Socs1^fl/fl mice (30) were backcrossed to C57BL/6 mice obtained from Charles River Laboratories for more than ten generations. Lrat^Cre mice were obtained from Dr. C. Österreicher (University of Vienna) (29) and rederived by cesarian section into the specific pathogen-free facility in our animal colony. R26^ZsGreen reporter mice (Jax mice: 007906; B6. Cg-Gt(ROSA)26Sor tm6(CAG-ZsGreen1) Hze/J; also known as Ai6 mouse) (31) were obtained from the Jackson Laboratory. The R26^ZsGreen reporter mouse harbors a targeted mutation at the Rosa 26 locus (Gt (ROSA)26S) with a loxP-flanked STOP cassette, which prevents the transcription of a CAG promoter-driven enhanced green fluorescent protein ZsGreen1. Lrat^Cre mice were bred with the R26^ZsGreen reporter to verify Lrat^Cre -induced ZsGreen expression in HSCs. Socs1^fl/fl mice were bred with Lrat^Cre mice to generate Socs1^fl/flLrat^Cre mice lacking SOCS1 expression in HSCs (Socs1^ΔHSC ). Mice were housed in ventilated cages with 12 hours day/night cycle and fed with normal chow ad libitum. All experiments on mice were carried out during daytime with the approval of the Université de Sherbrooke Ethics Committee for Animal Care and Use (Protocol ID: 2018-2083).

To induce liver fibrosis, carbon tetrachloride (CCl₄; Sigma-Aldrich, Oakville, ON) diluted at 1:3 ratio in corn oil was administered via intra-peritoneal route (0.5 μL/g body weight) in 8-10-week-old mice twice a week for five weeks (32). Only male mice were used in this study due to the protective effect of estrogens on inflammatory cytokine production in female mice (33–35). Three days after the last injection, mice were euthanized, and serum and liver tissues were collected. Liver tissues were processed for histology, protein and mRNA expression and flow cytometry analyses of intrahepatic leukocytes, as detailed below.

Serum alanine transferase (ALT) levels were measured using a kinetic assay kit from Pointe Scientific Inc. (Brussels, Belgium) following manufacturer’s instructions. Hydroxyproline content in liver tissue homogenates was measured as described previously (26).

Pieces of liver tissues were fixed in 4% paraformaldehyde overnight and stored in 70% ethanol until they were processed for paraffin embedding following standard methods. Sections of formalin-fixed paraffin embedded liver tissues (5 µm) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) or Sirius red as previously described (26). Digital images of stained sections were acquired using a Nanozoomer Digital Pathology (NDP) slide scanner and analyzed using the NDP.view2 software (Hamamatsu Photonics, Japan). Quantification of Sirius red staining areas was done using the Image J software (National Institutes of Health, Bethesda, MD, USA) from twenty randomly selected fields from three to five mice in each group.

Immunohistochemical (IHC) staining of liver sections for alpha smooth muscle actin (αSMA) was done as previously described to detect myofibroblasts (26) ( Supplementary Table S1 ). Macrophages were detected by immunofluorescence (IF) staining. Deparaffinized tissue sections were incubated overnight with CD68 antibody ( Supplementary Table S1 ) followed by a AlexaFluor-488-conjugated secondary antibody (Invitrogen/ThermoFisher Scientific) for 1 h at room temperature in the dark. Nuclei were stained with Hoechst 33342 (Thermo Fisher; Cat# 62249) for 5 min at room temperature. The stained slides were washed and mounted in Vectashield (Vector Laboratories; Cat# H-1900) antifading medium. IHC images were captured using the NDP slide scanner and IF images acquired using Axioskop 2 fluorescence microscope (Carl Zeiss Canada Ltd, Toronto, Canada). Staining intensity of αSMA and the proportion of CD68 positive cells were quantified in three randomly selected fields from three to five mice in each group using the NIH ImageJ software.

RNA extraction, cDNA preparation and gene expression analysis by RT-qPCR were carried out as described previously (26). All RT-qPCR primers ( Supplementary Table S2 ) showed more than 90% efficiency and displayed a single melting curve. Expression levels of specific genes were normalized for the housekeeping gene Rplp0 (36B4) within each experimental group and expressed as fold induction compared to the control group.

Liver tissue lystaes were prepared from snap frozen samples using a tissue homogenizer bead mill (MM 400; Retsch, Hann, Germany) and protein concentration determined as previously described (26). Thirty μg of total protein from each sample were separated on SDS-polyacrylamide gels, blotted on to PVDF membrane and probed for the indicated proteins using the primary antibodies listed in Supplementary Table S3 . HRP-conjugated mouse and rabbit secondary antibodies and enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Pittsburg, PA) were used to reveal the western blot bands. Images were captured using the VersaDOC 5000 imaging system (Bio-Rad).

HSCs were isolated from 12-weeks old mice by equilibrium density gradient centrifugation of liver cells released by collagenase digestion, following published methods with some modifications (36, 37). Mice were anesthetized by intraperitoneal administration of Ketamine-Xylazine mixture (Ketamine-87 mg/kg; Xylazine-13 mg/kg - in normal saline), placed on supine position, the liver was exposed and the inferior vena cava canulated as described by Mederacke et al. (37). The livers were perfused with 0.5 mM EGTA in HEPES-buffered Hank’s balanced salt solution (HHBSS: NaCl 140 mM, KCl 5.4 mM, Na₂HPO₄ 0.34 mM, NaHCO₃ 4.2 mM, KH₂PO₄ 0.44 mM; MgCl₂ 0.4 mM, HEPES 10 mM, D-glucose 100 mg/L) without calcium, maintained at 42°C in a water bath, for 2 min using a peristaltic pump, with the portal vein severed to flush out erythrocytes. The liver parenchyma was digested by perfusion with freshly prepared Type IV collagenase (Worthington Biochemical; Cat # LS004186; 18 mg of 325 collagen digestion units) in 50 mL of prewarmed HHBSS containing 1.5 mM CaCl₂ (wash buffer). Tissue digestion was stopped when the liver became amorphous and collapsed (~7 min). The digested liver was carefully removed and aseptically transferred to a Petri dish. The liver tissue was teased apart using forceps to release cells into suspension, which was passed through 70 μm nylon filter strainer (BD Falcon) to remove tissue debris. Hepatocytes were sedimented by centrifuging the cell suspension at 50 g for 5 min at 4°C. Nonparenchymal cells were pelleted down by centrifuging the supernatant at 500 g for 10 min at 4°C. The cell pellet was resuspended in 5 mL 20% OptiPrep™ (Axis Shield; 60% stock diluted with wash buffer), transferred to a 15 mL tube, slowly overlaid with 5 mL 11.5% OptiPrep and then 2 mL wash buffer, and centrifuged at 1500 g for 17 min at 4°C without brake. An opaque layer formed at the interface between 11.5% OptiPrep and the wash buffer was carefully collected with a Pasteur pipette and the cells were washed at 500 g for 5 min at 4°C. The cells were resuspended in DMEM containing 10% FCS and counted.

For IF microscopy, HSCs were seeded on coverslips in a 12 well plate at 1 ×10⁵ cells/well in DMEM-10% FCS and cultured with the indicated cytokines and growth factors. Control and stimulated cells on coverslips were washed in phosphate-buffered saline (PBS), fixed in ice-cold methanol for 5 min at room temperature, followed by washing in PBS three times each for 5 min. The coverslips were incubated in PBST (PBS with 0.2% Triton X-100) containing 5% BSA for 1 h to block non-specific Ab binding. This was followed by incubation with primary Ab diluted in PBST-1% BSA in a humidified chamber for 1 h at room temperature or overnight at 4°C. Subsequent steps were similar to those described for IF microscopy of tissue sections.

For gene expression studies, control HSCs were lysed immediately after isolation in RNAlater (ThermoFisher Scientific). For stimulation with cytokines and growth factors, primary HSCs were cultured in 35 mm Petri dishes (0.5 ×10⁶ cells/well) and cultured in DMEM-10% FCS in the presence of IL-6 (10 ng/mL), TGFβ (5 ng/mL) or PDGFB (20 ng/mL) (all from R&D Sytems, Minniapolis, MN). After for 24 h incubation, the cells were lysed in RNAlater for gene expression analysis as previously described (26).

IHLs were isolated by a four steps protocol involving (i) controlled collagenase digestion of minced liver tissue using the GentleMACS tissue dissociator device (Miltenyi Biotech), (ii) microfiltration and sedimentation of hepatocytes and IHLs by differential centrifugation, (iii) Percoll gradient centrifugation of sedimented IHLs to remove fatty debris, and (iv) magnetic selection of hematopoietic cells using anti-CD45 antibody to clarify the resuspended Percoll-sedimented cells as detailed elsewhere (38). This IHL isolation procedure developed for fatty liver tissues is applicable to fibrotic and normal livers. The cells were resuspended in PBS containing 2% fetal bovine serum (FBS) for flow cytometry analysis.

Aliquots of IHLs were incubated in 100 μL of Fc Block diluted in PBS-2%FBS for 10 min on ice. After washing in PBS-2%FBS, the cells are incubated with a panel of fluorochrome conjugated antibodies ( Supplementary Table S4 ) diluted in PBS-2%FBS. The cells were washed and fluorescence data were acquired using the CytoFlex flow cytometer (Beckman Coulter). The data was analyzed using the FlowJo software (BD Biosciences).

To induce HCC, 2 weeks old male mice were injected via intra peritoneal route diethylnitrosamine (DEN; Millipore-Sigma; 25 mg/Kg body weight) followed by bi-weekly injections of CCl₄ (0.5 ml/Kg) starting at 8 weeks of age for 14 consecutive weeks (39). Mice were euthanized after 22 weeks and tumor development assessed by counting the number of nodules and measuring the liver/body weight ratio.

The data were compiled on an Excel spreadsheet (Microsoft 365) and Prism V9.3.1 software (GraphPad, La Jolla, CA, USA) was used to plot graphs and for statistical analysis. p values <0.05 were considered significant.

Cytokines and growth factors produced by stressed hepatocytes and activated macrophages are the key drivers of liver fibrosis and represent potential therapeutic targets (4, 12). The loss of SOCS1, a key regulator of inflammatory cytokine and growth factor signaling in the liver, promotes liver fibrosis (25, 26, 40). Primary HSCs isolated from SOCS1-deficient mice display increased proliferation in response to IL-6, PDGF, EGF, TGFα and HGF, suggesting a cell-intrinsic role of SOCS1 in regulating HSC activation (26). To directly assess the role of SOCS1 in regulating HSCs responses during liver fibrosis, we crossed Socs1^fl/fl mice with Lrat^Cre deleter mice, which ablates floxed genes specifically in HSCs in the liver (28, 29). We confirmed the expression of Lrat promoter driven Cre expression in HSCs using the ROSA26-ZsGreen reporter mice (31). Cryosections of livers from Lrat^Cre ROSA26-ZsGreen mice showed a ZsGreen expression pattern that is consistent with the distribution of HSCs ( Supplementary Figure S1A ), which was confirmed by immunostaining for the HSC marker desmin ( Supplementary Figure S1B ) as well as by verifying ZsGreen expression in primary HSCs ( Supplementary Figure S1C-E ).

Liver fibrosis was induced in Socs1^ΔHSC and Socs1^fl/fl control mice by intraperitoneal administration of CCl₄ twice a week for 5 weeks. Sirius red staining of the liver sections of these mice showed increased collagen deposition with prominent bridging fibrosis pattern compared to limited septal fibrosis observed in Socs1^fl/fl control mice ( Figure 1A ). Quantification of the staining area and intensity showed significantly elevated levels of collagen deposition in HSC-specific SOCS1-deficient mice livers that was confirmed by hepatic hydroxyproline content ( Figure 1B, C ), Masson’s trichrome staining and western blot ( Supplementary Figure S2 and Figure 1D ). However, the increase in serum ALT levels following CCl₄ treatment was comparable between Socs1^ΔHSC and Socs1^fl/fl control mice ( Figure 1E ), suggesting that increased fibrosis in Socs1^ΔHSC mice resulted from increased fibrogenic response caused by the loss of SOCS1 in HSCs rather than from increased liver damage. This was confirmed by immunohistochemical staining of αSMA in myofibroblasts, which showed intense staining and significantly increased staining area in Socs1^ΔHSC mice compared to Socs1^fl/fl controls ( Figure 1F, G ). Consistent with this data, CCl₄-treated Socs1^ΔHSC mice livers showed increased expression of Acta2 and Col1a1 genes and αSMA and collagen 1 protein expression ( Figure 1H, D ). The fibrotic livers of Socs1^ΔHSC mice showed significantly elevated expression of Col3a1, the antifibrotic matrix metalloproteinase 2 (Mmp2) (41) and tissue inhibitor of MMPs 1 (Timp1) genes compared to Socs1^fl/fl mice livers ( Figure 1H ). Moreover, expression of genes coding for proinflammatory cytokines IL-6 and IL-1β, profibrogenic TGFβ and the myofibroblast growth factor PDGFB, which were markedly induced in Socs1^fl/fl mice livers, was significantly elevated in the fibrotic livers of Socs1^ΔHSC mice ( Figure 1I ). These data indicated that SOCS1 expression in HSCs plays a crucial role in regulating hepatic fibrogenic response induced by chemical agents.

As TGFβ is a key driver of ECM deposition (42, 43), we examined TGFβ signaling pathway components in the fibrotic livers of Socs1^ΔHSC and Socs1^fl/fl mice. Socs1^ΔHSC mice displayed increased phosphorylation of SMAD2 and SMAD3 in both CCl₄ and oil treated groups ( Figure 2A ). On the other hand, increased SMAD3 phosphorylation was observed in the livers of Socs1^fl/fl mice only after CCl₄ treatment, whereas SMAD2 phosphorylation level was not altered ( Figure 2A ). Similarly, phosphorylation of the MAP kinase ERK1/2 was prominent in both in both CCl₄ and oil treated groups of Socs1^ΔHSC mice but occurred only after CCl₄ treatment in control mice ( Figure 2A ). These observations suggested increased responsiveness of SOCS1-deficient HSCs to TGFβ and growth factor signaling that promoted their fibrogenic response. To test this possibility, primary HSCs enriched from Socs1^ΔHSC and Socs1^fl/fl mice were exposed to inflammatory (IL-6), fibrogenic (TGFβ) and growth stimulating (PDGFB) cytokines and the induction genes that promote myofibroblast differentiation (Acta2) and matrix production (Col1a1) and remodelling (Mmp2, Timp1) was evaluated. TGFβ strongly induced Acta2, Col1a1, Mmp2 and Timp1 genes in control HSCs and the induction of all but Mmp2 were further amplified significantly by SOCS1 deficiency ( Figure 2B ). Whereas IL-6 caused a discernible increase in the expression of Acta2, Col1a1 and Mmp2 genes in control and SOCS1-deficient HSCs, PDGFB, the most potent growth factor for HSCs (18, 44), did not change the expression of these fibrogenic genes. Notably, SOCS1-deficient HSCs showed discernibly elevated basal expression of Acta2 and Timp1 genes ( Figure 2B ), suggesting production of autocrine fibrogenic mediators in SOCS1-deficient HSCs. Indeed, TGFβ stimulation upregulated Tgfb and Pdgfb genes in control HSCs that was significantly amplified by SOCS1 deficiency ( Figure 2B ). Immunofluorescence staining of αSMA following TGFβ stimulation showed profound increase in SOCS1-deficient HSCs compared to control HSCs ( Figure 2C ). These results indicated that SOCS1 is a critical to regulator of HSC activation by limiting their responsiveness to TGFβ stimulation and TGFβ-induced autocrine mediator production.

Hematoxylin and eosin-stained liver sections of CCl₄-treated mice revealed increased inflammatory cell infiltration in Socs1^ΔHSC mice in the periportal area and around the central vein compared to Socs1^fl/fl controls ( Figure 3A ). This observation suggested that SOCS1 deficiency in HSCs enhances hepatic inflammatory response during fibrogenesis. In support of this notion, the expression of the Ccl2 chemokine gene, which is strongly induced by CCL₄ treatment in control mice, was further increased in HSC-specific SOCS1-deficient mice ( Figure 3B ). However, the induction of Ccl5 (RANTES) and Cx3cl1 (Fractalkine) genes was comparable between Socs1^ΔHSC and Socs1^fl/fl mice livers. We also observed that TGFβ strongly induced the Ccl2 gene in primary HSCs that was significantly elevated in SOCS1-deficient HSCs ( Figure 2B ). As Ccl2 encodes the macrophage chemoattractant protein 1 (MCP1/CCL2), we examined the distribution of macrophages in the CCL₄-treated livers. The fibrotic livers of Socs1^fl/fl mice harbored significantly more CD68+ cells, possibly representing Kupffer cells arising from recruited macrophages (45), and their numbers increased further in Socs1^ΔHSC mice compared to Socs1^fl/fl mice ( Figure 3C, D ). Flow cytometry analysis of intrahepatic leukocytes revealed that the proportion and number of total CD45+CD11b+ myeloid cells and CD11b+Ly6G+ polymorphonuclear neutrophils, which were comparable between vehicle-treated Socs1^ΔHSC and Socs1^fl/fl mice, significantly increased following fibrosis induction and this increase was further augmented by SOCS1 deficiency in HSCs ( Figure 3E–G ). These data indicated that SOCS1 expression in HSCs is crucial to control immune cell recruitment and to regulate fibrosis-associated inflammatory response during liver fibrosis.

The increased numbers of myeloid cells ( Figure 3E, F ) and the heightened induction of Il6, Il1b, Tgfb and Pdgfb genes in CCl₄-treated livers of Socs1^ΔHSC mice ( Figure 1I ), suggested potent activation of monocyte-derived macrophages. To test this hypothesis, we evaluated the expression level of proinflammatory macrophage marker Ly6C (45) on CD45+CD11b+ cells ( Figure 4A ). The fibrotic livers of Socs1^fl/fl mice livers harbored increased proportion and number of Ly6C^hi proinflammatory macrophages that were further increased in the livers of Socs1^ΔHSC mice ( Figure 4B ). Even though Ly6C^lo anti-inflammatory macrophages were significantly reduced in frequency in the fibrotic livers of both Socs1^fl/fl and Socs1^ΔHSC mice, absolute number of this macrophage subset was not significantly altered ( Figure 4C ). Proinflammatory macrophages are also characterized by the upregulation the chemokine receptor CCR2 (45, 46). When the expression of CCR2 and Ly6C was examined on CD11b+Ly6G- cells, we observed a significant increase in the proportion and number of Ly6C^hiCCR2+ cells in CCl₄-treated livers of Socs1^fl/fl mice that was further increased in Socs1^ΔHSC mice ( Figure 4D, E ). The proportion of Ly6C^hi cells that did not express CCR2 significantly decreased in both Socs1^fl/fl and Socs1^ΔHSC mice, although their absolute numbers of were not significantly affected ( Figure 4F ).

During fibrosis progression proinflammatory macrophages transition to restorative macrophages that promote tissue repair and fibrosis resolution after cessation of the inflammatory stimuli (45). The pro-resolution macrophages are characterized by the expression of the CX3CR1 chemokine receptor. Segregation of the macrophage population based on the expression of Ly6C and CX3CR1 revealed that the proportion of Ly6C^loCX3CR1+ pro-resolution macrophages did not change in the livers of CCl₄-treated Socs1^fl/fl and Socs1^ΔHSC mice although their numbers showed a discernible, though not significant, increase in both groups ( Figure 5A, B ). On the other hand, the proportion and number of Ly6C^hi cells that also expressed CX3CR1 was markedly upregulated in the fibrotic livers of Socs1^fl/fl and Socs1^ΔHSC mice, with a significant increase in both frequency and number ( Figure 5A, C ). Next, we analyzed the co-expression of CCR2 and CX3CR1 within CD11b+Ly6G−Ly6C^hi pro-inflammatory macrophage population. We observed a marked increase in the frequency of Ly6C^hiCCR2+CX3CR1+ cells in both Socs1^fl/fl and Socs1^ΔHSC mice with significantly elevated number of these cells in Socs1^ΔHSC mice ( Figure 5D, E ). These results suggest that the increased inflammatory response in Socs1^ΔHSC mice prevents these intermediate or transitional macrophages from acquiring the CX3CR1+Ly6C^loCCR2− restorative macrophage phenotype.

The fibrotic livers of Socs1^ΔHSC mice also contained an increased frequency and number of CD11b+CD11c+ myeloid dendritic cells (DC), whereas the number of plasmacytoid DCs were not affected ( Supplementary Figure S3 ). The livers of CCl₄-treated Socs1^ΔHSC mice also harbored an elevated number of CD8+ T lymphocytes that displayed an activated effector (CD69+), effector memory (CD44^hiCD62L^lo) and central memory (CD44^hiCD62L^hi) phenotype, whereas the numbers of CD4 T and NK cells and the activation status of CD4+ T cells were not affected ( Supplementary Figures S4, S5 ).

Liver fibrosis driven by activated HSC and the associated inflammation driven by macrophages are important drivers of HCC development and progression (10, 47, 48). To determine if increased liver fibrosis in Socs1^ΔHSC mice promotes HCC development, we administered DEN to 2 weeks old mice followed by CCl₄ treatment beginning at 8 weeks of age and continued for 14 weeks to induce and sustain liver fibrosis ( Figure 6A ). Examination of the livers at the end of this treatment period revealed increased liver body weight ratio in Socs1^ΔHSC mice than in control mice, with increased number of liver tumor nodules that showed histological features of hepatocellular carcinoma ( Figure 6B–D ).