Abstract
Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.
Highlights
A central part of many inflammatory diseases is the progression into chronicity
Both CD4+ and CD8+ synovial fluid mononuclear cells (SFMCs) had increased programmed death 1 (PD-1) expression compared to both rheumatoid arthritis (RA) Peripheral blood mononuclear cells (PBMCs) and healthy controls (HC) PBMCs (Figure 1A)
We analyzed these receptors on the CD4+ PD-1+ SFMCs and found them co-expressed with PD-1 (Figures 1C,D)
Summary
A central part of many inflammatory diseases is the progression into chronicity. T cell exhaustion is a state of cellular dysfunction that is described in infections, cancers, and autoimmune diseases [1,2,3,4]. The exhausted T cells counteract the ability of the immune system to clear the infection or the tumor cells. In a prolonged inflammatory response, the exhausted T cells develop into a hyporesponsive state and are, associated with the progression of chronicity [6]. The function of exhausted T cells in autoimmune diseases is less clear. These cells have recently been suggested to be associated with a better prognosis [4]. Exhausted cells are mainly described within the CD8+ T cell subpopulation [7], but exhausted CD4+ T cells [8] and B cells [9, 10] are described in relation to chronic inflammatory diseases
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