New Insights From Single-Cell Sequencing Data: Synovial Fibroblasts and Synovial Macrophages in Rheumatoid Arthritis

Cheng, Liyun; Wang, Yanyan; Wu, Ruihe; Ding, Tingting; Xue, Hongwei; Gao, Chong; Li, Xiaofeng; Wang, Caihong

doi:10.3389/fimmu.2021.709178

Cited by 53 publications

(28 citation statements)

References 111 publications

(144 reference statements)

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“…In comparison to bulk RNA, scRNA-seq analysis enables high-throughput analysis of cellular transcriptome expression at the single-cell level, opening a new window into the study of multicellular biological heterogeneity. In RA, scRNA-seq has allowed researchers to identify synovial cell types and study cell differentiation and development around cell subpopulations to explore novel therapeutic targets [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis

Wang

Gong

2022

Computational and Mathematical Methods in Medicine

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Objectives. This study aimed to investigate the potential role of synovial fibroblasts (SFs) in the development of rheumatoid arthritis (RA) to identify potential molecular targets and provide a theoretical basis for the treatment of RA. Methods. GSE109449, a fibroblast transcriptome dataset of synovial tissue from RA and osteoarthritis (OA), were obtained from the GEO database. After standard cell quality control, this single-cell transcriptome data was used to perform routine single-cell analysis processes. After completing dimensionality reduction, clustering, and cell subset identification of fibroblasts, the SCENIC analysis helped calculate the significant gene regulatory networks in fibroblasts and their subsets. From these computed gene regulatory networks, the regulon in which follistatin-like protein 1 (FSTL1) resides was extracted and used to analyze the transcriptional regulatory status of fibroblasts. Finally, the gene set enrichment analysis (GSEA) was used to calculate the respective enriched gene sets of IRF1 and FSTL1. Results. Three SF subgroups were identified from the single-cell transcriptome analysis; SF subset 3 was more abundant in RA than in OA ( p < 0.001 ). From the SCENIC analysis, we obtained 269 regulons and the corresponding gene regulatory networks in SF from the RA datasets. Next, we screened and obtained a regulon-containing FSTL1, where IRF1 was the major transcription factor. The top five regulons in SF subset 3 were TWIST1, MECOM, KLF6, MAFB, and RUNX1. Among the 3 SF subsets, IRF1 regulon was ranked the highest in SF subset 3. Differential analysis of pseudobulk RNA-seq showed that IRF1 was up-regulated in RA compared to OA. Between the three SF subgroups, IRF1 and FSTL1 expression was more up-regulated in SF subset 3 compared to the other two subgroups. Conclusions. IRF1 was found to regulate the invasiveness of SFs by regulating FSTL1, which may influence the disease progression of RA.

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Section: Introductionmentioning

confidence: 99%

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis

Wang

Gong

2022

Computational and Mathematical Methods in Medicine

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“…OA-FLS show a much lower basal expression of p75NTR and of proNGF, which are only marginally up-regulated by IL-1b. A vast body of evidence shows that OA-FLS and RA-FLS are characterized by differences in the proportion (37) of fibroblast subsets with non-overlapping functions which play distinct roles in the pathogenesis of OA and RA: in RA there is a prevalence of immune effector fibroblasts that sustain inflammation through the production of chemokines and cytokines, and of bone effector fibroblasts that mediate joint damage through the production of matrix metalloproteinases (37)(38)(39)(40). RA-FLS have a pro-inflammatory and aggressive phenotype (41) and a number of epigenetic changes have been suggested to account for these abnormalities (42).…”

Section: Discussionmentioning

confidence: 99%

Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

et al. 2022

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We have recently provided new evidence for a role of p75NTR receptor and its preferential ligand proNGF in amplifying inflammatory responses in synovial mononuclear cells of chronic arthritis patients. In the present study, to better investigate how activation of the p75NTR/proNGF axis impacts synovial inflammation, we have studied the effects of proNGF on fibroblast-like synoviocytes (FLS), which play a central role in modulating local immune responses and in activating pro-inflammatory pathways. Using single cell RNA sequencing in synovial tissues from active and treatment-naïve rheumatoid arthritis (RA) patients, we demonstrated that p75NTR and sortilin, which form a high affinity receptor complex for proNGF, are highly expressed in PRG4pos lining and THY1posCOL1A1pos sublining fibroblast clusters in RA synovia but decreased in RA patients in sustained clinical remission. In ex vivo experiments we found that FLS from rheumatoid arthritis patients (RA-FLS) retained in vitro a markedly higher expression of p75NTR and sortilin than FLS from osteoarthritis patients (OA-FLS). Inflammatory stimuli further up-regulated p75NTR expression and induced endogenous production of proNGF in RA-FLS, leading to an autocrine activation of the proNGF/p75NTR pathway that results in an increased release of pro-inflammatory cytokines. Our data on the inhibition of p75NTR receptor, which reduced the release of IL-1β, IL-6 and TNF-α, further confirmed the key role of p75NTR activation in regulating inflammatory cytokine production. In a set of ex vivo experiments, we used RA-FLS and cultured them in the presence of synovial fluids obtained from arthritis patients that, as we demonstrated, are characterized by a high concentration of proNGF. Our data show that the high levels of proNGF present in inflamed synovial fluids induced pro-inflammatory cytokine production by RA-FLS. The blocking of NGF binding to p75NTR using specific inhibitors led instead to the disruption of this pro-inflammatory loop, reducing activation of the p38 and JNK intracellular pathways and decreasing inflammatory cytokine production. Overall, our data demonstrate that an active proNGF/p75NTR axis promotes pro-inflammatory responses in synovial fibroblasts, thereby contributing to chronic synovial inflammation, and point to the possible use of p75NTR inhibitors as a novel therapeutic approach in chronic arthritis.

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“…FLSs were recently proven to be key players of observed inflammation in RA context 17,18 . These cells locate inside joints in the synovium and are involved in pannus formation, a hallmark pathological change in patients with RA [19][20][21] . Recent advances in the treatment of rheumatologic disorders have resulted in a reduction of access to synovial tissues by investigators 22 .…”

Section: Discussionmentioning

confidence: 99%

Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis

Zafari

Rafiei

Faramarzi

et al. 2021

Rev. Assoc. Med. Bras.

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OBJECTIVE:Cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical researches.Primary cells represent the normal physiological condition of human cells. Fibroblasts are the most common native cells of connective tissue that play a crucial role in the entire pathogenesis of various disorders, such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), which overlie the loose connective tissue of the synovial sublining, are known to be the central mediators of joint damage. The most routine approach for the isolation of FLS is an enzymatic digestion of synovial tissue. This experimental study is designed to introduce an easy, fast, and high-throughput method compared with enzymatic digestion for isolation of FLS. METHODS:The synovial tissue and synovial fluid (SF) samples were collected from eight patients with RA who underwent routine knee replacement surgery. Synovial tissue was incubated with collagenase VIII enzyme, while SF was washed with a similar volume of phosphatebuffered saline. The cells were further subcultured and stored based on the standard protocols. The purity of isolated synoviocytes was confirmed using flow cytometry analysis.RESULTS: Isolation of FLS from SF was more successful with a faster rate, 3-5 days after culture. The morphological assessment and flow cytometry analysis confirmed the purity of SF-derived cells in passage 4.CONCLUSIONS: SF could be a more accessible source of FLS than synovial tissue. Obtaining primary FLS from SF is a simple, fast, and cost-effective way to have a large-scale cell during a short time.

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New Insights From Single-Cell Sequencing Data: Synovial Fibroblasts and Synovial Macrophages in Rheumatoid Arthritis

Cited by 53 publications

References 111 publications

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis

Pro Nerve Growth Factor and Its Receptor p75NTR Activate Inflammatory Responses in Synovial Fibroblasts: A Novel Targetable Mechanism in Arthritis

Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis

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