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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

Effect of macrophages depletion in the liver fibrosis amelioration mediated by AdIGF-I-MSCs. a Representative images of Sirius Red stained-liver sections from fibrotic mice treated with saline, saline + LipClod, AdIGF-I-MSCs or AdIGF-I-MSCs + LipClod 2 weeks after treatments. b Morphometric analyses of Sirius Red-positive area. c mRNA expression levels of Col1A2 and d α-SMA in liver samples. ANOVA Tukey’s post-test; **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. saline; σp < 0.05 vs AdIGF-I-MSCs + LipClod; τp < 0.05, ττp < 0.01, τττp < 0.001, ττττ < 0.0001 vs. saline + LipClod
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Fig6: Effect of macrophages depletion in the liver fibrosis amelioration mediated by AdIGF-I-MSCs. a Representative images of Sirius Red stained-liver sections from fibrotic mice treated with saline, saline + LipClod, AdIGF-I-MSCs or AdIGF-I-MSCs + LipClod 2 weeks after treatments. b Morphometric analyses of Sirius Red-positive area. c mRNA expression levels of Col1A2 and d α-SMA in liver samples. ANOVA Tukey’s post-test; **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. saline; σp < 0.05 vs AdIGF-I-MSCs + LipClod; τp < 0.05, ττp < 0.01, τττp < 0.001, ττττ < 0.0001 vs. saline + LipClod

Mentions: We have herein shown that application of AdIGF-I-MSCs induces a shift in hMø toward an antifibrogenic/pro-regenerative phenotype. We have also shown that macrophages likely play a role in the reduction of HeSCs activation found in our therapeutic model. With these results, we wondered whether hMø might be relevant in the AdIGF-I-MSCs-mediated liver fibrosis amelioration. With this aim, TAA was chronically applied to 6–8-week-old BALB/c mice, three times a week, for 8 weeks. At the sixth week, animals were depleted from hMø by intravenous application of liposome-encapsulated clodronate, or saline as control (not shown). The day after, AdIGF-I-MSCs or vehicle were intravenously injected, and 2 weeks later animals were sacrificed and liver samples analyzed after Sirius Red staining (see Figure S3c). As shown in Fig. 6a-b, depletion of macrophages resulted in a significant abrogation of the therapeutic effect as seen by quantification of collagen deposits. No significant changes were found in liver fibrosis outcome when macrophage-depleted animals were treated with vehicle (control). Consistently, liver COL1A2 mRNA expression levels were found increased in AdIGF-I-MSCs-treated animals injected with clodronate (Fig. 6c). Furthermore, the effect of the therapeutic condition on the reduced activation of HeSCs was abrogated after partial depletion of macrophages as suggested by quantification of α-SMA mRNA expression levels (Fig. 6d). In conclusion, these results implicate hMø in the liver fibrosis amelioration achieved by AdIGF-I-MSCs infusion.Fig. 6


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
Effect of macrophages depletion in the liver fibrosis amelioration mediated by AdIGF-I-MSCs. a Representative images of Sirius Red stained-liver sections from fibrotic mice treated with saline, saline + LipClod, AdIGF-I-MSCs or AdIGF-I-MSCs + LipClod 2 weeks after treatments. b Morphometric analyses of Sirius Red-positive area. c mRNA expression levels of Col1A2 and d α-SMA in liver samples. ANOVA Tukey’s post-test; **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. saline; σp < 0.05 vs AdIGF-I-MSCs + LipClod; τp < 0.05, ττp < 0.01, τττp < 0.001, ττττ < 0.0001 vs. saline + LipClod
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Fig6: Effect of macrophages depletion in the liver fibrosis amelioration mediated by AdIGF-I-MSCs. a Representative images of Sirius Red stained-liver sections from fibrotic mice treated with saline, saline + LipClod, AdIGF-I-MSCs or AdIGF-I-MSCs + LipClod 2 weeks after treatments. b Morphometric analyses of Sirius Red-positive area. c mRNA expression levels of Col1A2 and d α-SMA in liver samples. ANOVA Tukey’s post-test; **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. saline; σp < 0.05 vs AdIGF-I-MSCs + LipClod; τp < 0.05, ττp < 0.01, τττp < 0.001, ττττ < 0.0001 vs. saline + LipClod
Mentions: We have herein shown that application of AdIGF-I-MSCs induces a shift in hMø toward an antifibrogenic/pro-regenerative phenotype. We have also shown that macrophages likely play a role in the reduction of HeSCs activation found in our therapeutic model. With these results, we wondered whether hMø might be relevant in the AdIGF-I-MSCs-mediated liver fibrosis amelioration. With this aim, TAA was chronically applied to 6–8-week-old BALB/c mice, three times a week, for 8 weeks. At the sixth week, animals were depleted from hMø by intravenous application of liposome-encapsulated clodronate, or saline as control (not shown). The day after, AdIGF-I-MSCs or vehicle were intravenously injected, and 2 weeks later animals were sacrificed and liver samples analyzed after Sirius Red staining (see Figure S3c). As shown in Fig. 6a-b, depletion of macrophages resulted in a significant abrogation of the therapeutic effect as seen by quantification of collagen deposits. No significant changes were found in liver fibrosis outcome when macrophage-depleted animals were treated with vehicle (control). Consistently, liver COL1A2 mRNA expression levels were found increased in AdIGF-I-MSCs-treated animals injected with clodronate (Fig. 6c). Furthermore, the effect of the therapeutic condition on the reduced activation of HeSCs was abrogated after partial depletion of macrophages as suggested by quantification of α-SMA mRNA expression levels (Fig. 6d). In conclusion, these results implicate hMø in the liver fibrosis amelioration achieved by AdIGF-I-MSCs infusion.Fig. 6

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