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Effects of Interleukin-17A on Osteogenic Differentiation of Isolated Human Mesenchymal Stem Cells.

Osta B, Lavocat F, Eljaafari A, Miossec P - Front Immunol (2014)

Bottom Line: These levels decreased in combination with IL-17A at 6 h only.However, IL-17 decreased the TNF-α-induced BMP2 inhibition.Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics and Inflammation Research Unit EA 4130, Department of Clinical Immunology and Rheumatology, Edouard Herriot Hospital, University of Lyon 1 , Lyon , France.

ABSTRACT

Objectives: Rheumatoid arthritis (RA) is characterized by defective bone repair and excessive destruction and ankylosing spondylitis (AS) by increased ectopic bone formation with syndesmophytes. Since TNF-α and IL-17A are involved in both diseases, this study investigated their effects on the osteogenic differentiation of isolated human bone marrow-derived mesenchymal stem cells (hMSCs).

Methods: Differentiation of hMSCs into osteoblasts was induced in the presence or absence of IL-17A and/or TNF-α. Matrix mineralization (MM) was evaluated by alizarin red staining and alkaline phosphatase (ALP) activity. mRNA expression was measured by qRT-PCR for bone morphogenetic protein (BMP)-2 and Runx2, genes associated with osteogenesis, DKK-1, a negative regulator of osteogenesis, Schnurri-3 and receptor activator of nuclear factor kappa B ligand (RANKL), associated with the cross talk with osteoclasts, and TNF-α receptor type I and TNF-α receptor type II (TNFRII).

Results: TNF-α alone increased both MM and ALP activity. IL-17A alone increased ALP but not MM. Their combination was more potent. TNF-α alone increased BMP2 mRNA expression at 6 and 12 h. These levels decreased in combination with IL-17A at 6 h only. DKK-1 mRNA expression was inhibited by TNF-α and IL-17A either alone or combined. Supporting an imbalance toward osteoblastogenesis, RANKL expression was inhibited by TNF-α and IL-17A. However, TNF-α but not IL-17 alone decreased Runx2 mRNA expression at 6 h. In parallel, TNF-α but not IL-17 alone increased Schnurri-3 expression with a synergistic effect with their combination. This may be related to an increase of TNFRII overexpression.

Conclusion: IL-17 increased the effects of TNF-α on bone matrix formation by hMSCs. However, IL-17 decreased the TNF-α-induced BMP2 inhibition. Synergistic interactions between TNF-α and IL-17 were seen for RANKL inhibition and Schnurri-3 induction. Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA. Conversely, in the absence of osteoclasts, this could promote ectopic bone formation as in AS.

No MeSH data available.


Related in: MedlinePlus

Effects of IL-17A and/or TNF-α on extracellular matrix mineralization are shown. hMSCs were plated at a density 5 × 103 cell/cm2 and cultured for 21 days (A) in the absence (column 1) or presence (column 2) of osteogenic factors, 1 ng/ml TNF-α (column 3) or 50 ng/ml IL-17A (column 4), or both (column 5) were added or not to cultures. Plates were then stained with alizarin red, which colors calcium deposits in the extracellular matrix. (B) Results of day 17 were analyzed using the Wilcoxon test. *p  < 0.05; **p  < 0.005 vs. induction medium alone.
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Figure 1: Effects of IL-17A and/or TNF-α on extracellular matrix mineralization are shown. hMSCs were plated at a density 5 × 103 cell/cm2 and cultured for 21 days (A) in the absence (column 1) or presence (column 2) of osteogenic factors, 1 ng/ml TNF-α (column 3) or 50 ng/ml IL-17A (column 4), or both (column 5) were added or not to cultures. Plates were then stained with alizarin red, which colors calcium deposits in the extracellular matrix. (B) Results of day 17 were analyzed using the Wilcoxon test. *p  < 0.05; **p  < 0.005 vs. induction medium alone.

Mentions: A key characteristic of MSCs is their ability to differentiate into cells of different tissue lineages. Mineralization of the extracellular matrix is a marker of hMSCs differentiation into osteoblasts. To evaluate the effects of IL-17A and/or TNF-α on MM, hMSCs were cultured for 21 days in a medium supplemented with osteogenic factors with and without cytokines. Alizarin red staining was used to visualize mineralization. As shown in Figure 1A, column 2, culture of MSC with osteogenic factors alone induced a weak MM, which appeared at day 17 and reached its maximum level at day 21. Addition of IL-17A alone did not modify this mineralization (Figure 1A, column 3; Figure 1B). Addition of TNF-α enhanced this mineralization at day 17 (Figure 1A, column 4; Figure 1B, *p  < 0.05), which was further enhanced in the presence of the two cytokines (Figure 1A, column 5; Figure 1B, **p  < 0.005). At day 21, maximum levels were observed in each condition except in the negative control. Thus, these results show that TNF-α but not IL-17A alone enhanced bone mineralization, which was further potentiated but without acceleration by IL-17A.


Effects of Interleukin-17A on Osteogenic Differentiation of Isolated Human Mesenchymal Stem Cells.

Osta B, Lavocat F, Eljaafari A, Miossec P - Front Immunol (2014)

Effects of IL-17A and/or TNF-α on extracellular matrix mineralization are shown. hMSCs were plated at a density 5 × 103 cell/cm2 and cultured for 21 days (A) in the absence (column 1) or presence (column 2) of osteogenic factors, 1 ng/ml TNF-α (column 3) or 50 ng/ml IL-17A (column 4), or both (column 5) were added or not to cultures. Plates were then stained with alizarin red, which colors calcium deposits in the extracellular matrix. (B) Results of day 17 were analyzed using the Wilcoxon test. *p  < 0.05; **p  < 0.005 vs. induction medium alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4151036&req=5

Figure 1: Effects of IL-17A and/or TNF-α on extracellular matrix mineralization are shown. hMSCs were plated at a density 5 × 103 cell/cm2 and cultured for 21 days (A) in the absence (column 1) or presence (column 2) of osteogenic factors, 1 ng/ml TNF-α (column 3) or 50 ng/ml IL-17A (column 4), or both (column 5) were added or not to cultures. Plates were then stained with alizarin red, which colors calcium deposits in the extracellular matrix. (B) Results of day 17 were analyzed using the Wilcoxon test. *p  < 0.05; **p  < 0.005 vs. induction medium alone.
Mentions: A key characteristic of MSCs is their ability to differentiate into cells of different tissue lineages. Mineralization of the extracellular matrix is a marker of hMSCs differentiation into osteoblasts. To evaluate the effects of IL-17A and/or TNF-α on MM, hMSCs were cultured for 21 days in a medium supplemented with osteogenic factors with and without cytokines. Alizarin red staining was used to visualize mineralization. As shown in Figure 1A, column 2, culture of MSC with osteogenic factors alone induced a weak MM, which appeared at day 17 and reached its maximum level at day 21. Addition of IL-17A alone did not modify this mineralization (Figure 1A, column 3; Figure 1B). Addition of TNF-α enhanced this mineralization at day 17 (Figure 1A, column 4; Figure 1B, *p  < 0.05), which was further enhanced in the presence of the two cytokines (Figure 1A, column 5; Figure 1B, **p  < 0.005). At day 21, maximum levels were observed in each condition except in the negative control. Thus, these results show that TNF-α but not IL-17A alone enhanced bone mineralization, which was further potentiated but without acceleration by IL-17A.

Bottom Line: These levels decreased in combination with IL-17A at 6 h only.However, IL-17 decreased the TNF-α-induced BMP2 inhibition.Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA.

View Article: PubMed Central - PubMed

Affiliation: Immunogenomics and Inflammation Research Unit EA 4130, Department of Clinical Immunology and Rheumatology, Edouard Herriot Hospital, University of Lyon 1 , Lyon , France.

ABSTRACT

Objectives: Rheumatoid arthritis (RA) is characterized by defective bone repair and excessive destruction and ankylosing spondylitis (AS) by increased ectopic bone formation with syndesmophytes. Since TNF-α and IL-17A are involved in both diseases, this study investigated their effects on the osteogenic differentiation of isolated human bone marrow-derived mesenchymal stem cells (hMSCs).

Methods: Differentiation of hMSCs into osteoblasts was induced in the presence or absence of IL-17A and/or TNF-α. Matrix mineralization (MM) was evaluated by alizarin red staining and alkaline phosphatase (ALP) activity. mRNA expression was measured by qRT-PCR for bone morphogenetic protein (BMP)-2 and Runx2, genes associated with osteogenesis, DKK-1, a negative regulator of osteogenesis, Schnurri-3 and receptor activator of nuclear factor kappa B ligand (RANKL), associated with the cross talk with osteoclasts, and TNF-α receptor type I and TNF-α receptor type II (TNFRII).

Results: TNF-α alone increased both MM and ALP activity. IL-17A alone increased ALP but not MM. Their combination was more potent. TNF-α alone increased BMP2 mRNA expression at 6 and 12 h. These levels decreased in combination with IL-17A at 6 h only. DKK-1 mRNA expression was inhibited by TNF-α and IL-17A either alone or combined. Supporting an imbalance toward osteoblastogenesis, RANKL expression was inhibited by TNF-α and IL-17A. However, TNF-α but not IL-17 alone decreased Runx2 mRNA expression at 6 h. In parallel, TNF-α but not IL-17 alone increased Schnurri-3 expression with a synergistic effect with their combination. This may be related to an increase of TNFRII overexpression.

Conclusion: IL-17 increased the effects of TNF-α on bone matrix formation by hMSCs. However, IL-17 decreased the TNF-α-induced BMP2 inhibition. Synergistic interactions between TNF-α and IL-17 were seen for RANKL inhibition and Schnurri-3 induction. Such increase of Schnurri-3 may in turn activate osteoclasts leading to bone destruction as in RA. Conversely, in the absence of osteoclasts, this could promote ectopic bone formation as in AS.

No MeSH data available.


Related in: MedlinePlus