Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease influenced by both genetic and environmental factors. It has been postulated that a high-risk genetic background, in combination with epigenetic marks and environmental exposures, leads to a cascade of events inducing synovitis and consequent destructive arthritis. The clinical picture of joint involvement in RA is the result of chronic inflammation of the synovium, characterised by interactions of resident cells such as fibroblast-like synoviocytes (FLS) with cells of the innate (e.g. macrophages, dendritic cells, mast cells and NK cells, neutrophils) and adaptive immune system (e.g. B and T lymphocytes). Currently, our understanding of the role of innate and adaptive immunity in the pathogenesis of RA is expanding. The concept of how immune responses contribute to the disease has dramatically evolved over the last 50 years. Shedding some light on the different aspects of RA pathogenesis will help to identify new targets for the development of disease-modifying therapies. Thus, in this review we report new insights in RA pathogenesis, resulting from a literature research date published in the last year.

One year in review 2017: pathogenesis of rheumatoid arthritis.

The Rheumatoid Arthritis-Associated Citrullinome

Purpose of reviewDysregulated citrullination is a key element that drives the production and maintenance of antibodies to citrullinated proteins, a hallmark in rheumatoid arthritis (RA). This article reviews recent literature on the origin of citrullinated antigens in RA.Recent findingsThe study of

Rheumatoid arthritis and citrullination.

Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from pre-existing and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness, and uncovered an unanticipated suppressive role for KRT17. We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.

https://science.sciencemag.org/content/sci/early/2021/01/21/science.abc1944.full.pdf

Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts.

Roles of peroxiredoxins in cancer, neurodegenerative diseases and inflammatory diseases

Long noncoding RNAs (lncRNAs) have recently received wide attention as key molecules that mediate a variety of physiological and pathological processes by regulating gene expression; however, knowledge of lncRNAs in rheumatoid arthritis (RA) is limited. Thus, we investigated the lncRNA expression profile in fibroblast-like synoviocytes (FLSs) from patients with RA and explored the function of abundantly expressed lncRNAs. LncRNA and mRNA microarrays were performed to identify differentially expressed lncRNAs in RA FLSs compared with normal FLSs. Quantitative polymerase chain reaction (qPCR) was used to validate the results, and correlation analysis was used to analyze the relationship between these aberrantly expressed lncRNAs and clinical characteristics. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic value of the lncRNAs identified. According to the gene expression profiles, 135 lncRNAs were differentially expressed between RA and normal FLSs. Furthermore, qPCR data showed that lncRNA ENST00000483588 was up-regulated and that three lncRNAs (ENST00000438399, uc004afb.1, and ENST00000452247) were down-regulated in RA FLSs. The expression level of ENST00000483588 was positively correlated with the level of C-reactive protein and the Simplified Disease Activity Index score. Moreover, the areas under the ROC curve were 0.85, 0.92, 0.97, and 0.92 for ENST00000483588, ENST00000438399, uc004afb.1, and ENST00000452247, respectively. The results indicate that the dysregulation of ENST00000483588, ENST00000438399, uc004afb.1, and ENST00000452247 may be involved in the pathological processes of RA and that these lncRNAs may have potential value for the diagnosis and assessment of the disease activity of RA.

/pdf/long-noncoding-rna-expression-profile-in-fibroblast-like-4c4ir82zab.pdf

Long noncoding RNA expression profile in fibroblast-like synoviocytes from patients with rheumatoid arthritis

The objective of the study is to investigate potential citrullinated autoantigens as targets of anti-citrullinated protein antibodies (ACPAs) response in synovial fluids (SFs) of patients with rheumatoid arthritis (RA). SFs from six RA patients and six osteoarthritis (OA) patients as controls were collected. The citrullinated proteins in SFs were extracted by immunoprecipitation with rabbit anti-citrulline antibodies. Matrix-assisted laser desorption/ionization time of flight mass spectrometry/time of flight mass spectrometry (MALDI-TOF/TOF) mass spectrometry was subsequently performed to discover a characteristic neutral loss to finally determine citrullinated autoantigens. A total of 182 citrullinated peptides and 200 citrullinated sites were identified in RA SFs, while 3 citrullinated peptides and 4 citrullinated sites were identified in OA SFs. The 182 citrullinated peptides from RA SFs and the 3 citrullinated peptides from OA SFs were derived from 83 and 3 autoantigens, respectively. Eighty-three autoantigens except protein-arginine deiminase type-2 (PADI2) and protein-arginine deiminase type-2 (PADI4) were over-citrullinated compared with controls, and the citrullinated sites of PADI2 and PADI4 were different in two groups. Interestingly, citrullinated histone H3.3 (H3F3A) was found in OA controls, but not in RA groups. The differential citrullinated proteins identified in RA SFs suggested potential autoantigens were targeted for ACPAs response and might contribute to the induction and perpetuation of complement activation and joint inflammation in RA.

/pdf/identification-of-citrullinated-peptides-in-the-synovial-4fn62gs0yh.pdf

Identification of citrullinated peptides in the synovial fluid of patients with rheumatoid arthritis using LC-MALDI-TOF/TOF.

Objectives The purpose of this study was to identity protein expression patterns of fibroblast-like synoviocytes (FLSs) derived from the synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis. Methods Two-dimensional gel electrophoresis (2-DE) in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) was used to visualise and identify differential cellular protein expression profiles in FLSs between RA and control groups. Western-blot analysis was performed to further verify selected differentially-expressed proteins. Results A total of 1633 and 1603 protein spots were examined in synovial FLSs of RA patients and controls, respectively. Ninety-two spots in the RA group were statistically over- or under-expressed compared with controls. Among them, 33 proteins over-expressed by more than 3-fold were then identified by MALDI-TOF-MS analysis. These proteins included enzymatic and structural proteins (e.g. PKM1/M2, α-enolase, ERp60, lamin-A/C), signal transduction proteins (e.g. annexin 11, peroxiredoxin 1, TrpRS), heat-shock/chaperone proteins (e.g. TCP-1, GRP75, HspB5, Bip) and some unknown protein species. Three proteins, namely α-enolase, GRP75 and PKM2, were verified by Western blot and the results were found to be consistent with proteomic analysis. Conclusions The differentially expressed proteins identified in RA synovial FLSs might be candidate RA-associated proteins and may prove to be promising diagnostic indicators or new therapeutic targets for RA.

Proteomic analysis of synovial fibroblast-like synoviocytes from rheumatoid arthritis.

Objectives: Inhibitor of differentiation 3 (Id3) protein has been implicated in the control of multiple cell death signalling pathways and in aetiology of numerous diseases. The aims of this study were to construct a recombinant eukaryotic expression vector (pEGFP/Id3), containing human Id3 (hId3) fused with enhanced green fluorescent protein (EGFP), and to determine effects of ectopic Id3 overexpression, on human lung adenocarcinoma cell (A549) proliferation.



Materials and methods:  Human Id3 cDNA was inserted into pEGFP-N1 vector to yield the recombinant eukaryotic expression vector pEGFP/Id3. Cells were transfected with pEGFP or pEGFP/Id3, and proliferation of EGFP-expressing cells was monitored by flow cytometry (FCM) and confocal fluorescence microscopy. RT-PCR, immunoblotting and immunocytochemistry were used to assess Id3 mRNA transcription and protein expression. Apoptosis was evaluated by Annexin V/7-AAD staining and FCM, while nuclear morphology of apoptotic cells was examined using Hoechst 33258 staining.



Results:  Over 4 days transfection with pEGFP, the proportion of EGFP-positive A549 cells peaked at approximately 60% by 48 h and remained stable over the next 48 h. In contrast, the proportion of EGFP-positive cells in cultures transfected with pEGFP/Id3 decreased from a peak of 60% at 48 h to <5% at 96 h, suggesting that Id3 expression inhibited cell proliferation or survival. Annexin V/7-AAD and Hoechst 33258 staining revealed significantly higher rates of apoptosis in pEGFP/Id3-transfected cells.



Conclusion:  Overexpression of Id3 triggered apoptosis in A549 human lung adenocarcinoma cells, implicating Id3 in negative control of tumour growth. These Id3-induced pro-apoptotic signalling pathways require further study, but this preliminary investigation suggests that Id3 regulation could be exploited in anti-tumour therapies.

Overexpression of Id3 induces apoptosis of A549 human lung adenocarcinoma cells.

The inhibitor of DNA-binding/differentiation 3 (Id3) protein is a helix-loop-helix transcription factor and may have an important role in cell proliferation and differentiation. This study was to evaluate the effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity. Short interference RNA suppression of Id3 (miRId3) in A549 cells was used to investigate the functional role(s) of Id3. Next, we used in vitro wound-healing assay and trans-well assay to study the effects of overexpressed Id3 on migration and invasion of A549 cells. Furthermore, to explore the influence of overexpressed Id3 on in vivo tumorigenesis, adenoviruses containing Id3 gene (Ad-Id3) and empty vector (Ad-LacZ) were generated. Co-transfection of pcDNA/miRId3 and pEGFP/Id3 into A549 cells reversed the Id3-induced cell proliferation inhibition and apoptosis. Upon Id3 transfection, A549 cells displayed decreased migratory and invasive capabilities, however, co-transfection of miRId3 and Id3 into A549 cells reversed the Id3-induced inhibitions of migratory and invasive capabilities. Three groups of nude mice were inoculated with Ad-LacZ, Ad-Id3 transfectants and untransfected A549 cells, respectively. Twenty-eight days after inoculation, tumors induced by Ad-Id3 transfectants grew much more slowly compared with Ad-LacZ transfectants and control group. This study provides for the first time both in vitro and in vivo proofs that forced expression of Id3 in lung adenocarcinoma cells reduces tumor growth rate and may be a potential target for tumor suppression.

Wei Yu

Papers

Long noncoding RNA expression profile in fibroblast-like synoviocytes from patients with rheumatoid arthritis

Identification of citrullinated peptides in the synovial fluid of patients with rheumatoid arthritis using LC-MALDI-TOF/TOF.

Proteomic analysis of synovial fibroblast-like synoviocytes from rheumatoid arthritis.

Overexpression of Id3 induces apoptosis of A549 human lung adenocarcinoma cells.

Effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity.