remarkably inhibited by Ccr2 knockout (Figure [155]7A).

Figure 7.

   [156]Figure 7
   [157]Open in a new tab

   Suppression of STAT1/NFκB/ERK signaling pathway by CVC treatment in
   murine liver and macrophages. (A) Ccr2^+/+ or Ccr2^-/- mice were
   injected with 2 doses of CCl[4] or olive oil. Protein levels of STAT1,
   p-STAT1, NFκB, p-NFκB, ERK, and p-ERK were analyzed by WB. N=6/group.
   (B) Raw264.7 murine macrophages were treated with DSMO, CVC, the STAT1
   inhibitor nifuroxazide, the NFκB inhibitor celastrol, and the MEK
   inhibitor AZD6244 for 2 hours followed by stimulation with LPS for
   another 6 hours. WB was applied to determine the protein levels of
   STAT1, p-STAT1, NFκB, p-NFκB, ERK, and p-ERK (B). n=3/group. *p<0.05,
   **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

   To explore the molecular interconnective effects of CVC on these
   signaling pathways, the Raw264.7 murine macrophage lineage was applied
   in vitro. LPS is a typical stimulator of inflammatory macrophages.
   Compared with vehicle treatment, LPS stimulation significantly
   increased the protein levels of p-STAT1, p-NFκB, and p-ERK (Figure
   [158]7B). The enhanced protein levels of p-STAT1, p-NFκB, and p-ERK
   were remarkably inhibited in CVC-treated cells (Figure [159]7B).
   Furthermore, treatment with the STAT1 inhibitor nifuroxazide, NFκB
   inhibitor celastrol, and MEK inhibitor AZD6244 also reduced the protein
   levels of p-STAT1, p-NFκB, and p-ERK, respectively (Figure [160]7B).
   These results suggest that the blockade of CCR2 may attenuate
   inflammatory macrophage and neutrophil accumulation and liver fibrosis
   by inactivating the JAK-STAT, TNF-NFκB, and MAPK-ERK signaling
   pathways.

   In scRNA-seq of macrophages, Xaf1, Slfn4, Slfn8, Ifi213, and Il1β were
   suppressed by CVC. We next verified whether CVC could inhibit the
   expression of these genes via the JAK-STAT1, TNF-NFκB, and MAPK-ERK
   signaling pathways in Raw264.7 murine macrophages. As expected, the
   mRNA levels of Xaf1, Slfn4, Slfn8, Ifi213, and Il1β in macrophages were
   significantly elevated under LPS stimulation compared to
   vehicle-treated cells (Figure [161]8A). However, these elevated genes
   were remarkably downregulated by CVC treatment (Figure [162]8A).
   Additionally, the enhanced mRNA levels of Xaf1, Slfn4, Slfn8, Ifi213,
   and Il1β induced by LPS were also inhibited by the STAT1 inhibitor
   nifuroxazide, NFκB inhibitor celastrol, and MEK inhibitor AZD6244
   (Figure [163]8A). Consistently, the increased protein levels of XAF1
   and SLFN8 induced by LPS were also inhibited by the STAT1, NFκB and MEK
   inhibitors (Figure [164]8B).

Figure 8.

   [165]Figure 8
   [166]Open in a new tab

   (A-B) The mRNA levels of Xaf1, Slfn4, Slfn8, Ifi213, and Il1β were
   analyzed by qPCR (A). The protein levels of XAF1 and SLFN8 were
   quantified by WB (B). (C) Raw264.7 murine macrophages were treated with
   LPS followed by co-cultured with primary murine HSCs for 48 hours. HSC
   activation was detected by IF of αSMA and Collagen I. n=3/group,
   *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

   A transwell assay was then performed to validate the antifibrotic
   effect of macrophage CCR2 inhibition with CVC on HSC activation.
   Raw264.7 murine macrophages were plated into the top well with
   corresponding treatments, and primary murine HSCs were plated into the
   bottom of the transwell apparatus. LPS-treated macrophages increased
   HSC activation, as determined by IF of αSMA and Collagen I in HSCs.
   However, this HSC activation was abrogated when CVC was added to
   macrophages (Figure [167]8C). In summary, CVC inhibits liver fibrosis
   by repressing gene transcription via the CCR2-STAT1/NFκB/ERK signaling
   pathway.

Insufficient CVC in regulating intracellular signaling in human fibroblasts
and macrophages

   The use of CVC in mouse models appears controversial with recent
   clinical data. Thus, we validated the effects of CVC on intracellular
   signaling in human macrophage (THP-1) and HSC (LX2) cell lines. In
   human THP-1 macrophages, LPS stimulation significantly increased the
   protein levels of p-STAT1, p-NFκB, and p-ERK (Supporting [168]Figure
   S11A). However, only p-NFκB was significantly suppressed by CVC, while
   p-ERK was even increased by CVC compared with LPS-treated THP-1 cells
   (Supporting [169]Figure S11A). Furthermore, no significant changes in
   the protein levels of p-STAT1, p-NFκB, and p-ERK were observed among
   the vehicle, LPS, and LPS+CVC groups in LX2 human HSC cell lines
   (Supporting [170]Figure S12A). In addition, the increased IL1β induced
   by LPS was not abrogated by CVC in either THP-1 or LX2 cells
   (Supporting [171]Figure S11B, Supporting [172]Figure S12B). In summary,
   the controversial results of CVC in mouse and human clinical data might
   be attributed to insufficient regulation of intracellular signaling in
   human fibroblasts and macrophages.

Discussion

   Chronic inflammation induced by immune cells activates HSCs, leads to
   ECM deposition, reconstructs hepatic lobules, and eventually promotes
   liver fibrosis.[173]^5^, [174]^13^, [175]^32^, [176]^33 Although many
   clinical practices and basic research have been performed, effective
   antifibrotic drugs remain deficient. The pharmacological inhibition of
   CCR2 with CVC has recently been used for the clinical treatment of
   NASH.[177]^25 However, the beneficial effects of CVC on human fibrosis
   were questioned in a recent phase III clinical trial.[178]^29
   Investigations that explore the cellular and molecular mechanisms of
   CVC in liver fibrosis may help to explain the controversial clinical
   and experimental data. Unlike previously reported antifibrotic effects
   of CVC in NASH and ALD, we discovered a new role for CVC in
   ameliorating CCl[4]-induced liver fibrosis by restoring the
   proinflammatory microenvironment at the single-cell level. We
   demonstrated a new cellular mechanism by which CVC inhibits ECM
   accumulation in liver fibrosis by suppressing the hepatic accumulation
   of inflammatory FSCN1^+ macrophages and HERC6^+ neutrophils.
   Furthermore, we verified a novel molecular mechanism by which CVC
   reduces profibrotic gene transcription by inactivating the
   STAT1/NFκB/ERK signaling pathways (Figure [179]9). Collectively, this
   study clarified the new role and novel mechanism of CVC in attenuating
   liver fibrosis by restoring the proinflammatory microenvironment.

Figure 9.

   [180]Figure 9
   [181]Open in a new tab

   Proposed mechanisms. CVC alleviates liver fibrosis by inhibiting
   hepatic infiltration of FSCN1^+ macrophages and HERC6^+ neutrophils.
   CVC can transcriptionally inhibit Xaf1, Slfn4, Slfn8, Ifi213, and Il1β
   by inactivating the STAT1, NFκB, and ERK signaling pathways.

   CCR2 is a marker gene depicting immunocyte infiltration and fibrotic
   severity in preclinical research.[182]^19^, [183]^20^, [184]^34
   Activated CCR2 causes M1-polarized macrophage recruitment and
   ECM-producing HSC activation, ultimately leading to liver
   fibrosis.[185]^12^, [186]^21^, [187]^35 Consistently, we verified the
   upregulation of CCR2 in murine and human fibrotic livers. Recent
   studies have demonstrated that pharmacological inhibition of CCR2 by
   CVC decreases hepatic steatosis and fibrosis [188]^15^, [189]^25^,
   [190]^26 Furthermore, CCR2 inhibition by CVC can improve insulin
   resistance and steatohepatitis in NASH patients.[191]^27^, [192]^28
   Whether CVC can also improve liver fibrosis in other models remains
   unknown. We verified the antifibrotic effects of CVC by reducing ECM
   accumulation on CCl[4]-induced liver fibrosis from both prevention and
   treatment perspectives. Our results indicate that pharmacological
   inhibition of CCR2 by CVC might become a key novel strategy for liver
   cirrhosis therapy without causing other organ injuries.

   During liver injury, classical monocytes accumulate in the sinusoid and
   differentiate into CCR2^+ (Ly6C^high) macrophages, eventually
   exacerbating liver fibrosis[193]^12^, [194]^36. Consistently, our
   previous study explored whether LSEC-mediated aberrant angiocrine
   signals can recruit CCR2^+ MDM accumulation in CCl[4]-induced liver
   fibrosis.[195]^12 In addition, CCR2 is also expressed in T/NK cells and
   neutrophils, and neutrophils also lead to CCR2-mediated macrophage
   infiltration and HSC activation[196]^37^, [197]^38. Previous studies on
   CVC treatment focused on inhibiting the recruitment of Ly6C^high
   monocytes in murine NASH and ALD models.[198]^15^, [199]^25 However,
   whether other clusters of immune cells also contribute to the
   antifibrotic effects of CVC remains unclear. Using scRNA-seq, we
   identified the immune cell landscape of how CVC improves liver fibrosis
   by restoring the proinflammatory microenvironment. CVC predominantly
   inhibited the hepatic accumulation of inflammatory FSCN1^+ macrophages
   and HERC6^+ neutrophils in prevention and treatment administration.
   Moreover, Ccr2 knockout also ameliorated the abovementioned
   inflammatory macrophage and neutrophil infiltration during early liver
   injury. As CVC is a dual CCR2/CCR5 inhibitor, it is presumable that CVC
   mainly inhibits inflammatory macrophage and neutrophil accumulation via
   CCR2. Using scRNA-seq and experimental validation, we have clarified
   that the antifibrotic effect of CVC is attributed to abrogating the
   proinflammatory microenvironment.

   Once activated by the chemokine ligand CCL2, CCR2 can stimulate
   multiple downstream signaling pathways in cancers, including the
   PI3K-Akt, RAC-GTPase, and PKC signaling pathways.[200]^39 However, the
   downstream signaling pathways and profibrotic genes activated by CCR2
   in the context of liver fibrosis have not been fully explored. Using
   scRNA-seq, bulk RNA sequencing, and experimental validation in vivo and
   in vitro, we identified that the JAK-STAT1, TNF-NFκB, and MAPK-ERK
   signaling pathways contribute to the antifibrotic effects of CVC. For
   profibrotic genes activated by CCR2, CVC can reverse alcohol-related
   increases in hepatic TNFα, IL1β, IL-6, and CCL2 in an ALD murine
   model.[201]^25 In this study, we have displayed a hallmark of
   CVC-mediated profibrotic genes during liver fibrosis. Xaf1, Slfn4,
   Slfn8, Ifi213, and Il1β are the common hub profibrotic genes
   downregulated by CVC both in vivo and in vitro. CVC can inhibit the
   transcription of these profibrotic hub genes by inactivating the STAT1,
   NFκB, and ERK signaling pathways in the murine liver and macrophages.
   However, except for Il1β, which has well-known functions during liver
   fibrosis,[202]^40^, [203]^41 Xaf1, Slfn4, Slfn8, and Ifi213 are
   profibrotic genes with unknown functions. Thus, further investigations
   are urgently needed to explore how inhibition of Xaf1, Slfn4, Slfn8,
   and Ifi213 by CVC can attenuate liver fibrosis. In contrast, CVC is
   insufficient in regulating these intracellular signaling pathways in
   human fibroblasts and macrophages, which may explain the controversial
   clinical and experimental data.

   In conclusion, we clarify the cellular and molecular mechanisms of the
   antifibrotic effects of CVC by restoring the proinflammatory
   microenvironment. CVC ameliorates ECM deposits and liver fibrosis by
   suppressing the hepatic accumulation of inflammatory FSCN1^+
   macrophages and HERC6^+ neutrophils. The antifibrotic effects of CVC
   have been attributed to transcriptionally inhibiting hub profibrotic
   genes by inactivating the STAT1/NFκB/ERK signaling pathways. These
   results elucidate the antifibrotic strategy with CVC for the clinical
   treatment of liver cirrhosis.

Supplementary Material

   Supplementary methods, figures and tables.
   [204]Click here for additional data file.^ (8.5MB, pdf)

Acknowledgments