The hepatocyte epidermal growth factor receptor (EGFR) pathway regulates the cellular interactome within the liver fibrotic niche

Liver fibrosis is the consequence of chronic liver injury in the presence of an inflammatory component. Although the main executors of this activation are known, the mechanisms that lead to the inflammatory process that mediates the production of pro‐fibrotic factors are not well characterized. Epidermal growth factor receptor (EGFR) signaling in hepatocytes is essential for the regenerative processes of the liver; however, its potential role in regulating the fibrotic niche is not yet clear. Our group generated a mouse model that expresses an inactive truncated form of the EGFR specifically in hepatocytes (ΔEGFR mice). Here, we have analyzed the response of WT and ΔEGFR mice to chronic treatment with carbon tetrachloride (CCl4), which induces a pro‐inflammatory and fibrotic process in the liver. The results indicated that the hallmarks of liver fibrosis were attenuated in CCl4‐treated ΔEGFR mice when compared with CCl4‐treated WT mice, coinciding with a faster resolution of the fibrotic process and ameliorated damage. The absence of EGFR activity in hepatocytes induced changes in the pattern of immune cells in the liver, with a notable increase in the population of M2 macrophages, more related to fibrosis resolution, as well as in the population of lymphocytes related to eradication of the damage. Transcriptome analysis of hepatocytes, and secretome studies of extracellular media from in vitro experiments, allowed us to elucidate the specific molecular mechanisms regulated by EGFR that mediate hepatocyte production of both pro‐fibrotic and pro‐inflammatory mediators; these have consequences for the deposition of extracellular matrix proteins, as well as for the immune microenvironment. Overall, our study uncovered novel mechanistic insights regarding EGFR kinase‐dependent actions in hepatocytes that reveal its key role in chronic liver damage. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Fibrosis is a highly conserved and coordinated, initially protective, response to tissue injury.The interaction among multiple pathways determines whether fibrosis is self-limiting and homeostatic or whether it is uncontrolled and excessive [1].When a chronic injury takes place in the liver, mobilization of lymphocytes and other inflammatory cells occurs, thus setting the stage for persistence of an inflammatory response.Macrophages produce pro-fibrotic mediators that are responsible for the activation of quiescent hepatic stellate cells (HSCs) to a myofibroblast (MFB) phenotype.MFBs are the principal source of extracellular matrix (ECM) protein accumulation and prominent mediators of fibrogenesis [2].
The epidermal growth factor receptor encoded by the gene EGFR (previously known as ERBB1) is a classical tyrosine kinase receptor that belongs to the family of ErbB receptors, and it is highly expressed in the liver.The EGFR pathway regulates hepatocyte proliferation, playing important roles in liver regeneration and hepatocellular carcinogenesis [3].But the EGFR signaling system has also been identified as a key player in all stages of the liver's response to injury, from early inflammation and hepatocellular proliferation to fibrogenesis and neoplastic transformation [4].The role of EGFR in chronic liver injury and fibrosis is also relevant for human pathophysiology, because EGF, EGFR, and phospho-EGFR levels increase in the liver of cirrhotic or metabolic dysfunction-associated steatotic liver (MAFLD) patients [3].A direct role of EGFR, or its ligands, in HSC activation and subsequent ECM modification has been suggested, concomitant with regulation of steatosis, in different in vitro and in vivo animal models of liver fibrosis and MAFLD [5][6][7][8][9].However, different studies have pointed out that the EGFR signaling might be protective in other processes of liver injury and fibrosis [10,11].
With the aim of better understanding the specific role of the hepatocyte EGFR pathway in liver physiology and pathology, some years ago we generated a transgenic mouse model expressing a truncated form of human EGFR specifically in hepatocytes, which acts as dominant negative mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions [12].We found that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine (DEN)-induced tumors, which correlated with a strong attenuation and delay in the DEN-induced inflammatory process [12].We also found that after chronic feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which induced cholestatic damage, ΔEGFR mice presented with a more efficient regeneration, concomitant with a shift from a pro-fibrotic to a restorative inflammatory response [13].Previous studies had suggested that proinflammatory stimuli could mediate transactivation of the EGFR pathway, which would contribute to carcinogenesis in inflammatory liver diseases [14,15].However, our data pointed to a role for the EGFR pathway in hepatocytes, upstream of the inflammatory process.In this context, further investigations were required to comprehensively study the direct role of EGFR in hepatocytes in regulating inflammation during chronic liver injury.
Accordingly, the aim of this study was to elucidate whether the catalytic activity of the EGFR in hepatocytes could participate in regulating the inflammatory/ pro-fibrotic environment in response to a chronic liver insult.

In vivo experimental procedures
The novel transgenic mouse model that expresses a hepatocyte-specific truncated form of human EGFR (supplementary material, Figure S1A), which acts as dominant negative mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions, was used [12].Chronic liver injury was induced by treatment with carbon tetrachloride (CCl 4 ; Sigma-Aldrich, St Louis, MO, USA).For this, 0.48 g/kg body weight CCl 4 , 1:10 v/v in mineral oil (MO), or vehicle (MO), was administered to 8-week-old C57BL/6 male mice by intraperitoneal injection (3 μl/g body weight) twice a week for 4 or 8 weeks.At these timepoints, animals were sacrificed, and blood and liver tissue were collected (supplementary material, Figure S1B).The transgenic ΔEGFR mouse line is maintained in heterozygosity; therefore wild-type (WT) and ΔEGFR mice belong to the same strain.The WT and ΔEGFR mice used for experiments belonged to the same litters.We proved that ΔEGFR mice continued expressing the truncated human transgene following the CCl 4 treatment (supplementary material, Figure S1C).To confirm that CCl 4 was similarly metabolized in both groups, we analyzed the expression of Cyp2b10, whose expression increased similarly after the treatment in WT and ΔEGFR livers (supplementary material, Figure S1D).Moreover, since CCl 4 is a Cyp2e1 suicide substrate whose binding leads to its inactivation and degradation [16], we analyzed its expression, observing a similar decrease in both cases.These results suggest that CCl 4 was equally metabolized in all experimental groups (supplementary material, Figure S1D).
Detailed information about materials and methods and statistical analysis may be found in Supplementary materials and methods.
The hepatocyte EGFR pathway regulates the liver fibrotic niche

Inactivation of EGFR in hepatocytes ameliorates CCl 4 -induced liver damage and fibrosis
We first examined the consequences of inactivating EGFR signaling in hepatocytes on the liver damage progression after 4 or 8 weeks of CCl 4 chronic treatment.As observed in Figure 1A, serum levels of transaminases increased after 8 weeks of treatment in WT mice (no differences were observed at 4 weeks of treatment; results not shown).However, no significant increase was observed in ΔEGFR mice.Histological analysis also revealed less damage in the liver parenchyma after 8 weeks of treatment (Figure 1B), correlating with decreased levels of alpha-smooth muscle actin (α-SMA), characteristic of activated MFB (Figure 1C,D).In ΔEGFR livers, the expression and deposits of α-SMA increased after 4 weeks but decreased at 8 weeks, reflecting resolution of fibrosis.Specific analysis of the process of fibrosis revealed that the increase in the deposits of collagen fibers, caused by the CCl 4 treatment, was significantly lower in ΔEGFR livers (Figure 2A).The expression of different collagen genes, such as Col1a1 or Col3a1, increased significantly after 4 weeks of treatment; however, increases in expression were lower in ΔEGFR livers, which was particularly relevant for Col3a1 (Figure 2B).It was very interesting to find differences in lysyl oxidase-related genes (responsible for the interchain polypeptide cross-linking of collagens), which were significantly increased in WT but not in ΔEGFR livers after 4 weeks of CCl 4 treatment (Figure 2B).ΔEGFR mice presented different patterns in the expression of metalloprotease mRNAs, particularly Mmp2 and Mmp9, with a potential role in the resolution of fibrosis, which were significantly increased in ΔEGFR, but not in WT livers, after 8 weeks of treatment (Figure 2C).The expression of Timp1 (TIMP metallopeptidase inhibitor 1) was significantly lower in ΔEGFR livers after 4 weeks of CCl 4 treatment (Figure 2C), which would indicate a higher metalloprotease activity.Expression of other members of the family, such as Timp3, did not change.
We could not find relevant differences among WT and ΔEGFR livers in the expression of genes related to the transforming growth factor-beta (TGF-β) pathway, as one of the major pro-fibrotic factors.Thus, although the increase in some of its ligands appeared to be delayed in ΔEGFR mice (supplementary material, Figure S2A), phosphorylation of Smad3, a hallmark of the activation of the pathway, was significantly increased after 8 weeks of treatment in both WT and ΔEGFR livers (supplementary material, Figure S2B).No clear differences were observed in the expression of other pro-fibrotic genes, such as Ccn2 (CTGF) or Pdgfb (supplementary material, Figure S3).

ΔEGFR livers showed significant differences in macrophage populations in the CCl 4 model of liver injury
With the aim of comparing the inflammatory response in WT and ΔEGFR mice treated with CCl 4 , we first analyzed the expression of Adgre1 at the mRNA and protein levels (immunohistochemistry of F4/80 in tissue sections), as well as the expression of general inflammatory cytokines, such as Tnf or Il1b (supplementary material, Figure S4).No significant differences could be observed.Next, we isolated and analyzed by flow cytometry the intrahepatic immune monocyte/macrophage populations after 4 weeks of treatment.No increase in the percentage of resident macrophages was observed (Figure 3A).However, ΔEGFR livers presented a decrease in the late recruited macrophages (correlating with the less parenchymal damage observed in Figure 1) and the most significant result was the shift in the M1/M2 balance towards M2 polarity (Figure 3C), which correlated with a significantly lower ratio between the expression of the pro-inflammatory cytokine Il12b, characteristic of M1 macrophages, and the antiinflammatory cytokine Il10, characteristic of M2 polarization, detected in ΔEGFR livers (Figure 3D).Regarding lymphocyte populations, a shift in the Cd4/Cd8 ratio was observed in favor of Cd4 lymphocytes in ΔEGFR livers (supplementary material, Figure S5), concurring with significantly lower levels of naïve and IL17 + Cd4 + lymphocytes, and a relevant increase in the Treg cell subpopulation (supplementary material, Figure S5).

The catalytic activity of EGFR regulates the hepatocyte gene transcriptome in response to CCl 4 -induced liver damage
To deepen our understanding of underlying molecular mechanisms by which the EGFR pathway in hepatocytes could be controlling the fibrotic niche interactome, we next performed RNA sequencing (RNA-seq) analysis in hepatocytes isolated from WT and ΔEGFR mice treated with either MO or CCl 4 for 4 weeks (supplementary material, Figure S6A).The most relevant results are summarized in Figure 4.When the changes in gene expression caused by CCl 4 treatment versus control samples in both WT and ΔEGFR hepatocytes were compared, 1,258 genes were specifically differentially expressed in the WT comparison, whereas only 209 genes were differentially expressed in the one from ΔEGFR (Figure 4A).Transcription factor (TF) activity was inspected by functional enrichment analysis, based on the differentially expressed genes of each comparison.Treated hepatocytes from WT mice showed 265 TF binding sites significantly enriched among those genes, which did not appear in ΔEGFR mice (Figure 4B and see details in supplementary material, Figure S6B).Many of the TFs whose activity appeared modulated in WT hepatocytes were related to proliferation and differentiation (EGR, Myc, E2F, Ets-related, KLFs, etc.), stress pathways (HIF-1, p53), and metabolism (PPARa, SREBP, CREB-related).The most significant difference in the ΔEGFR hepatocytes was the increase in the activity of PPARg, as well as GLI or NF1.In addition to the expected differences in the activation of the EGFR pathway (supplementary material, Figure S7A), relevant differences in tissue remodeling, collagen metabolic processes, or regulation The hepatocyte EGFR pathway regulates the liver fibrotic niche 485  The hepatocyte EGFR pathway regulates the liver fibrotic niche 487 488 E Gonzalez-Sanchez, J Vaquero et al of inflammation and cytokine production pathways were identified in hepatocytes from WT but not from ΔEGFR mice (Figure 4C).The cellular response to xenobiotic stimulus was activated in WT and ΔEGFR mice (supplementary material, Figure S7A), which corroborates that the response to CCl 4 has not been affected by silencing EGFR in hepatocytes.Among the genes specifically regulated in hepatocytes from CCl 4 -treated WT mice that did not appear modulated in ΔEGFR hepatocytes, we found genes related to fibrogenesis, such as members of the Pdgf family, metalloproteases (Mmp15 or Mmp14), and lysyl oxidases or lysyl oxidase-like genes (Lox and Loxl1), among others (Figure 4D), as well as genes involved in regulation of inflammation, such as cytokines/chemokines or genes related to TNF receptor signaling (Figure 4D).A volcano plot showing differences in the expression of genes when comparing ΔEGFR versus WT hepatocytes isolated after 4 weeks of CCl 4 treatment revealed a significant decrease in the expression of inflammatory genes, such as the cytokines Cxcl2 or Cxcl14 or the chemokine receptor Ccrl2 (Figure 4E, left).In fact, Gene Ontology (GO) enrichment analysis also revealed that ΔEGFR cells presented a significant decrease in inflammatory pathways (Figure 4F).The volcano representation also revealed differences in genes related to metabolism, such as Ppard, Dbp, or Thrsp (Figure 4E, left).In most of the cases, CCl 4 treatment induced changes in gene expression in WT hepatocytes, which were not observed in ΔEGFR hepatocytes (Figure 4E, right).
It is also worth mentioning that hepatocytes from CCl 4treated WT mice, but not those from ΔEGFR mice, showed relevant changes in the expression of genes related to cell cycle progression and cell proliferation (Figure 4D), which would indicate higher hepatocyte regeneration in the WT than in the ΔEGFR mice.In parallel, hepatocyte identity genes, such as Hnf4a (Figure 4D), or genes related to liverspecific functions (supplementary material, Figure S7B), were downregulated.Interestingly, the ΔEGFR hepatocytes from CCl 4 -treated mice showed higher expression of mitochondrial metabolism-related pathways, such as fatty acid catabolic processes, mitochondrial respiratory chain, aerobic electron transport chain, or ATP synthesis than those isolated from the WT mice (supplementary material, Figure S7C), which indicated that they are metabolically more efficient.Relative upregulation of TGF-β pathwayrelated genes, as well as epithelial-mesenchymal transition (EMT) gene signature (as defined in supplementary material, Table S5), was observed in hepatocytes after CCl 4 treatment, but changes were similar in WT and ΔEGFR hepatocytes (supplementary material, Figures S8 and S9).
Overall, our results indicate that in response to a chronic insult, the EGFR pathway in hepatocytes modulates some of the transcriptional program changes.

The catalytic activity of EGFR in hepatocytes regulates the inflammatory and pro-fibrotic secretome in response to CCl 4 -induced liver damage
We previously generated immortalized hepatocytes from WT and ΔEGFR mice [12], which are a useful tool for in vitro approaches.Here, we have analyzed how the secretome of these cells may affect the fibrotic cell interactome in in vitro experiments, focusing on the effects on macrophages.We incubated the WT and ΔEGFR hepatocytes with heparin-binding EGF-like growth factor (HB-EGF), a ligand of the EGFR, to activate the pathway (Figure 5A,B).We next followed the experimental set-up depicted in Figure 5C to generate conditioned medium that was used to analyze the effects on the phenotype of a mouse macrophage cell line (RAW264.7 cells).As observed in Figure 5D, the conditioned medium of WT and ΔEGFR hepatocytes produced opposite effects on the expression profile of genes associated with an M1 phenotype (pro-inflammatory/fibrotic), such as Il12b, or M2 (resolution of fibrosis), such as Il10.The ratio Il12b/Il10 significantly increased only in RAW264.7 cells incubated with the conditioned medium from HB-EGF-treated WT hepatocytes.
To analyze whether the EGFR pathway would also be regulating the human macrophage phenotype, we used a human liver tumor cell line, Hep3B, and a human monocyte cell line, THP-1.Using a similar experimental approach (supplementary material, Figure S10A), conditioned medium of Hep3B cells untreated or treated with HB-EGF was collected to analyze the effects on THP-1 cells.The results indicated that the conditioned medium from HB-EGF-treated Hep3B cells increased monocyte adhesion (supplementary material, Figure S10B) and the expression of proinflammatory genes (M1 phenotype), downregulating the expression of anti-inflammatory-related genes (M2 phenotype) (supplementary material, Figure S10C).The Il12b/Il10 ratio significantly increased in THP-1 cells after the incubation with the conditioned medium from HB-EGF-treated Hep3B cells (supplementary material, Figure S10D).
Overall, our results indicate that hepatocytes may actively participate by secreting factors that regulate the inflammatory microenvironment in the fibrotic niche.To validate this, we next performed a proteomic analysis of conditioned medium from hepatocytes obtained following the same experimental approach presented in Figure 5C.The most significant results are presented in Figure 5E.WT hepatocytes responded to HB-EGF by inducing the secretion of Cxcl10, Cxcl12, and Cxcl16, and this increase was not observed for ΔEGFR hepatocytes, reinforcing the role of hepatocytes in the secretion of inflammatory cytokines under the control of the EGFR pathway.Moreover, it was also very interesting to observe that WT hepatocytes responded to HB-EGF by inducing the secretion of different proteins involved in fibrosis, among them, different members of collagens, or proteins involved in their maturation, such as Loxl1.ΔEGFR hepatocytes secreted much lower levels of all these proteins, and as expected, no changes were observed in response to HB-EGF.Indeed, the secretome analysis also revealed a role for hepatocytes in the production of ECM proteins.
The hepatocyte EGFR pathway regulates the liver fibrotic niche

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The EGFR pathway is activated during the progression of fibrosis in humans, correlating with the expression of specific fibrotic and inflammatory genes
To analyze the translational relevance of the results presented above, data from gene expression in a cohort of liver biopsies from HCC-naïve MASLD (metabolic dysfunction-associated steatotic liver disease, previously named NAFLD) patients were accessed through GEO accession number GSE193066 (see Supplementary materials and methods).We observed that the relative enrichment of EGF/EGFR signaling (WikiPathways) was significantly increased during progression of liver fibrosis (Figure 6A).The relative enrichment of EGFR target genes (using the Gene Transcription Regulation Database publicly available at https://www.gsea-msigdb.org/gsea/msigdb/geneset_page.jsp?geneSetName=EGF R_TARGET_GENES) also presented a tendency, almost significant, to increase at advanced stages of fibrosis.Interestingly, the expression of fibrosis-related genes, such as collagen genes (COL1A1, COL3A1, COL4A1, COL5A1), LOXL1, MMP2, MMP9, or TIMP1 presented a significant positive correlation with the EGF/EGFR signaling pathway (Figure 6B).Regarding genes related to the immune system, it was relevant that CXCL10, which was found to be regulated by the EGFR pathway in the secretome analysis in hepatocytes (Figure 5E), showed increased expression at advanced stages of fibrosis (Figure 6C), and the expression of CXCL10 and CXCL12 correlated with the EGF/EGFR signaling pathway (Figure 6D).A differential pattern was also found in the expression of CD4 and CD8A (supplementary material, Figure S11A), which correlated with the differential pattern of Cd8 + and Cd4 + lymphocytes found in the ΔEGFR livers after CCl 4 treatment (supplementary material, Figure S5).Furthermore, the expression of CD8A positively correlated with the EGF/EGFR signaling (supplementary material, Figure S11B).

Discussion
Development of liver fibrosis is frequently the consequence of chronic hepatocyte damage, HSC activation and proliferation, and changes in ECM production and accumulation [1].This process is accompanied by amplified inflammatory responses, with recruitment and activation of macrophages to the site of injury to orchestrate the process of wound healing and tissue repair [17,18].Unidentified fibrosis can evolve into more severe consequences over time, such as cirrhosis and liver cancer [19].Improving the knowledge about the intercellular interactions in the fibrotic niche is necessary to better understand this process and uncover potential therapeutic targets.This work demonstrates that the EGFR pathway in hepatocytes is required for an efficient progression of the fibrotic process in response to CCl 4 treatment in mice.Attenuation of the EGFR kinase activity in hepatocytes reduces the parenchymal liver damage and the extension of fibrotic areas, correlating with a decrease in the expression of Col3a1 and Loxl1, an increase in Mmp9, and a decrease in Timp1.Previous studies had supported a role for the EGFR signaling pathway in the initiation and development of liver fibrosis [20][21][22].However, in most of these works, the attention was mainly focused on the role of the EGFR pathway in hepatic steatosis and/or on HSC/MFB activation and proliferation.The main work that analyzed the role of the hepatocyte ERBB tyrosine kinases comes from Dr Russell's group, and suggested that EGFR-ERBB3 heterodimeric signaling in damaged hepatocytes may play a relevant role in liver fibrosis [9].Our study constitutes a step forward for deepening insight into the molecular mechanisms that are regulated by the EGFR in hepatocytes during chronic liver damage.
The results shown here strongly indicate that after CCl 4 treatment, ΔEGFR livers present significant differences in the immune landscape, favoring the antiinflammatory (M2) phenotype in macrophages.Despite tremendous progress, numerous challenges remain in deciphering the full spectrum of macrophage activation and its implication in either promoting liver disease progression or repairing injured liver tissue [23].While in homeostatic conditions resident macrophages can be identified in the liver, in recent years numerous studies have provided compelling evidence that in inflammatory conditions there is considerably more heterogeneity among hepatic macrophages [24,25].Experimental animal models indicate that macrophages are critical for fibrosis regression because they play key roles in repairing the necrotic lesions, not only by removing necrotic tissues but also by inducing cell death-resistant hepatocytes and exerting anti-inflammatory actions [26,27].Although the role of M2-like macrophages in liver fibrosis is still controversial, strong evidence supports the theory that they could exert protection against injury, among other actions, by inhibiting necroptosis and conferring apoptosis resistance to hepatocytes [28,29].Furthermore, M2 macrophage-derived exosomes significantly inhibit activation of HSCs [30].The EGFR catalytic activity in hepatocytes also provoked differences in the T-cell populations, with an increase in the Treg lymphocytes and differences in the proportion of Cd8 + and Cd4 + cells, decreasing the naïve and IL17 + cells.It has been proposed that by inhibiting pro-fibrotic Th17 and Cd8 + T cells, Treg cells may help to maintain a healthy inflammatory environment, regulating the aberrant activation and functions of immune effector cells [31].Overall, our results strongly indicate that the activation of the EGFR pathway in hepatocytes during chronic liver injury may have consequences on the inflammatory environment, regulating macrophage plasticity and favoring a high Th17/Treg cell ratio.
Analysis of the transcriptional signature of hepatocytes from fibrotic WT and ΔEGFR livers revealed the role of the EGFR pathway in regulating the expression of genes that promote fibrogenesis, such as different The hepatocyte EGFR pathway regulates the liver fibrotic niche

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E Gonzalez-Sanchez, J Vaquero et al mRNA forms of the Pdgf gene, as well as genes such as Lox and Loxl1 coding for lysyl oxidases involved in collagen chain trimerization.Different genes related to inflammatory processes, such as genes related to the TNF pathway (Tnfrsf12, Tnfrf11a, Tnfsfm13), the proinflammatory dermokine (Dmkn), or regulators of the T-cell-mediated response, such as Cd276, also appeared upregulated specifically in WT livers from CCl 4 -treated mice.Overall, the EGFR pathway modulates a transcriptomic program in hepatocytes that contributes to the generation of a pro-inflammatory and fibrotic environment, which justifies the strong differences observed between WT and ΔEGFR mice after CCl 4 treatment.Correlating with this, numerous TFs appeared regulated in CCl 4 -treated WT but not in ΔEGFR hepatocytes.An aspect that caught our attention was the strong difference in the expression of genes related to proliferation and differentiation, which were upregulated and downregulated, respectively, in WT but not in ΔEGFR hepatocytes after CCl 4 treatment.
Transient hepatocellular dedifferentiation can occur as part of the regenerative mechanisms triggered in response to acute liver injury.However, persistent downregulation of key identity genes is now accepted as a strong determinant of organ dysfunction in chronic liver disease [32], by stably reprogramming the critical balance of transcription factors responsible for hepatocyte identity, among them, HNF4α [32,33].Hepatocytes from CCl 4 -treated ΔEGFR hepatocytes maintained Hnf4a expression and did not show changes in the expression of proliferation-differentiation genes, which may suggest that damage to hepatocytes is much less and/or that the EGFR pathway is required for this gene transcription reprogramming.In parallel, ΔEGFR hepatocytes displayed higher expression of genes related to mitochondrial metabolism pathways, which might confer greater efficient energy production capacity.Our analysis of the secretomes of WT and ΔEGFR hepatocytes, either untreated or treated with HB-EGF, demonstrated that activation of the EGFR pathway alters the hepatocyte secretome, conferring the capacity to modulate macrophage polarity, promoting an antiinflammatory phenotype.Proteomic analysis of the conditioned media revealed that the secretion of chemokines, such as Cxcl10, Cxcl12, or Cxcl16, was significantly increased by HB-EGF in WT hepatocytes but not in ΔEGFR hepatocytes.These cytokines have been previously related to the progression of chronic liver injury.Intrahepatic CXCL10 has been strongly associated with liver fibrosis in different contexts in human liver pathologies [34,35], through regulation of macrophage-associated inflammation [36].CXCL12, the ligand of CXCR4, can be involved in multiple pathological mechanisms in fibrosis, such as inflammation, immunity, EMT, and angiogenesis [37].Furthermore, our secretome analyses indicated a role for hepatocytes in producing ECM proteins under the control of the EGFR pathway.It is worth highlighting that Loxl1 appeared in all the studies presented in this paper: in the in vivo experiments, in the transcriptomic analysis performed in hepatocytes, as well as in the secretome.It is very clear that hepatocytes contribute to the expression, production, and secretion of lysyl oxidases required for the final formation of the collagen fibers, and the EGFR pathway regulates this process.
Our data reveal that the findings obtained in the mouse model could be translated to human patients suffering from a fibrotic process.The analysis of transcriptomic data in a cohort of liver biopsies from HCC-naïve MASLD patients demonstrated that the EGFR signaling pathway increases in advanced stages of fibrosis and correlates with the expression of fibrotic and inflammatory genes identified in the in vivo and in vitro experiments.Expression of LOXL1, mentioned above as a clear candidate to be under the control of the EGFR pathway, as well as TIMP1, which has also been identified in some of the experimental analyses in the mouse model, showed a strong correlation with the EGF/EGFR signaling pathway.Also, it is worth highlighting CXCL10, whose expression shows a strong increase in advanced stages of fibrosis, positively correlated with the EGF/EGFR signaling pathway.Altogether, data from the human samples may indicate that genes that play essential roles during liver fibrosis/ inflammation may be under the control of the EGFR pathway.
In conclusion, the data presented here lead us to propose a model where the EGFR pathway in hepatocytes controls liver fibrosis and inflammation in response to a chronic insult on the liver, through remodeling the hepatocytes' gene transcriptome/secretome, and which has consequences on the deposition of ECM as well as on the immune microenvironment (Figure 6E).

Figure 1 .
Figure 1.The impairment of EGFR catalytic activity in hepatocytes attenuates CCl 4 -induced liver damage and myofibroblast activation.(A and B) Serum concentrations of alanine-and aspartate-aminotransferases (ALT and AST, respectively) (A) and hematoxylin and eosinstained liver sections (B) were analyzed to assess CCl 4 -induced liver damage.(C and D) Alpha-smooth muscle actin (α-SMA) expression was evaluated by immunostaining (quantification on the right) (C) and western blotting (densitometric analysis respect to β-actin on the right) (D), and data are expressed as fold-change versus mineral oil (MO).n = 4-7 animals per group.*p < 0.05, **p < 0.01 using Student's t-test.Scale bars: 500 and 100 μm (magnified views).

Figure 6 .
Figure 6.The EGFR pathway is activated in human fibrosis.(A) Boxplots of relative enrichment (GSVA score) of EGF/EGFR signaling and EGFR target genes across the fibrosis stages.(B) Pearson correlation of fibrosis-related genes' expression with EGF/EGFR signaling relative enrichment.(C) Boxplots of CXCL10/CXCL12 across the fibrosis stages.(D) Pearson correlations between CXCL10/CXCL12 gene expression and the EGF/EGFR signaling relative enrichment.Each dot is a sample (color indicates the fibrosis stage).(E) Schematic diagram depicting the major findings of the study.Created with BioRender.com.Data were analyzed using Kendall's tau test (p values were adjusted for multiple testing).
Animal protocols were approved by the Animal Experimentation Ethics Committee of the UCM and the Animal Welfare Division of the Environmental Affairs Council of the Government of Madrid (Proex 262.6/21).