IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells.

OBJECTIVE: Mesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. METHODS: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. RESULTS: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA), which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. CONCLUSIONS: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation

IL-17 does not affect SAMD2 activation, but attenuates SOX9 phosphorylation induced by chondrogenic culture.

<p>Human MSCs were cultured in monolayer (DMEM containing 5% FBS) with indicated concentrations of IL-17 throughout the culture period. After 16 hr culture with serum-starved medium (0% FBS), cells were stimulated with TGF-β3 (10 ng/mL) for 15 min and A, whole cell lysates or B, cytoplasmic (upper panel) and nuclear (lower panel) fraction were analyzed for SMAD2 and phospho-SMAD2 expression by western blotting. β-actin and TBP were used as loading controls. C, Human MSCs were cultured on cover-glass slides and SMAD2 localization was determined by immunofluorescence microscopy. Original magnification×20. Scale bars represent 100 µm. D, Human MSCs were cultured as aggregates in chondrogenic induction medium supplemented with TGF-β3 and aggregate lysates were evaluated at the indicated time points by western blot analysis for total and phosphorylated SOX9. β-actin was used as a loading control. (A, B, C and D) Data are representative of two independent experiments with similar findings. E, IL-17 or 10 µM H89 was added at the indicated concentrations and analysis carried out at day 2 and 7 by western blotting (top: day 2, bottom: day 7). F, Densitometric analysis on day 7 was performed with CS Analyzer, version 3.0 (bottom). Values represent the mean±SD of three independent experiments. *<i>P<</i>0.05, compared to no cytokine, by Dunnett’s multiple comparison test. NS: not significant.</p

IL-17 suppressed the expression of chondrogenic marker genes.

<p>Human MSCs were cultured as aggregates in TGF-β3-containing medium with the indicated concentrations of IL-17. After 14 days, type II collagen (<i>COL2A1</i>), aggrecan (<i>ACAN</i>), type X collagen (<i>COL10A1</i>), and alkaline phosphatase (<i>ALP</i>) mRNA levels were determined by real-time PCR and expressed relative to β-actin expression level. Values are mean±SD of 3 aggregates from 1 of 3 independent experiments with similar findings.</p

IL-17 inhibits TGF-β3-induced chondrogenic differentiation.

<p>A, Human mesenchymal stem cells (MSCs) were cultured in pellets, with or without TGF-β3 (10 ng/mL). Macro-images of the aggregates cultured with IL-17, TNF-α or IL-1β at the indicated concentrations for 14 days. Scale bar represents 1 mm. B, Aggregates cultured in the presence of the indicated cytokines for 21 days were fixed and paraffin embedded, then sections were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. (A and B) Data are representative of two independent experiments with similar findings. C, Wet weight of aggregates treated with IL-17 for 14 days. Values are mean±SD of 6–7 aggregates per group from three independent experiments with similar tendencies. ^##<i>P<</i>0.01, compared to no cytokine (−TGF-β3), by Student’s <i>t</i>-test. *<i>P</i><0.05; **<i>P<</i>0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. D, Sulfated glycolaminoglycan (sGAG) content in aggregates treated with IL-17 for 21 days. Values are mean±SD of 3–4 aggregates per group from two independent experiments with similar findings. ^##<i>P<</i>0.01, compared to no cytokine (−TGF-β3), by Student’s <i>t</i>-test. **<i>P<</i>0.01, compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test. E, IL-17 receptor A (<i>IL-17RA</i>) mRNA levels in aggregates were determined by real-time PCR for the indicated time points. Values are normalized to β-actin expression and expressed as mean±SD of 6 aggregates per group from three independent experiments with similar tendencies. **<i>P<</i>0.01, compared to day 0 (undifferentiated MSC), by Dunnett’s multiple comparison test.</p

PKA activation is required for chondrogenic differentiation of human MSCs.

<p>A, Paraffin sections from aggregates cultured in the presence of 10 µM H89 for 21 days were stained with Safranin O and anti-type II collagen antibody. Original magnification×10. Scale bars represent 500 µm. B, <i>COL2A1, ACAN, COL10A1</i>, and <i>ALP</i> mRNA levels in aggregates treated with the indicated concentrations of H89 for 21 days were determined by real-time PCR. Values are mean±SD of three aggregates from 1 of 2 independent experiments with similar findings. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079463#pone-0079463-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079463#pone-0079463-g002" target="_blank">2</a> for the definition of other symbols.</p

Spontaneous Differentiation of Human Mesenchymal Stem Cells on Poly-Lactic-Co-Glycolic Acid Nano-Fiber Scaffold.

INTRODUCTION:Mesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints. METHODS:MSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro. RESULTS:Healthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts. CONCLUSIONS:Our PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction

Epstein-Barr virus-induced gene 3 commits human mesenchymal stem cells to differentiate into chondrocytes via endoplasmic reticulum stress sensor.

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α

IL-17 treatment reduces PKA activity during chondrogenesis.

<p>A, Human MSCs were cultured as aggregates with TGF-β3 in the presence of IL-17 (100 ng/mL) or H89 (10 µM). After 7 days, 3 aggregates were pooled and lysed in each group, and PKA activity within the soluble protein fraction was determined using PepTag non-radioactive PKA assay (top). Recombinant PKA catalytic subunit (2 µg/mL) was used as a positive control and water was used as a negative control. Densitometric analyses of the band intensities were performed and results were expressed as the test band intensity relative to that of the “no TGF-β3” sample (bottom). B, The DNA content of three aggregates in each group was measured after 7-day culture. Values shown in A and B are mean±SD of three independent experiments. **<i>P<</i>0.01 compared to no cytokine (+TGF-β3), by Dunnett’s multiple comparison test.</p