Candidate Markers for Stratification and Classification in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune, inflammatory disease, characterized by synovitis in small- and medium-sized joints and, if not treated early and efficiently, joint damage, and destruction. RA is a heterogeneous disease with a plethora of treatment options. The pro-inflammatory cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of RA, and TNF inhibitors effectively repress inflammatory activity in RA. Currently, treatment decisions are primarily based on empirics and economic considerations. However, the considerable interpatient variability in response to treatment is a challenge. Markers for a more exact patient classification and stratification are lacking. The objective of this study was to identify markers in immune cell populations that distinguish RA patients from healthy donors with an emphasis on TNF signaling. We employed mass cytometry (CyTOF) with a panel of 13 phenotyping and 10 functional markers to explore signaling in unstimulated and TNF-stimulated peripheral blood mononuclear cells from 20 newly diagnosed, untreated RA patients and 20 healthy donors. The resulting high-dimensional data were analyzed in three independent analysis pipelines, characterized by differences in both data clean-up, identification of cell subsets/clustering and statistical approaches. All three analysis pipelines identified p-p38, IkBa, p-cJun, p-NFkB, and CD86 in cells of both the innate arm (myeloid dendritic cells and classical monocytes) and the adaptive arm (memory CD4 + T cells) of the immune system as markers for differentiation between RA patients and healthy donors. Inclusion of the markers p-Akt and CD120b resulted in the correct classification of 18 of 20 RA patients and 17 of 20 healthy donors in regression modeling based on a combined model of basal and TNF-induced signal. Expression patterns in a set of functional markers and specific immune cell subsets were distinct in RA patients compared to healthy individuals. These signatures may support studies of disease pathogenesis, provide candidate markers for response, and non-response to TNF inhibitor treatment, and aid the identification of future therapeutic targets.

A comparison framework and guideline of clustering methods for mass cytometry data

“…Mass cytometry, which is achieved with Cytometry by Time-Of-Flight (CyTOF), is an advanced experimental technology that measures levels of multiple proteins (up to 40) in a large amount (usually several millions) of cells [2]. The supreme ability to access a large panel of proteins simultaneously makes CyTOF useful in drug optimization [3], vaccine development [4], and disease marker discovery [5]. Compared to the well-known technology of single-cell RNA-sequencing (scRNA-seq) [6–8], which processes on average tens of thousands to hundreds of thousands of cells, CyTOF achieves a higher throughput (on average up to millions of cells) and classifies cells from a mixture into distinct subtypes based on expression levels of their surface antigen.…”

Combination of subtherapeutic anti-TNF dose with dasatinib restores clinical and molecular arthritogenic profiles better than standard anti-TNF treatment

“…To further study the potential repositioning of dasatinib and bosutinib for the treatment of arthritis, we took advantage of the hTNFTg mice (Tg197), which spontaneously develop human TNF-driven arthritis pathology (16), similar to that observed in a subset of human RA patients responsive to anti-hTNF treatment (42). In Tg197 mice, synovial broblasts (SFs) play a key pathogenic role (43), similar to the human RA pathology and we show here that both TKIs have a suppressive effect on parameters of the activated arthritogenic phenotype of Tg197-derived SFs.…”

Progress and applications of mass cytometry in sketching immune landscapes

“…17 Combined with lineage markers and stimulations or inhibitions, the signaling states and cellular responsiveness are comprehensively evaluated. 16,18,19…”

“…Many studies have used CyTOF to examine the pathogenesis of autoimmune diseases (Table 4). 18,36,[106][107][108][109][110][111] Several researchers focused on the immunome perturbations in patients with rheumatoid arthritis, a chronic autoimmune, inflammatory disease depicted with synovitis in small-and medium-sized joints, 18,[106][107][108][109] exploring both the peripheral blood 18,107 and synovium tissue samples. 106,108 In one study, 106 the investigators examined synovitis of patients with rheumatoid arthritis through a 36-plex CyTOF panel specific to activated T cells.…”