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Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

ABSTRACT: In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8058094 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

Wang Yilin Y Khan Aneesah A Antonopoulos Aristotelis A Bouché Laura L Buckley Christopher D CD Filer Andrew A Raza Karim K Li Kun-Ping KP Tolusso Barbara B Gremese Elisa E Kurowska-Stolarska Mariola M Alivernini Stefano S Dell Anne A Haslam Stuart M SM Pineda Miguel A MA

Nature communications 20210420 1

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glyco ...[more]

PMID: 33879788

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Project description:Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Project description:BackgroundPrevious studies have indicated that endoplasmic reticulum (ER) stress may actively promote the pathogenesis of rheumatoid arthritis (RA) by evoking autophagy. However, the underlying mechanism remains largely unknown. This study aimed to explore the mechanism of the ER stress-autophagy pathway in regulating the phenotype transformation of rheumatoid arthritis synovial fibroblasts (RASFs).MethodsSynovial tissue was obtained from RA and osteoarthritis (OA) patients during joint replacement surgery. ER stress/autophagy signature markers were examined in synovial tissue by real-time quantitative polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Phenotype transformation of RASFs, including increased cell proliferation and invasion capability, was measured by CCK-8 assay and transwell invasion assay. Signaling pathways were further investigated and inositol requiring enzyme 1 (IRE1) was down-regulated in RASFs by transfecting specific short hairpin RNA-ERN1 (shRNA-ERN1) carried by lentiviral vectors.ResultsThe expression of ER stress/autophagy pathway-associated proteins, including GRP78, IRE1, protein kinase R-like endoplasmic reticulum kinase (PERK), and LC3, was significantly increased in RA synovium compared with OA synovium. After stimulation with tumor necrosis factor alpha (TNF-α) in vitro, the proliferation and invasion ability of RASFs were upregulated, while this phenomenon could be inhibited by 4-PBA (ER stress inhibitor) or 3-MA (autophagy inhibitor). The expression of IRE1 and p-JNK in particular, occurred in an obviously time-dependent manner after stimulation with TNF-α. Moreover, the proliferation and invasion of RASFs were inhibited after transfection with sh-RNA-ERN1 to downregulate IRE1 expression.ConclusionsER stress triggered autophagy via the IRE1/JNK pathway to regulate the phenotype transformation of RASFs, indicating an important role of the ER stress-autophagy pathway in the pathological process of synovitis in RA.

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Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

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Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

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