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Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation.

ABSTRACT: The joint synovium consists of a heterogeneous cell population, chiefly comprised of macrophages, and fibroblast-like synoviocytes (FLS). An inter-species co-culture model was developed to examine interactions between these cells. Equine FLS and the canine macrophage line DH82 were differentially labeled using fluorescent markers and results from direct co-culture compared with those from both indirect co-culture, and conditioned media experiments. The transcript expression of IL-1?, IL-6, ADAMTS4, and ADAMTS5 in each cell type were determined using species-specific qPCR assays. Lipopolysaccharide stimulation of EFLS rapidly increased IL-1?, IL-6, ADAMTS4, and ADAMTS5 mRNAs. The induction of ADAMTS5 was significantly reduced when equine FLS were cultured with DH82 cells directly or indirectly. Exposure of equine FLS to denatured conditioned media also significantly reduced ADAMTS5 induction. DH82 cells increased interleukin-1? expression substantially following LPS stimulation. However, knockdown of interleukin-1? in DH82 cells, or inhibition of NF-?B in equine FLS prior to co-culture did not change the inhibitory effect on equine FLS ADAMTS5 gene expression. This work indicates that macrophages can influence FLS gene expression through a soluble mediator, and modulate the expression of an enzyme critical in osteoarthritis pathology during inflammatory stimulation. © 2018 The Authors. Journal of Orthopaedic Research® Published by WileyPeriodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 9999:1-8, 2018.

SUBMITTER: Morgan RE

PROVIDER: S-EPMC6120467 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation.

Morgan Rhiannon E RE Clegg Peter D PD Hunt John A JA Innes John F JF Tew Simon R SR

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20180309

The joint synovium consists of a heterogeneous cell population, chiefly comprised of macrophages, and fibroblast-like synoviocytes (FLS). An inter-species co-culture model was developed to examine interactions between these cells. Equine FLS and the canine macrophage line DH82 were differentially labeled using fluorescent markers and results from direct co-culture compared with those from both indirect co-culture, and conditioned media experiments. The transcript expression of IL-1β, IL-6, ADAMT ...[more]

PMID: 29521434

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Project description:The in vitro invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) have been shown to correlate with disease severity and radiographic damage. We recently determined that FLSs obtained from pristane-induced arthritis (PIA)-susceptible DA rats are also highly invasive in the same in vitro assay through Matrigel. The transfer of alleles derived from the arthritis-resistant F344 strain at the arthritis severity locus Cia5d (RNO10), as in DA.F344(Cia5d) congenics, was enough to significantly and specifically reduce the invasive properties of FLSs. This genetically controlled difference in FLS invasion involves increased production of soluble membrane-type 1 matrix metalloproteinase (MMP) by DA, and is dependent on increased activation of MMP-2. In the present study we aimed to characterize the pattern of gene expression that correlates with differences in invasion in order to identify pathways regulated by the Cia5d locus.Synovial tissues were collected from DA and DA.F344(Cia5d) rats 21 days after the induction of PIA. Tissues were digested and FLSs isolated. After a minimum of four passages, FLSs were plated on Matrigel-covered dishes at similar densities, followed by RNA extraction. Illumina RatRef-12 expression BeadChip arrays were used. Expression data were normalized, followed by t-test, logistic regression, and cluster analysis. Real-time PCR was used to validate the microarray data.Out of the 22,523 RefSeq gene probes present in the array, 7,665 genes were expressed by the FLSs. The expression of 66 genes was significantly different between the DA and DA.F344(Cia5d) FLSs (P < 0.01). Nineteen of the 66 differentially expressed genes (28.7%) are involved in the regulation of cell cycle progression or cancer-associated phenotypes, such as invasion and contact inhibition. These included Cxcl10, Vil2 and Nras, three genes that are upregulated in DA and known to regulate MMP-2 expression and activation. Nine of the 66 genes (13.6%) are involved in the regulation of estrogen receptor signaling or transcription. Five candidate genes located within the Cia5d interval were also differentially expressed.We have identified a novel FLS invasion associated gene expression signature that is regulated by Cia5d. Many of the genes found to be differentially expressed were previously implicated in cancer cell phenotypes, including invasion. This suggests a parallel in the behavior of arthritis FLSs and cancer cells, and identifies novel pathways and genes for therapeutic intervention and prognostication.

Project description:BackgroundThe heterogeneity of osteoarthritis (OA) significantly limits the effectiveness of pharmacological treatments in an unselected patient population. In this context, the identification of OA subtypes is meaningful for the development of therapies that target specific types of OA pathogenesis.MethodsExpression array profiles of 70 OA and 36 control synovial samples were extracted from the GEO database. Unsupervised consensus clustering was performed based on the most variable genes to identify OA subclusters. Next, Joint samples from OA patients were obtained. We divided the OA patient into two subpopulations according to synovial ADCY7 levels. Synovium and cartilage samples from different OA subpopulations were evaluated. In addition, we established a high-fat diet (HFD)-induced rat OA model. We evaluated OA progression, lipid metabolism, synovitis and fibroblast-like synoviocytes (FLS) function in this HFD-induced OA model.Results70 OA patients were categorized into three distinct subclusters. We noted that one subcluster was characterized by synovial lipid metabolism disorder GO terms. We further identified the most noticeable KEGG pathway "Regulation of lipolysis in adipocytes" in this subcluster as well as the most significantly differentially expressed gene, ADCY7. We found that the ADCY7 high expressing group (32.6%) exhibited features of synovial inflammatory lipolysis epithelial-mesenchymal transition (EMT) tendency, as well as faster join space narrowing. The HFD induced OA-like degeneration in rat joints. We observed similar synovial inflammatory lipolysis and EMT in FLS, characterized by higher proliferative and invasive activity and elevated proinflammatory and procatabolic properties. ADCY7 was highly expressed in the synovium of the HFD-OA model rats and the inhibition of ADCY7 effectively attenuated these HFD-induced degenerative changes as well as synovial inflammatory lipolysis and FLS dysfunction. In HFD-FLSs, ADCY7 promoted the phosphorylation of PKA as well as its downstream lipid droplet-associated protein PLIN1 and hormone-sensitive lipase (HSL). The inhibition of PKA largely alleviated ADCY7-mediated HFD-FLS dysfunction.ConclusionsWe described a synovial EMT and lipid metabolism disorder in the pathogenesis of OA. This novel mechanism may represent a currently undefined OA subtype. ADCY7 is a potential molecular marker of this pathomechanism.The translational potential of this articleUtilizing synovial samples from OA patients, we identified a subpopulation with high ADCY7 expression. This may represent a currently undefined OA subtype and explain the clinical phenomenon of more severe synovial inflammation in obese OA patients. In addition, we established an HFD-induced OA rat model and found an upregulation of ADCY7 in the synovium. We confirmed that the inhibition of ADCY7 could effectively attenuate HFD-induced degenerative changes as well as the inflammatory lipolysis and FLS dysfunction observed in the rat model. This suggests that ADCY7 and its downstream pathways are potential pharmacological targets for treating this lipid-metabolism-disorder-related OA mechanism.

Dataset Information

Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation.

Publications

Interaction with macrophages attenuates equine fibroblast-like synoviocyte ADAMTS5 (aggrecanase-2) gene expression following inflammatory stimulation.

Similar Datasets

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