Tineke Anna de Jong

Cellular senescence, a state of proliferation arrest, is implicated in the pathogenesis of age-related diseases such as rheumatoid arthritis (RA). The pathogenesis of RA, characterized by immune dysregulation and systemic autoimmunity preceding clinical onset of disease, may involve early accumulation of senescent lymph node (LN) fibroblasts driving immune tolerance breakdown. This study aims to explore the hallmarks of senescence in LN fibroblasts during the earliest phases of RA and evaluate the effects of dasatinib. Human LN fibroblasts were isolated from inguinal LN needle biopsies from autoantibody-positive individuals at risk of developing RA (RA-risk individuals), RA patients, and seronegative healthy volunteers. Senescence hallmarks and the effects of dasatinib treatment were assessed using quantitative gene expression analysis, flow cytometry, microscopy, and live-cell imaging. Cell size, granularity, and autofluorescence were significantly greater in RA LN fibroblasts compared with controls. Altered gene expression of senescence-associated genes was observed in RA LN fibroblasts. Elevated senescence-associated β-galactosidase activity, more lipofuscin-positive granules, and DNA damage were observed in RA-risk and RA LN fibroblasts. Notably, RA(-risk) LN fibroblasts presented impaired DNA damage repair capacity. Dasatinib treatment significantly improved the size and ability of the LN fibroblast pool to repair DNA damage. We observed multiple senescence hallmarks in RA LN fibroblasts and, to a lesser extent, in RA-risk LN fibroblasts, which could be partially restored by senescent cell removal via dasatinib treatment. These findings suggest a role for senescent LN fibroblasts in RA pathogenesis and highlight the potential of dasatinib as a potential therapeutic intervention to mitigate senescence-associated defects in RA.

DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the “PRC2-AgeIndex,” defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.

Background Previous work has shown subtle infiltration of synovial T cells in the absence of overt synovial inflammation in individuals at risk of developing rheumatoid arthritis (RA). Objective To study the molecular changes in synovium preceding arthritis development in at risk individuals. Materials and methods We included sixty-seven individuals with arthralgia who were IgM rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positive and without any evidence of arthritis. All individuals underwent mini-arthroscopic synovial tissue sampling of a knee joint at baseline and were followed prospectively. An explorative genome-wide transcriptional profiling study was performed on synovial tissue using Agilent arrays (discovery cohort). Survival analysis was used to identify transcripts associated with arthritis after follow up. Expression levels of differentially expressed genes were validated using quantitative real-time PCR (qPCR). Immunohistochemistry was used to study gene candidates at the protein level in situ. Results In the discovery cohort, 6 of the 13 at risk individuals developed RA after a median follow-up time of 20 months (IQR 2 – 44; pre-RA). The 7 individuals who did not develop RA had a median follow-up time of 85 months (IQR 69 – 86). Using a False Discovery Rate of Conclusion Molecular changes can be detected in synovial tissues before clinical onset of arthritis. Alterations in the immune response genes and lipid metabolism are associated with development of arthritis.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.