IMT504 inhibits the progression of fibrogenesis in mice

Considering the immunomodulatory properties of IMT504, we wonder if this ODN could inhibit the progression of liver fibrogenesis. We first verified that TAA treatment led to established fibrosis in GLASTCreERT2; Rosa26Tom (Tx P2) mice after 8 weeks (Fig. 1A). This was assessed by measuring the percentage of Sirius red stained area and quantifying the area of α-smooth muscle actin immunoreactivity. Mice administered a single dose of IMT504, 2 weeks after TAA treatment onset, exhibited a significant reduction in fibrosis degree compared to the control group (Fig. 1B, C; Figure S1). This effect was even more pronounced when mice received 3 doses of IMT504, administered with a 2 week-interval between each dose (Fig. 1B, C; Figure S1). A proinflammatory liver microenvironment, typically associated with the activation of hepatic stellate cells, hinders liver regeneration and hepatocyte proliferation [11]. Supporting the notion that IMT504 plays an immunomodulatory role, an increase in hepatocyte proliferation was found, in relation with the number of IMT504 doses administered (Figure S2).

To delve into the early mechanisms triggered by IMT504 in the liver, wild-type littermates of GLASTCreERT2; Rosa26Tom (Tx P2) mice were treated with TAA for 2 weeks. These mice were subsequently injected with IMT504 (or vehicle) and were sacrificed after 1 day. Ample material from both the entire liver and parenchymatic fractions were collected, which were then processed for qPCR. IMT504 treatment exhibited a significant reduction in mRNA expression levels of profibrogenic markers (col1a1, α-SMA and TGF-β1) as well as pro-inflammatory markers (TNF-α, IL-1β, IL-6 and iNOS) (Fig. 1D). Furthermore, it led to an increase in tolerogenic markers (IL-10 and Arginase-1). Additionally, within the parenchymatic fraction, an upregulation of markers associated with hepatoblast/hepatocyte proliferation (HGF, PCNA, Notch2), hepatocyte function (IGF-I), DNA synthesis and repair quality control (Brca2, Myb, Abl1), and positive regulation of cell-cycle progression (Ccna1) was found. Conversely, the expression levels of Gadd45α (a marker of cellular stress, DNA damage and cell cycle arrest) were found to be downregulated. Interestingly, two markers of liver progenitor cells, TWEAK (TNF-like weak inducer of apoptosis) and CD133 [28, 29], were found to be overexpressed in the liver parenchymatic fraction of animals treated with the ODN.

To ascertain whether IMT504 could alleviate liver fibrosis in a different in vivo model with a distinct etiology, CD1 mice were subjected to bile-duct ligation (BDL). Five days after BDL, these mice were administered IMT504 or saline and were euthanized 9 days later. IMT504 treatment was associated with reduced collagen fiber deposits and myofibroblast activation, while also stimulating hepatocyte proliferation (Figure S3). Consequently, these findings confirm the ability of IMT504 to ameliorate liver fibrosis in different in vivo mouse models.

IMT504 facilitates liver fibrosis regression in mouse TAA model

We subsequently investigated whether IMT504 could also facilitate the regression of pre-existing fibrosis. To address this, a single dose of IMT504 or vehicle was administered to wild-type littermates of GLASTCreERT2; Rosa26Tom (Tx P2) mice after a 8-week TAA treatment (Fig. 2A). Following this intervention, the animals underwent a two-week recovery period without further TAA applications. After this period, the mice were humanely euthanized, and their livers were extracted and processed for analysis.

The administration of IMT504 significantly improved fibrosis regression: it led to a substantial reduction in both the Sirius Red stained-area (Fig. 2) and the area immunostained for α-smooth muscle actin, a marker of myofibroblast activation (Figure S4). Additionally, there was a significantly increase in the number of PCNA+ hepatocytes, indicating enhanced proliferation, in the IMT504-treated group (Figure S4). Furthermore, our analysis of mRNA expression levels at the end of the experiment revealed promising findings (Fig. 2). When compared to the vehicle control, IMT504 condition resulted in significant reductions of profibrogenic and proinflammatory markers. Some of them, such as TGF-β1 and IL-1β, even return to normal levels. Notably, IL-10 expression remained significantly upregulated, while iNOS expression was downregulated compared to control groups, including naïve mice. Hepatocyte proliferation and functional markers were also upregulated. Collectively, these data support the conclusion that IMT504 accelerates recovery from liver fibrosis.

To investigate the early mechanisms underlying IMT504-mediated acceleration of liver fibrosis regression, we conducted new analyses. Animals treated with TAA for 8 weeks were sacrificed 24 h after the application of IMT504, and their liver samples were processed for qPCR analysis. The results closely resembled those obtained when IMT504 was administered at 2 weeks of TAA treatment, specifically concerning profibrogenic, proinflammatory, hepatocyte proliferation, and hepatocyte progenitor markers (Figure S4).

IMT504 likely enhances the contribution of GLAST+ Wnt1+ BMSPs with endothelial-like cells and hepatocyte-like cells during liver fibrogenesis

Previously, we demonstrated that GLAST+ Wnt1+ BMSPs, a subpopulation of perisinusoidal stromal cells, become mobilized to the peripheral blood and may contribute to ELCs and HLCs following liver injury [9]. Given that IMT504 is known to induce the mobilization of BMSPs into the bloodstream [16], we sought to determine whether this ODN could also enhance the contribution of the subpopulation of GLAST+ Wnt1+ BMSPs to liver cells during fibrogenesis.

To address this, we crossed GLASTCreERT2 with Rosa26Tom heterozygous mice (Fig. 1A). The resulting double-transgenic offspring enable the identification of GLAST+ cells at the moment of tamoxifen (Tx) injection through the expression of the tdTomato (Tom) reporter gene. This allows us to identify GLAST+ Wnt1+ BMSPs in the bone marrow or, if mobilized through the bloodstream, in the organs where they are recruited, and to determine the cell types they give rise to. Adult GLASTCreERT2; Rosa26Tom mice (Tx postnatal day -P-2) were injected intraperitoneally (i.p.) with TAA for 8 weeks. Consistent with our hypothesis, animals that received a single dose of IMT504 at the second week of TAA treatment exhibited a higher incidence of Tom+ ELCs (Fig. 3; Movies S1 and S2) and HLCs (Fig. 4) compared to the vehicle control group. Moreover, this effect was significantly more pronounced in animals receiving three doses of IMT504 -administered at the 2nd, 4th and 6th weeks of TAA treatment- (Figs. 3 and 4; Movie S3). Interestingly, IMT504 was also found to promote the proliferation of GLAST-traced ELCs and HLCs in the liver after 8 weeks of TAA treatment (Figs. 3 and 4).

Fig. 3

IMT504 likely enhances the contribution of GLAST+ Wnt1+ BMSPs with ELCs in the liver during fibrogenesis. (A) Representative microphotographs showing ELCs Tom⁺ in GLASTCreERT2; Rosa26Tom mice (Tx P2) of different experimental groups. Scale bars: 20 μm. (B) Statistical comparisons of numbers of endothelial-like Tom+ cells; n = 4. (C) Representative flow cytometry plots showing the abundance of CD31+ Tom+ in the non-parenchyma enriched fraction of the liver. (D) Statistical comparisons of percentage of CD31+ Tom+ ELCs, in the non-parenchymatic liver fraction, from flow cytometry analyses; n = 5. (E) Statistical comparisons of percentage of Ki67+ cells among the total of CD31+ Tom+ ELCs, from flow cytometry analyses; n = 4. (B, D,E) *p < 0.05; **p < 0.01; ****p < 0.0001. Tukey`s multiple comparisons test

Fig. 4

IMT504 likely enhances the contribution of GLAST+ Wnt1+ BMSPs with HLCs in the liver during fibrogenesis. (A) Representative microphotographs showing Tom+ HLCs in GLASTCreERT2; Rosa26Tom mice (Tx P2) of different treatments. Scale bars: 20 μm. (B) Statistical comparisons of numbers of Tom+ HLCs; n = 4. (C) Representative flow cytometry plots showing the abundance of Albumin+ Tom+ in the parenchyma enriched fraction of the liver. (D) Statistical comparisons of the percentage of Albumin+ Tom+ HLCs, from flow cytometry analyses; n = 4. (E) Statistical comparisons of percentage of Ki67+ cells among the total of Albumin+ Tom+ HLCs, from flow cytometry analyses; n = 4. (B, D, E) ***p < 0.001; ****p < 0.0001; Tukey`s multiple comparison test

We then investigated whether GLAST+ Wnt1+ BMSPs can acquire the expression of ELC and HLC specific markers both in vitro and in vivo. To achieve this, we cultured a fraction enriched in this BMSP subpopulation for 7 days with conditioned media from hepatectomized liver, which led to the acquisition of ELC and HLC specific markers (Figure S5). Additionally, we injected the bone marrow mononuclear fraction obtained from Wnt1Cre; Rosa26Tom mice into athymic mice that had undergone hepatectomy 5 days prior. Five days post-transplant, flow cytometry analysis revealed that a fraction of Tom+ cells coexpressing CD44 and fibronectin had acquired ELC (Figure S6) and HLC specific markers (Figure S7). This suggests that GLAST+ Wnt1+ BMSPs may have the potential to differentiate into both ELCs and HLCs.

In addition, we found that CD44+ Tom+ cells in the bone marrow and peripheral blood coexpress CD133, a well-known progenitor marker (Figure S8A, B). CD133 is also a liver progenitor marker in cells that coexpress albumin [28]. Therefore, our data may suggest that GLAST+ Wnt1+ BMSPs are likely to acquire ELC and HLCs specific markers and contribute to liver progenitors. Given the possibility that some GLAST+ endothelial and/or hepatocyte progenitors may reside in the liver, we analyzed the incidence of CD31+ Tom+ cells, and the coexpression of CD133 and albumin in Tom+ cells, by flow cytometry. Virtually no Tom+ ELCs were found in the liver of naïve or IMT504-treated double-transgenic mice injected with tamoxifen at P60 (Figure S8F). And with regards to the liver parenchymatic fraction, in some, but not all, naïve and IMT504-treated GLASTCreERT2; Rosa26Tom (TxP2) mice a minor fraction of CD133+ Albumin+ Tom+ cells was found (0.012 ± 0.0169% and 0.0237 ± 0.0336%; naïve and IMT504, respectively; of total parenchymatic cells). A similar pattern was observed when CRE recombination (Tom expression) was induced at P60, although the incidence of CD133+ Albumin+ Tom+ was reduced (0.0004 ± 0.0008% and 0.0011 ± 0.0001%; naïve vs. IMT504) (Figure S8C, G). In contrast, significantly higher numbers of CD31+ Tom+ ELCs and CD133+/Ep-CAM+ Albumin+ Tom+ HLCs were observed in the liver of all TxP2- and Tx P60-injected mice treated with TAA and IMT504 (Figure S8C, G). Overall, these findings suggest that although some GLAST+ liver progenitors may eventually exist in the liver, non-resident Tom+ cells (likely GLAST+ Wnt1+ BMSPs) give rise to Tom+ ELCs, HLCs and liver progenitors following injury, with IMT504 expanding these subpopulations.

IMT504 enhances the incidence within the bone marrow of GLAST+ Wnt1+ BMSPs in the context of fibrosis

Previously, we demonstrated that TAA reduces the capacity of GLAST+ Wnt1+ BMSPs to form dense colonies in a colony-forming unit assay during early liver fibrogenesis [9]. We asked whether IMT504 treatment could expand the GLAST+ Wnt1+ BMSPs pool and ultimately restore their ability to form dense colonies again. This could partially explain the increased contribution of GLAST+ Wnt1+ BMSP-derived cells to liver regeneration mediated by IMT504. To address this, we systemically administered IMT504 to Wnt1Cre; Rosa26Tom mice previously treated with TAA during 2 weeks. Eighteen hours later, we performed a colony-forming unit-fibroblasts (CFU-Fs) assay using the BM-MNCs fraction. A significant increased incidence of Tom+ CFU-Fs was observed in the IMT504-treated group compared to the naïve control (Fig. 5A, B). Similarly, when IMT504 was applied to TAA-treated mice, the number of Tom+ CFU-Fs increased compared to TAA-treated animals receiving a vehicle injection (Fig. 5A, B). Interestingly, the increase in CFU-Fs induced by IMT504 was only seen in the Tom+ BMSP subpopulation (Fig. 5A, B). Notably, IMT504 was found to restore the capacity of GLAST+ Wnt1+ BMSPs to form dense colonies (Fig. 5C).

Fig. 5

In vivo effect of IMT504 on Wnt1 or GLAST-traced bone marrow mononuclear cells. (A) Statistical comparisons of numbers of CFU-Fs, Tom+ or Tom−, obtained from BM-MNCs of Wnt1Cre; Rosa26Tom which were or not treated with TAA during 2 weeks, and were s.c. injected or not with IMT504. (B) Statistical comparisons of percentage of CFU-Fs Tom+. (A, B) Dunn`s multiple comparison test; n = 6. (C) Microphotographs showing representative colonies obtained from naïve, TAA 2w and TAA 2w + IMT504 treated mice. Scale bar: 50 μm. (D, left) Statistical comparisons of percentage of CD44+ Tom+ cells within the bone marrow after 2 weeks of TAA and/or IMT504 treatments, as measured by flow cytometry. (D, right) Statistical comparisons of percentage of Ki67+ cells among the total of CD44+ Tom+ cells within the bone marrow from GLASTCreERT2; Rosa26Tom (Tx P2) after 2 weeks of TAA and IMT504/saline treatments, as measured by flow cytometry. (E) Statistical comparisons of percentage of CD44+ Tom+ cells within the peripheral blood after 2 weeks of TAA and IMT504/saline treatments, as measured by flow cytometry. (D, E) Turkey`s multiple comparison test; n = 4. (A, B,D, E) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

As Wnt signaling is frequently associated with proliferation and mobilization of progenitor cells [30,31,32,33], we sought to determine whether IMT504 could de novo activate Wnt1 in Tom− stromal progenitor cells. BM-MNCs from Wnt1Cre; Rosa26Tom mice were plated in 6 well-plates and Tom+ and Tom− colonies were quantified once colonies formed (control, 0 h). Subsequently, these cultures were incubated with IMT504 for 2 h, and the percentage of colonies was re-quantified 24 and 48 h later. As shown in Figure S9, there were no significant changes in the proportion of Tom+ colonies. From this result, we conclude that IMT504 is unlikely to induce de novo activation of the Wnt1 promoter in previously Tom− BMSPs.

We then analyzed the incidence of GLAST+ Wnt1+ BMSPs in the BM and peripheral blood of GLASTCreERT2; Rosa26Tom mice (Tx P2 or P60) treated with TAA during 2 weeks and injected with IMT504 or vehicle 24 h before, by flow cytometry. As expected, the frequency of CD44+ Tom+ cells notably rose in the bone marrow after IMT504 treatment (Fig. 5D, left; Figure S8D; Figure S10A). Furthermore, liver injury prompted the mobilization of GLAST+ Wnt1+ BMSPs toward the peripheral blood, a phenomenon significantly potentiated by IMT504 (Fig. 5E; Figure S8E). Hence, IMT504 appears to stimulate both the expansion and mobilization of GLAST+ Wnt1+ BMSPs in vivo. Similar results were found in bone marrow and peripheral blood samples obtained from mice treated with TAA during 8 weeks and three doses of IMT504 (Figure S11).

In summary, these results suggest that IMT504 induces the expansion of GLAST+ Wnt1+ BMSPs and restores their capacity to form dense colonies in the context of TAA-mediated early fibrogenesis. These findings lead us to conclude that IMT504 can promote the expansion of GLAST+ Wnt1+ BMSPs in vivo, likely contributing to the increase incidence of Tom+ ELCs and HLCs during liver fibrogenesis.

IMT504 stimulates the proliferation of GLAST + Wnt1 + BMSPs with the induction of Wnt signaling pathway

To assess whether IMT504 could stimulate the proliferation of GLAST+ Wnt1+ BMSPs in vivo, we conducted flow cytometry analyses and quantified the percentage of GLAST+ Wnt1+ BMSPs expressing Ki67 in the BM of GLASTCreERT2; Rosa26Tom mice (Tx P2) treated with or without TAA over a 2-week period, followed by euthanasia one day after the administration of a single dose of IMT504 or vehicle. IMT504 was able to augment the proportion of GLAST+ Wnt1+ BMSPs when applied to naïve mice (Fig. 5D, right). As anticipated, TAA treatment diminished the proliferative capacity of GLAST+ Wnt1+ BMSPs, a phenotype which was rescued by IMT504 (Fig. 5D, right; Figure S10B). Additionally, at one day post-treatment, IMT504 not only increased the abundance of CD31+ Tom+ ELCs following 2 weeks of TAA treatment (Figure S10C), but also augmented the proportion of Ki67+ proliferating Tom+ cells within the CD31+ cell population (Figure S10D). These features were also found in the fraction of Albumin+ Tom+ HLCs (Figure S10E, F). These findings corroborate the data presented in Figs. 3 and 4, suggesting that increased proliferation of Tom+ ELCs and Tom+ HLCs occur shortly after the application of the oligodeoxynucleotide.

To further confirm the role of IMT504 in promoting the proliferation of GLAST+ Wnt1+ BMSPs, we established primary cultures from GLASTCreERT2; Rosa26Tom BM-MNCs and separately expanded fractions enriched in Tom+ and Tom− cells, allowing them to reach at least 10 passages (P10) in vitro. By P7, 79.97 ± 1.29% of cells in the Tom+ cultures expressed the reporter gene, whereas virtually no Tom+ cells were detected in Tom− cultures (not shown). To assess the impact of IMT504 on the proliferation of P8 GLAST+ Wnt1+ BMSPs, Tom+ and Tom− fractions were pretreated with IMT504 for 2 h. After 4 h, flow cytometry analysis revealed a significantly increase in Ki67+ proliferating BMSPs, rising from ~ 30% to ~ 75%, exclusively in the Tom+ cells incubated with IMT504 (Fig. 6A, B). This was confirmed in P8 GLAST+ Wnt1+ BMSPs derived from Wnt1Cre; Rosa26Tom mice (Fig. 6B).

Fig. 6

Effect of IMT504 on the proliferation of P8 GLAST+ Wnt1+ BMSPs (A) Representative histograms from flow-cytometry analyses of Tom+ cells, treated or not with IMT504, immunolabeled for Ki67, and (B) statistical comparisons in between Tom+ and/or Tom- cells of different experimental conditions obtained from different double-transgenic mice; left, Tukey´s multiple comparisons test (n = 6); right: t-student (n = 5). (C) Statistical comparisons showing changes in Wnt1, β-catenin and Cyclin-D1 mRNA expression levels, obtained by qPCR analyses, in between vehicle-treated Tom+ and Tom− cells. (D) Statistical comparisons showing changes in Wnt1, β-catenin and Cyclin-D1 expression levels, obtained by qPCR analyses, in between cells which were treated with vehicle (Ctrl) or IMT504. (E) Experimental schematic figure, representative histograms from flow-cytometry analyses of Tom+ cells immunolabeled for Ki67 and statistical comparisons in between different experimental conditions. (C-E) Tukey´s multiple comparisons test; n = 4. (B-E) *p < 0.05; **p < 0.01; ***p < 0,001; ****p < 0.0001

Given our ability to trace GLAST+ Wnt1+ BMSPs owing to their early expression of Wnt1, and considering that Wnt signaling is known to induce the proliferation and mobilization of various progenitors, we then asked whether GLAST+ Wnt1+ BMSPs express Wnt1 and Wnt signaling pathway or target markers in the adult. Semiconfluent P8 Tom+ and Tom− cells were deprived from serum during 22–40 h, and samples were processed for qPCR analysis. Notably, mRNA expression levels of Wnt1, β-catenin and the canonical Wnt signaling target gene Cyclin-D1 [34] were higher in Tom+ compared to Tom− BMSPs (Fig. 6C). Moreover, their expression levels further increased in Tom+ cells when subjected to longer incubation times in serum-deprived conditions, whereas they remained unchanged in Tom− cells.

Since Wnt1 signaling pathway is specifically active in GLAST+ Wnt1+ BMSPs, we asked whether Wnt signaling might be modulated by IMT504 in these progenitors. To explore this, P8 Tom+ and Tom− cells were incubated with IMT504 or with vehicle for 2 h, and were allowed to recover for 4–22 h in a serum deficient culture medium. Subsequently, samples were collected and processed for qPCR analysis. While Wnt1 mRNA expression levels remained unchanged in Tom− cells, a 400x increase in this marker was observed in Tom+ cells after 2 + 4 h, and it was further upregulated in the 2 + 22 h IMT504-treatment condition (Fig. 6D). Similar patterns were observed for beta-catenin and Cyclin-D1. Additionally, other genes positively regulated by Wnt, including Axin2, Lef1 and Sp5, exhibited similar patterns of overexpression, along with a minor although significant increase in the ligand Wnt3a (Figure S12).

To analyze whether Wnt signaling is involved in the observed induction of GLAST+ Wnt1+ BMSPs proliferation by IMT504, we treated these cells with IMT504 and either Dkk1 (a Wnt antagonist) or IWR1 (a Wnt inhibitor). This treatment blocked the proliferative effect of IMT504 (Fig. 6E). Furthermore, the ability of IMT504 to induce Wnt signaling in GLAST+ Wnt1+ BMSPs was confirmed by incubating the cells with CHIR, a specific Wnt activator (57.83 ± 2.8 vs. 33.41 ± 1,19; CHIR vs. DMEM; p < 0.0001; n = 3; t-student test). In conclusion, our results suggest that IMT504 stimulates the proliferation of GLAST + Wnt1 + BMSPs likely through the induction of the Wnt signaling pathway.

IMT504 promotes the mobilization of in vitro expanded GLAST + Wnt1 + BMSPs

To determine if IMT504 could enhance the motility of P8 GLAST+ Wnt1+ BMSPs, we conducted an in vitro Boyden chamber assay. As expected, IMT504-treated Tom+ cells exhibited the highest motility score (Fig. 7). Interestingly, when GLAST+ Wnt1+ BMSPs were treated with IMT504, they displayed the ability to migrate even in the absence of chemoattractants to the same extent as Tom− cells in the presence of conditioned media from LX2 cells, a condition known to induce the migration of stromal progenitor cells [35]. Furthermore, Tom+ cells displayed an overall higher motility capacity compared to Tom− with the latter showing limited mobilization ability.

Fig. 7

Effect of IMT504 on the mobilization of P8 GLAST+ Wnt1+ BMSPs. (A) Representative images showing DAPI staining on cells which have passed through membrane pores during 4 h in a Boyden chamber assay; scale bars: 50 μm. (B) Statistical comparisons of different experimental conditions; Tukey`s multiple comparisons test; n = 5. (C) Statistical comparisons showing changes in E-cadherin expression levels, as measured by qPCR, in between Tom+ and/or Tom− cells which were pretreated or not with IMT504; n = 3. (B, C) *p < 0,05; **p < 0,01; ***p < 0.001; ****p < 0.0001; Tukey´s multiple comparisons test

It is well established that Wnt canonical signaling pathway can influence cell motility by downregulating E-cadherin expression levels [36]. In line with this, incubating P8 GLAST + Wnt1 + BMSPs with IMT504 and allowing them to recover for 4 h resulted in reduced E-cadherin mRNA expression levels compared to control Tom+ cells. These levels were further reduced when Tom+ cells were collected 22 h after IMT504 stimulation (Fig. 7C). In contrast, no significant changes were observed in Tom− cells, whether or not they received IMT504 treatment. Interestingly, control Tom+ cells exhibited a significant reduction in E-cadherin expression levels when cultured for 40 h compared to 22 h (Fig. 7C and Figure S13). Additionally, IMT504 induced an overexpression of Wnt5a mRNA levels in Tom+ cells (Figure S13), suggesting the potential involvement of non-canonical Wnt signaling pathways in their increased migratory behavior of GLAST + Wnt1 + BMSPs, which requires further investigation. Collectively, these findings indicate that in vitro expanded GLAST + Wnt1 + BMSPs possess greater motility capacity than other BMSP subpopulations, and IMT504 significantly enhances their mobilization.

Effect of IMT504 and conditioned media derived from in vitro expanded GLAST + Wnt1 + BMSPs on the expression profile of various liver cell types

We finally asked if IMT504 and/or the CM obtained from IMT504 treated Tom+ cells could directly and specifically change the mRNA expression profile of diverse liver cells involved in fibrogenesis. First, primary cultured macrophages that were previously isolated from fibrotic livers were incubated with IMT504 or CM from IMT504-treated Tom+ or Tom− cells or subjected to other control conditions. Only IMT504 or CM from IMT504-treated Tom+ cells were able to alter the expression profile of these macrophages. Macrophages changed from an inflammatory into a tolerogenic phenotype, characterized by reduction in TNF-α and iNOS levels, and an increase in IL-10 and/or Arginase-1 mRNA levels. Additionally, they acquired a proregenerative phenotype, marked by an increase in HGF, with the IMT504 treatment showing a more pronounced effect compared to CM from IMT504-treated Tom+ cells (Fig. 8).

Fig. 8

Effect of IMT504 or conditioned media (CM) from GLAST+ Wnt1+ BMSPs or Tom− BMSPs treated with IMT504 or vehicle (veh) on the expression pattern of different liver cell populations. (A) Statistical comparisons of TNF-α, IL-10, arginase 1, iNOS and HGF mRNA levels in primary cultured macrophages obtained from fibrotic livers (treated with thioacetamide for 6 weeks) after different experimental treatments, as measured by qPCR. (B) Statistical comparisons of collagen 1a1, alpha-smooth muscle actin and TGF-β mRNA levels in CFSC-2G cells. (C) Statistical comparisons of PCNA, HGF, IGF-I and β-catenin mRNA levels in primary cultured hepatocytes from healthy livers. *p < 0,05; **p < 0,01; ***p < 0.001; ****p < 0.0001; Tukey´s multiple comparisons test; n = 3

Under the same experimental conditions, IMT504 and CM from IMT504-treated Tom+ cells were the only stimuli capable of reducing the activation profile of CFSC-2G rat hepatic stellate cells (Fig. 8). Furthermore, they upregulated HGF and IGF-I mRNA expression levels in primary cultured hepatocytes obtained from naïve CD1 mice (Fig. 8), without causing changes in PCNA or β-catenin mRNA expression levels in the latter cell type.

Based on these results, we can conclude that IMT504 has the ability to directly modulate the expression profile of macrophages, hepatic stellate cells and hepatocytes. This suggests that IMT504 could serve as a potent tool for ameliorating liver fibrosis in mouse. Additionally, the increase prevalence of GLAST + Wnt1 + BMSP-derived cells caused by IMT504 treatment may also play a role in this context, through paracrine mechanisms.