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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

Antifibrotic effects of hepatic macrophages after MSCs treatment. Quantitative analyses of TGF-β1 (d), α-SMA (b), and COL1A2 (c) mRNA expression in hepatic stellate cells after 18 hours incubation with supernatants of hMø after in vitro treatments with DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) conditional medium. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01, ***p < 0.001; * vs. DMEM hMø supernatants
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Fig5: Antifibrotic effects of hepatic macrophages after MSCs treatment. Quantitative analyses of TGF-β1 (d), α-SMA (b), and COL1A2 (c) mRNA expression in hepatic stellate cells after 18 hours incubation with supernatants of hMø after in vitro treatments with DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) conditional medium. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01, ***p < 0.001; * vs. DMEM hMø supernatants

Mentions: Based on our results showing the induction of an anti-inflammatory/pro-regenerative profile in hMø shortly after MSCs treatment, we then asked whether factors secreted by macrophages could modulate HeSCs activation. For this purpose, the activated rat hepatic stellate cell line CFSC-2G was incubated 18 hours with conditioned media obtained from hMø preconditioned with MSCs supernatants or with DMEM. CFSC-2G cells were then washed and collected. Interestingly, TGF-β1, α-SMA and COL1A2 mRNA expression levels were downregulated in HeSCs treated with conditioned media from hMø preincubated with MSCs supernatants (Fig. 5a-c). From these data we can now conclude that hMø modulated by MSCs-released factors would likely be involved in the reduction of HeSCs activation previously described in our experimental model.Fig. 5


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
Antifibrotic effects of hepatic macrophages after MSCs treatment. Quantitative analyses of TGF-β1 (d), α-SMA (b), and COL1A2 (c) mRNA expression in hepatic stellate cells after 18 hours incubation with supernatants of hMø after in vitro treatments with DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) conditional medium. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01, ***p < 0.001; * vs. DMEM hMø supernatants
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Fig5: Antifibrotic effects of hepatic macrophages after MSCs treatment. Quantitative analyses of TGF-β1 (d), α-SMA (b), and COL1A2 (c) mRNA expression in hepatic stellate cells after 18 hours incubation with supernatants of hMø after in vitro treatments with DMEM (white bars), AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) conditional medium. ANOVA Tukey’s post-test, *p < 0.05; **p < 0.01, ***p < 0.001; * vs. DMEM hMø supernatants
Mentions: Based on our results showing the induction of an anti-inflammatory/pro-regenerative profile in hMø shortly after MSCs treatment, we then asked whether factors secreted by macrophages could modulate HeSCs activation. For this purpose, the activated rat hepatic stellate cell line CFSC-2G was incubated 18 hours with conditioned media obtained from hMø preconditioned with MSCs supernatants or with DMEM. CFSC-2G cells were then washed and collected. Interestingly, TGF-β1, α-SMA and COL1A2 mRNA expression levels were downregulated in HeSCs treated with conditioned media from hMø preincubated with MSCs supernatants (Fig. 5a-c). From these data we can now conclude that hMø modulated by MSCs-released factors would likely be involved in the reduction of HeSCs activation previously described in our experimental model.Fig. 5

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