IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

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AB0036 APPA (APOCYNIN AND PAEONOL) REDUCES ROS PRODUCTION AND SENESCENCE IN HUMAN ARTICULAR CHONDROCYTES

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2027 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1051.1-1051

Author(s):

M. Fernandez-Moreno ◽

N. Larkins ◽

A. Reynolds ◽

T. Hermida Gómez ◽

F. J. Blanco

Keyword(s):

Gene Expression ◽

Research And Development ◽

Articular Chondrocytes ◽

Human Articular Chondrocytes ◽

Ros Production ◽

Tnf Α ◽

Inflammatory Stimuli ◽

And Cartilage ◽

Pro Inflammatory Cytokines ◽

Inflammatory Effect

Background:Disease modification is not yet possible for osteoarthritis (OA). Mitochondrial ROS and pro-inflammatory cytokines are involved in the pathogenesis of OA and are potential therapeutic targets. APPA, a combination of apocynin (AP) and paeonol (PA), has the potential capacity to modulate synthesis of pro-inflammatory stimuli.Objectives:To investigate the anti-inflammatory effect of APPA in human articular chondrocytes and cartilage.Methods:Tissue and chondrocytes from human OA cartilage were isolated. The effect of APPA on chondrocyte viability was analyzed using MTT. IL-1β 10 ng/mL and LPS 10 ng/mL were used as pro-inflammatory stimuli. ROS production was evaluated by flow cytometry using DCFH-DA and MitoSoxRed. The percentage of senescent cells was evaluated through the quantification of Fluorescein di-β-D-galactopyranoside (FDG) by flow cytometry. The effect of APPA on gene expression of pro-inflammatory cytokines (IL-8 and TNF-α) and enzymes degrading cartilage (MMP-13 and MMP-3) were analyzed in chondrocyte and cartilage by RT-PCR. Quantification of Toluidine Blue (TB) staining in cartilage was performed to evaluate proteoglycans content using software ImageJ/Fiji. Release of Glycosaminoglycan (GAGs) into the supernatant was quantified using BlyscanTM Glycosaminoglycan assay. Statistical analyses were performed with GraphPad Prism v6.Results:Chondrocytes, incubated in presence of APPA 10 µg/mL for 24 h had viability >85%, reduced cytoplasmic ROS (p=0.028) and mitochondrial anion superoxide production induced by LPS 10 ng/mL (p=0.057). Chondrocytes incubated in presence of APPA 10 µg/mL for 2 hours contained significantly fewer senescent cells (p=0.0079). APPA significantly reduced the gene expression induced by IL-1β 10 ng/mL in chondrocytes of IL-8, TNF-α, MMP-13 and MMP-3. Cartilage incubated with APPA 60 and 100 µg/mL for 48 h showed decreased the MMP-3 gene expression induced by IL-1β (p=0.021 and p<0.0001 respectively). Quantification of TB showed that APPA 60 and 100 µg/mL during 48h increased the proteoglycans in intermedial layer, which had been decreased through the incubation with IL-1β (p=0.0018 and p=0.018 respectively). Quantification of release GAGs into the supernatant decreased significantly when the cartilage explants were incubated for 48h in presence of APPA 100 µg/mL (p=0.028).Conclusion:APPA has a clear anti-inflammatory effect on human articular chondrocytes, and could reduce extracellular matrix degradation of cartilage. This could be mediated by the capacity to modulate ROS production and reduce senescence.Disclosure of Interests:Mercedes Fernandez-Moreno: None declared, Nicholas Larkins Shareholder of: I am a shareholder in AKL Research and Development Ltd, Alan Reynolds Shareholder of: I have share options in AKL Research and Development Ltd, Speakers bureau: I have not been a paid speaker for a pharma company - at least not since 2008 whichI think is outside the scope of this, Consultant of: The last time I was a paid consultant was in 2017 when I acted as a consultant for Avillion and Norgine, Employee of: I am also an employee of AKL Research and Development Ltd, Tamara Hermida Gómez: None declared, Francisco J. Blanco Speakers bureau: LillyPfizerSanofiGalapagos, Consultant of: LillyPfizerSanofiGalapagos, Grant/research support from: LillyMSDMerck SeronoPfizerPierre-FabraRocheSanofiServierUCBAbbvieAmgenBioibericaBristol MayerCelgeneCelltrionCellerixGrunenthalGebro PharmaAKL Research and Development Ltd

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Physioxia Has a Beneficial Effect on Cartilage Matrix Production in Interleukin-1 Beta-Inhibited Mesenchymal Stem Cell Chondrogenesis

Cells ◽

10.3390/cells8080936 ◽

2019 ◽

Vol 8 (8) ◽

pp. 936 ◽

Cited By ~ 4

Author(s):

Girish Pattappa ◽

Ruth Schewior ◽

Isabelle Hofmeister ◽

Jennifer Seja ◽

Johannes Zellner ◽

...

Keyword(s):

Articular Cartilage ◽

Beneficial Effect ◽

Articular Chondrocytes ◽

Interleukin 1 ◽

Cartilage Matrix ◽

Interleukin 1Β ◽

Low Oxygen ◽

Matrix Gene ◽

Matrix Production

Osteoarthritis (OA) is a degenerative condition that involves the production of inflammatory cytokines (e.g., interleukin-1β (IL-1β), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) that stimulate degradative enzymes, matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS) resulting in articular cartilage breakdown. The presence of interleukin-1β (IL-1β) is one reason for poor clinical outcomes in current cell-based tissue engineering strategies for treating focal early osteoarthritic defects. Mesenchymal stem cells (MSCs) are a potential cell source for articular cartilage regeneration, although IL-1β has been shown to inhibit in vitro chondrogenesis. In vivo, articular chondrocytes reside under a low oxygen environment between 2–5% oxygen (physioxia) and have been shown to enhance in vitro MSC chondrogenic matrix content with reduced hypertrophic marker expression under these conditions. The present investigation sought to understand the effect of physioxia on IL-1β inhibited MSC chondrogenesis. MSCs expanded under physioxic (2% oxygen) and hyperoxic (20%) conditions, then chondrogenically differentiated as pellets in the presence of TGF-β1 and either 0.1 or 0.5 ng/mL IL-1β. Results showed that there were donor variations in response to physioxic culture based on intrinsic GAG content under hyperoxia. In physioxia responsive donors, MSC chondrogenesis significantly increased GAG and collagen II content, whilst hypertrophic markers were reduced compared with hyperoxia. In the presence of IL-1β, these donors showed a significant increase in cartilage matrix gene expression and GAG content relative to hyperoxic conditions. In contrast, a set of MSC donors were unresponsive to physioxia and showed no significant increase in matrix production independent of IL-1β presence. Thus, physioxia has a beneficial effect on MSC cartilage matrix production in responsive donors with or without IL-1β application. The mechanisms controlling the MSC chondrogenic response in both physioxia responsive and unresponsive donors are to be elucidated in future investigations.

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Low-intensity ultrasound stimulates the viability and matrix gene expression of human articular chondrocytes in alginate bead culture

Journal of Biomedical Materials Research Part A ◽

10.1002/jbm.a.30816 ◽

2006 ◽

Vol 79A (4) ◽

pp. 858-864 ◽

Cited By ~ 47

Author(s):

Byung Hyune Choi ◽

Jeong-Im Woo ◽

Byoung-Hyun Min ◽

So Ra Park

Keyword(s):

Gene Expression ◽

Articular Chondrocytes ◽

Alginate Bead ◽

Human Articular Chondrocytes ◽

Low Intensity ◽

Matrix Gene ◽

Low Intensity Ultrasound

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Faculty Opinions recommendation of Transcriptional response of human articular chondrocytes treated with fibronectin fragments: an in vitro model of the osteoarthritis phenotype.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.739123584.793581017 ◽

2020 ◽

Author(s):

Peter Torzilli

Keyword(s):

In Vitro Model ◽

Articular Chondrocytes ◽

Transcriptional Response ◽

Human Articular Chondrocytes ◽

Fibronectin Fragments ◽

Vitro Model

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Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽

10.3390/nu13010123 ◽

2020 ◽

Vol 13 (1) ◽

pp. 123

Author(s):

Natalia K. Kordulewska ◽

Justyna Topa ◽

Małgorzata Tańska ◽

Anna Cieślińska ◽

Ewa Fiedorowicz ◽

...

Keyword(s):

Gene Expression ◽

Inflammatory Reaction ◽

Wide Spectrum ◽

Cell Monolayer ◽

In Vivo Studies ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

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LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Innate Immunity ◽

10.1177/1753425916669418 ◽

2016 ◽

Vol 22 (8) ◽

pp. 682-695 ◽

Cited By ~ 12

Author(s):

Qin Yang ◽

Maren J Pröll ◽

Dessie Salilew-Wondim ◽

Rui Zhang ◽

Dawit Tesfaye ◽

...

Keyword(s):

Gene Expression ◽

Alveolar Macrophages ◽

Methylation Status ◽

Gene Body ◽

Gene Body Methylation ◽

Tnf Α ◽

Pulmonary Alveolar Macrophages ◽

Cluster Of Differentiation ◽

Cd14 Gene

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

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Febrile-range temperature modifies cytokine gene expression in LPS-stimulated macrophages by differentially modifying NF-κB recruitment to cytokine gene promoters

AJP Cell Physiology ◽

10.1152/ajpcell.00346.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. C171-C181 ◽

Cited By ~ 35

Author(s):

Zachary A. Cooper ◽

Arundhati Ghosh ◽

Aditi Gupta ◽

Tapan Maity ◽

Ivor J. Benjamin ◽

...

Keyword(s):

Gene Expression ◽

Receptor Activation ◽

Cytokine Gene Expression ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Heat Shock Factor 1 ◽

Gene Promoters ◽

Cytokine Gene ◽

Tnf Α

We previously showed that exposure to febrile-range temperatures (FRT, 39.5–40°C) reduces LPS-induced TNF-α expression, in part through the direct interaction of heat shock factor-1 (HSF1) with the TNF-α gene promoter. However, it is not known whether exposure to FRT also modifies more proximal LPS-induced signaling events. Using HSF1-null mice, we confirmed that HSF1 is required for FRT-induced repression of TNF-α in vitro by LPS-stimulated bone marrow-derived macrophages and in vivo in mice challenged intratracheally with LPS. Exposing LPS-stimulated RAW 264.7 mouse macrophages to FRT reduced TNF-α expression while increasing IL-1β expression despite the two genes sharing a common myeloid differentiation protein-88 (MyD88)-dependent pathway. Global activation of the three LPS-induced signaling intermediates that lead to cytokine gene expression, ERK and p38 MAPKs and NF-κB, was not affected by exposing RAW 264.7 cells to FRT as assessed by ERK and p38 phosphorylation and NF-κB in vitro DNA-binding activity and activation of a NF-κB-dependent synthetic promoter. However, chromatin immunoprecipitation (ChIP) analysis demonstrated that exposure to FRT reduced LPS-induced recruitment of NF-κB p65 to the TNF-α promoter while simultaneously increasing its recruitment to the IL-1β promoter. These data suggest that FRT exerts its effects on cytokine gene expression in a gene-specific manner through distal effects on promoter activation rather than proximal receptor activation and signal transduction.

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