Tariq M. Haqqi

Immunopathogenesis of osteoarthritis

Even though osteoarthritis (OA) is mainly considered as a degradative condition of the articular cartilage, there is increasing body of data demonstrating the involvement of all branches of the immune system. Genetic, metabolic or mechanical factors cause an initial injury to the cartilage resulting in release of several cartilage specific auto-antigens, which trigger the activation of immune response. Immune cells including T cells, B cells and macrophages infiltrate the joint tissues, cytokines and chemokines are released from different kinds of cells present in the joint, complement system is activated, and cartilage degrading factors such as matrix metalloproteinases (MMPs) and prostaglandin E2 (PGE2) are released, resulting in further damage to the articular cartilage. There is considerable success in the treatment of rheumatoid arthritis using anti-cytokine therapies. In OA, however, these therapies did not show much effect, highlighting more complex nature of pathogenesis of OA. This needs the development of more novel approaches to treat OA, which may include therapies that act on multiple targets. Plant natural products have this kind of property and may be considered for future drug development efforts. Here we reviewed the studies implicating different components of the immune system in the pathogenesis of OA.

Modulation of Ten-Eleven Translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) Expression, α-Ketoglutarate (α-KG), and DNA Hydroxymethylation Levels by Interleukin-1β in Primary Human Chondrocytes

Background: Cytosine hydroxymethylation in the genomic DNA controls gene expression. Results: Treatment of primary chondrocytes with IL-1β alone or in conjunction with TNF-α inhibited TET1 and IDH expression and suppressed cytosine hydroxymethylation. Conclusion: IL-1β and TNF-α decrease DNA hydroxymethylation by suppressing the expression and activity of TET1 and IDH. Significance: Pro-inflammatory cytokines likely control gene expression by modulating the 5-hmC levels. 5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1–3 (TET1–3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1β and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1β and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1β alone or in combination with TNF-α. We observed a dramatic (10–20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2–3-fold) in TET3 expression in chondrocytes stimulated with IL-1β and TNF-α. IL-1β and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1β and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.

MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes.

Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin‐1β (IL‐1β) has been implicated as a principal instigator of OA, we sought to determine whether IL‐1β induces the expression of sonic HH (SHH) and its regulation by microRNAs (miRNAs) in human chondrocytes.

Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes

Abstract Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti‐inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL‐1&bgr;‐stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL‐6, COX‐2, PGE2, iNOS and NO in IL‐1&bgr;‐stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP‐13, MMP‐3, MMP‐9, and ADAMTS‐4 in OA chondrocytes, and blocked the release of s‐GAG and COL2A1 in IL‐1&bgr;‐stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL‐1&bgr;‐mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF‐&kgr;B activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO‐1‐SOD2‐NQO1‐GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP‐1/Nrf‐2 interaction by directly blocking the binding site of Nrf‐2 in the KEAP‐1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti‐inflammatory and chondroprotective potential of Wogonin in IL‐1&bgr;‐stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA. HighlightsWogonin suppressed the expression and production of inflammatory mediators.Wogonin suppressed the expression of matrix degrading proteases.Wogonin enhanced the expression of cartilage anabolic factors COL2A1 and ACAN.Wogonin modulated the redox homeostasis of OA chondrocytes by disturbing the balance between cellular ROS and GSH levels.Wogonin activated Nrf2 through induction of ROS/ERK/Nrf2/HO‐1‐NQO1 signaling axis.

MicroRNA-9 Promotion of Interleukin-6 Expression by Inhibiting Monocyte Chemoattractant Protein–Induced Protein 1 Expression in Interleukin-1β–Stimulated Human Chondrocytes

Enhanced expression of interleukin‐6 (IL‐6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein–induced protein 1 (MCPIP‐1) is a novel posttranscriptional regulator of IL‐6 expression and is targeted by microRNA‐9 (miR‐9). We investigated the expression of MCPIP‐1 in OA cartilage and explored whether targeting of MCPIP‐1 by miR‐9 contributes to enhanced IL‐6 expression in OA.

miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3′ untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 ‘seed sequence’, therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1β in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the ‘seed sequence’ located in the 3′ UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.

The Role of MicroRNAs and Their Targets in Osteoarthritis

Micro ribonucleic acid (microRNA) regulation and expression has become an emerging field in determining the mechanisms regulating a variety of inflammation-mediated diseases. Several studies have focused on specific microRNAs that are differentially expressed in cases of osteoarthritis. Furthermore, several targets of these miRNAs important in disease progression have also been identified. In this review, we focus on microRNA biogenesis, regulation, detection, and quantification with an emphasis on cellular localization and how these concepts may be linked to disease processes such as osteoarthritis. Next, we review the relationships of specific microRNAs to certain features and risk factors associated with osteoarthritis such as inflammation, obesity, autophagy, and cartilage homeostasis. We also identify certain microRNAs that are differentially expressed in osteoarthritis but have unidentified targets and functions in the disease state. Lastly, we identify the potential use of microRNAs for therapeutic purposes and also mention certain remedies that regulate microRNA expression.

Sucrose, But Not Glucose, Blocks IL1-β-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway.

Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin‐1β (IL‐1β) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediators expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin‐1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence‐based assay. We found that sucrose‐induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL‐1β‐induced apoptosis and mRNA expression of MMP‐13, COX‐2, and IL‐6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL‐1β induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629–639, 2017.

Interleukin-1β induced Stress Granules Sequester COX-2 mRNA and Regulates its Stability and Translation in Human OA Chondrocytes

Enhanced and immediate expression of cyclooxygenase-2 (COX-2) mRNA is observed in IL-1β-stimulated OA chondrocytes but the synthesis of protein found significantly delayed. Here we investigated the role of stress granules (SGs), ribonucleoprotein complexes that regulate mRNA translation, in the delayed translation of COX-2 mRNAs in IL-1β-stimulated OA chondrocytes. Stimulation of human chondrocytes with IL-1β activated the stress response genes and the phosphorylation of eIF2α that triggered the assembly of SGs. Using combined immunofluorescence staining of SGs markers and COX-2 protein, RNA fluorescence in situ hybridization and RNA immunoprecipitation, the COX-2 mRNAs were found sequestered in SGs in IL-1β-stimulated OA chondrocytes. No increase in COX-2 protein expression was observed during the persistence of SGs but enhanced expression of COX-2 protein was noted upon clearance of the SGs. Inhibition of SGs clearance blocked COX-2 mRNA translation whereas blocking the assembly of SGs by TIA-1 depletion resulted in rapid and increased production of COX-2 and PGE2. Our findings show for the first time assembly of SGs and sequestration of COX-2 mRNAs in human OA chondrocytes under pathological conditions. Post-transcriptional regulation of COX-2 mRNAs translation by SGs indicates a role in IL-1β-mediated catabolic response that could be therapeutically targeted in OA.

Histone Deacetylase Inhibitor Vorinostat (SAHA) Suppresses IL-1β–Induced Matrix Metallopeptidase-13 Expression by Inhibiting IL-6 in Osteoarthritis Chondrocyte

Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1β in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of MMP-13 was determined by treating chondrocytes with recombinant IL-6 or by IL6 knockdown using IL6-specific siRNA. We found that SAHA is a potent suppressor of IL-1β-induced MMP-13, tumor necrosis factor-α, and other catabolic marker expression in OA chondrocytes. Interestingly, SAHA rescued the COL2A1 and ACAN expression in OA chondrocytes that was down-regulated by IL-1β. Of importance is our finding that IL-6-stimulated MMP-13 expression was independent of IL-1β stimulation and was blocked by SAHA, suggesting that SAHA inhibits IL-6 signaling in OA chondrocytes. Taken together, our results suggest that SAHA could be used as a therapeutic agent for the management of OA.