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Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells

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ABSTRACT

Background: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hMø) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hMø in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hMø behavior in the context of liver fibrosis resolution.

Methods: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8 weeks). In vivo gene expression analyses, in vitro experiments using hMø isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of Mø were performed.

Results: One day after treatment, hMø from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hMø from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hMø, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hMø depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hMø abrogated such effects on the expression of the most highly regulated genes.

Conclusions: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0424-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

In vivo modulation of hepatic macrophages expression profile by MSCs. mRNA expression levels of the nitric oxide production mediators arginase-1 and iNOS (a) and IL-10 and pro-fibrogenic cytokines TGF-β1, IL-6, TNF-α, and IL-1bβ (b) in macrophages at 1 day after saline (white bars), AdGFP-MSCs (gray) and AdIGF-I-MSCs (black) treatments. c IL-12 protein levels in hepatic macrophages supernatants relative to macrophages/saline condition. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline condition; σp < 0.05 and σσp < 0.01 vs. macrophages/AdGFP-MSCs condition
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Fig2: In vivo modulation of hepatic macrophages expression profile by MSCs. mRNA expression levels of the nitric oxide production mediators arginase-1 and iNOS (a) and IL-10 and pro-fibrogenic cytokines TGF-β1, IL-6, TNF-α, and IL-1bβ (b) in macrophages at 1 day after saline (white bars), AdGFP-MSCs (gray) and AdIGF-I-MSCs (black) treatments. c IL-12 protein levels in hepatic macrophages supernatants relative to macrophages/saline condition. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline condition; σp < 0.05 and σσp < 0.01 vs. macrophages/AdGFP-MSCs condition

Mentions: Considering that factors secreted by AdIGF-I-MSCs were shown to induce IGF-I and HGF expression in hepatocytes and that IGF-I is known to counteract TGF-β1 signaling [18], we next asked whether MSCs might induce changes in relevant genes involved in macrophages activity in vivo. For this purpose, enriched fractions of hMø, with more than 90% of cells being F4/80+ [16], were obtained at 1 day after saline/cellular treatments (see Additional file 1: Figure S1a). Interestingly, systemic administration of AdIGF-I-MSCs induced significant changes in the mRNA expression levels of enzymes involved in nitric oxide (NO) production in acutely isolated hMø from fibrotic livers (Fig. 2a). For instance, arginase-1 was upregulated after AdIGF-I-MSCs when compared to controls (Fig. 2a). In addition, iNOS was downregulated in hMø isolated from AdGFP-MSCs-treated mice when compared to vehicle, and its mRNA levels were further reduced in AdIGF-I-MSCs condition. Moreover, the mRNA expression of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, was significantly downregulated after AdIGF-I-MSCs treatment when compared to vehicle (Fig. 2b). Furthermore, TNF-α and IL-6 mRNA expression levels were downregulated in the AdIGF-I-MSCs group when compared to control MSCs (Fig. 2b). However, hMø IL-10 mRNA expression levels were upregulated only after AdGFP-MSCs treatment. In addition, reduced IL-12 protein levels were found in the supernatant of AdGFP-MSCs hMø when compared to vehicle, and such changes were much more remarkable in AdIGF-I-MSCs hMø condition (Fig. 2c). It is worth noting that mRNA expression levels of TGF-β1, an anti-inflammatory but pro-fibrogenic cytokine, were significantly reduced in AdIGF-I-MSCs hMø in comparison with vehicle and AdGFP-MSCs groups. Altogether, these results suggest that in vivo applications of AdGFP-MSCs and AdIGF-I-MSCs differentially regulate the expression profile of hMø in the TAA liver fibrosis model.Fig. 2


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
In vivo modulation of hepatic macrophages expression profile by MSCs. mRNA expression levels of the nitric oxide production mediators arginase-1 and iNOS (a) and IL-10 and pro-fibrogenic cytokines TGF-β1, IL-6, TNF-α, and IL-1bβ (b) in macrophages at 1 day after saline (white bars), AdGFP-MSCs (gray) and AdIGF-I-MSCs (black) treatments. c IL-12 protein levels in hepatic macrophages supernatants relative to macrophages/saline condition. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline condition; σp < 0.05 and σσp < 0.01 vs. macrophages/AdGFP-MSCs condition
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Fig2: In vivo modulation of hepatic macrophages expression profile by MSCs. mRNA expression levels of the nitric oxide production mediators arginase-1 and iNOS (a) and IL-10 and pro-fibrogenic cytokines TGF-β1, IL-6, TNF-α, and IL-1bβ (b) in macrophages at 1 day after saline (white bars), AdGFP-MSCs (gray) and AdIGF-I-MSCs (black) treatments. c IL-12 protein levels in hepatic macrophages supernatants relative to macrophages/saline condition. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01 and ***p < 0.001 vs. macrophages/saline condition; σp < 0.05 and σσp < 0.01 vs. macrophages/AdGFP-MSCs condition
Mentions: Considering that factors secreted by AdIGF-I-MSCs were shown to induce IGF-I and HGF expression in hepatocytes and that IGF-I is known to counteract TGF-β1 signaling [18], we next asked whether MSCs might induce changes in relevant genes involved in macrophages activity in vivo. For this purpose, enriched fractions of hMø, with more than 90% of cells being F4/80+ [16], were obtained at 1 day after saline/cellular treatments (see Additional file 1: Figure S1a). Interestingly, systemic administration of AdIGF-I-MSCs induced significant changes in the mRNA expression levels of enzymes involved in nitric oxide (NO) production in acutely isolated hMø from fibrotic livers (Fig. 2a). For instance, arginase-1 was upregulated after AdIGF-I-MSCs when compared to controls (Fig. 2a). In addition, iNOS was downregulated in hMø isolated from AdGFP-MSCs-treated mice when compared to vehicle, and its mRNA levels were further reduced in AdIGF-I-MSCs condition. Moreover, the mRNA expression of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, was significantly downregulated after AdIGF-I-MSCs treatment when compared to vehicle (Fig. 2b). Furthermore, TNF-α and IL-6 mRNA expression levels were downregulated in the AdIGF-I-MSCs group when compared to control MSCs (Fig. 2b). However, hMø IL-10 mRNA expression levels were upregulated only after AdGFP-MSCs treatment. In addition, reduced IL-12 protein levels were found in the supernatant of AdGFP-MSCs hMø when compared to vehicle, and such changes were much more remarkable in AdIGF-I-MSCs hMø condition (Fig. 2c). It is worth noting that mRNA expression levels of TGF-β1, an anti-inflammatory but pro-fibrogenic cytokine, were significantly reduced in AdIGF-I-MSCs hMø in comparison with vehicle and AdGFP-MSCs groups. Altogether, these results suggest that in vivo applications of AdGFP-MSCs and AdIGF-I-MSCs differentially regulate the expression profile of hMø in the TAA liver fibrosis model.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cirrhosis is a major health problem worldwide and new therapies are needed. Hepatic macrophages (hM&oslash;) have a pivotal role in liver fibrosis, being able to act in both its promotion and its resolution. It is well-known that mesenchymal stromal cells (MSCs) can modulate the immune/inflammatory cells. However, the effects of MSCs over hM&oslash; in the context of liver fibrosis remain unclear. We previously described evidence of the antifibrotic effects of in vivo applying MSCs, which were enhanced by forced overexpression of insulin-like growth factor 1 (AdIGF-I-MSCs). The aim of this work was to analyze the effect of MSCs on hM&oslash; behavior in the context of liver fibrosis resolution.

Methods: Fibrosis was induced in BALB/c mice by chronic administration of thioacetamide (8&nbsp;weeks). In vivo gene expression analyses, in vitro experiments using hM&oslash; isolated from the nonparenchymal liver cells fraction, and in vivo experiments with depletion of M&oslash; were performed.

Results: One day after treatment, hM&oslash; from fibrotic livers of MSCs-treated animals showed reduced pro-inflammatory and pro-fibrogenic gene expression profiles. These shifts were more pronounced in AdIGF-I-MSCs condition. This group showed a significant upregulation in the expression of arginase-1 and a higher downregulation of iNOS expression thus suggesting decreased levels of oxidative stress. An upregulation in IGF-I and HGF expression was observed in hM&oslash; from AdIGF-I-MSCs-treated mice suggesting a restorative phenotype in these cells. Factors secreted by hM&oslash;, preconditioned with MSCs supernatant, caused a reduction in the expression levels of hepatic stellate cells pro-fibrogenic and activation markers. Interestingly, hM&oslash; depletion abrogated the therapeutic effect achieved with AdIGF-I-MSCs therapy. Expression profile analyses for cell cycle markers were performed on fibrotic livers after treatment with AdIGF-I-MSCs and showed a significant regulation in genes related to DNA synthesis and repair quality control, cell cycle progression, and DNA damage/cellular stress compatible with early induction of pro-regenerative and hepatoprotective mechanisms. Moreover, depletion of hM&oslash; abrogated such effects on the expression of the most highly regulated genes.

Conclusions: Our results indicate that AdIGF-I-MSCs are able to induce a pro-fibrotic to resolutive phenotype shift on hepatic macrophages, which is a key early event driving liver fibrosis amelioration.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0424-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus