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

View Article: PubMed Central - PubMed

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.


MSCs secrete factors which modulate the hepatic macrophages phenotype. mRNA expression levels of nitric oxide production mediators arginase-1 and iNOS (a) and of cytokines involved in the fibrogenic process (b) in macrophages preincubated 18 hours with DMEM (white bars), or AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) supernatants. Similar comparisons were done at the protein level for IL-6 and IL-10 (supernatants; c and d, respectively) and TNF-α (cytoplasmic extract; e). ANOVA Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. macrophages/DMEM; σp < 0.05, σσp < 0.01 and σσσσp < 0.0001 vs. macrophages/AdGFP-MSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5120504&req=5

Fig3: MSCs secrete factors which modulate the hepatic macrophages phenotype. mRNA expression levels of nitric oxide production mediators arginase-1 and iNOS (a) and of cytokines involved in the fibrogenic process (b) in macrophages preincubated 18 hours with DMEM (white bars), or AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) supernatants. Similar comparisons were done at the protein level for IL-6 and IL-10 (supernatants; c and d, respectively) and TNF-α (cytoplasmic extract; e). ANOVA Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. macrophages/DMEM; σp < 0.05, σσp < 0.01 and σσσσp < 0.0001 vs. macrophages/AdGFP-MSCs

Mentions: In order to evaluate if the previously shown effects of MSCs could be explained by paracrine mechanisms (mediated by soluble factors acting at short range), hMø were isolated from fibrotic livers (after 6 weeks of TAA treatment) and were incubated for 18 hours with conditioned media from AdIGF-I-MSCs or AdGFP-MSCs cultures or with DMEM (see Additional file 1: Figure S1b). Cells were washed and collected, or medium was replaced by serum-starved DMEM for 24 h and supernatant/cells collected. Consistent with previous results, mRNA expression levels of arginase-1 were increased in hMø incubated with AdIGF-I-MSCs supernatant when compared to controls, while treatments with both MSCs supernatants resulted in downregulation of iNOS gene expression (Fig. 3a). Furthermore, TGF-β1, IL-6, and TNF-α mRNA levels were also reduced in hMø after incubation with MSCs supernatants when compared to DMEM, whereas IL-10 gene expression remained unaffected (Fig. 3b). However, IL-10 secreted protein levels were increase in supernatants of hMø pretreated with MSCs conditioned media, when compared to DMEM control (Fig. 3c). Interestingly, levels of IL-6 and TNF-α proteins were reduced in Ad-IGF-I-MSCs condition when compared to AdGFP-MSCs and vehicle (Fig. 3d,e). Altogether, these data suggest that paracrine mechanisms mediate MSCs modulatory activity on the gene expression profile of hMø.Fig. 3


Involvement of hepatic macrophages in the antifibrotic effect of IGF-I-overexpressing mesenchymal stromal cells
MSCs secrete factors which modulate the hepatic macrophages phenotype. mRNA expression levels of nitric oxide production mediators arginase-1 and iNOS (a) and of cytokines involved in the fibrogenic process (b) in macrophages preincubated 18 hours with DMEM (white bars), or AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) supernatants. Similar comparisons were done at the protein level for IL-6 and IL-10 (supernatants; c and d, respectively) and TNF-α (cytoplasmic extract; e). ANOVA Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. macrophages/DMEM; σp < 0.05, σσp < 0.01 and σσσσp < 0.0001 vs. macrophages/AdGFP-MSCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120504&req=5

Fig3: MSCs secrete factors which modulate the hepatic macrophages phenotype. mRNA expression levels of nitric oxide production mediators arginase-1 and iNOS (a) and of cytokines involved in the fibrogenic process (b) in macrophages preincubated 18 hours with DMEM (white bars), or AdGFP-MSCs (gray) or AdIGF-I-MSCs (black) supernatants. Similar comparisons were done at the protein level for IL-6 and IL-10 (supernatants; c and d, respectively) and TNF-α (cytoplasmic extract; e). ANOVA Tukey’s post-test; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. macrophages/DMEM; σp < 0.05, σσp < 0.01 and σσσσp < 0.0001 vs. macrophages/AdGFP-MSCs
Mentions: In order to evaluate if the previously shown effects of MSCs could be explained by paracrine mechanisms (mediated by soluble factors acting at short range), hMø were isolated from fibrotic livers (after 6 weeks of TAA treatment) and were incubated for 18 hours with conditioned media from AdIGF-I-MSCs or AdGFP-MSCs cultures or with DMEM (see Additional file 1: Figure S1b). Cells were washed and collected, or medium was replaced by serum-starved DMEM for 24 h and supernatant/cells collected. Consistent with previous results, mRNA expression levels of arginase-1 were increased in hMø incubated with AdIGF-I-MSCs supernatant when compared to controls, while treatments with both MSCs supernatants resulted in downregulation of iNOS gene expression (Fig. 3a). Furthermore, TGF-β1, IL-6, and TNF-α mRNA levels were also reduced in hMø after incubation with MSCs supernatants when compared to DMEM, whereas IL-10 gene expression remained unaffected (Fig. 3b). However, IL-10 secreted protein levels were increase in supernatants of hMø pretreated with MSCs conditioned media, when compared to DMEM control (Fig. 3c). Interestingly, levels of IL-6 and TNF-α proteins were reduced in Ad-IGF-I-MSCs condition when compared to AdGFP-MSCs and vehicle (Fig. 3d,e). Altogether, these data suggest that paracrine mechanisms mediate MSCs modulatory activity on the gene expression profile of hMø.Fig. 3

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.