Lymphomonocytic Extracellular Vesicles Influence Fibroblast Proliferation and Collagen Production in Systemic Sclerosis

Abstract

Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by fibrosis, immune dysregulation, and vascular abnormalities. Extracellular vesicles (EVs), secreted by immune cells, have been implicated in modulating fibroblast activity and are actively involved in SSc pathogenesis. This study aims to determine whether lymphomonocytic-derived EVs influence fibroblast proliferation and collagen synthesis in SSc. Fibroblasts from healthy donors (HDFs) and SSc patients (SScHDFs) were exposed to EVs derived from Jurkat and U937 cell lines stimulated under pro-inflammatory conditions using tumor necrosis factor-α (TNFα) or phorbol 12-myristate 13-acetate + ionomycin (PMA + IONO). Proliferation was assessed using CCK-8 assays, while collagen production was quantified via ELISA. Our findings demonstrate that EVs derived from PMA + IONO-stimulated Jurkat and U937 cells significantly reduced fibroblast proliferation in a dose-dependent manner. Notably, SScHDFs exhibited lower baseline proliferation and a diminished overall response to EV treatment. Collagen production was markedly reduced in both fibroblast types following exposure to PMA + IONO-stimulated EVs, whereas TNFα-stimulated EVs affected only HDFs. These findings suggest that EVs from activated immune cells modulate fibroblast function in SSc, potentially contributing to disease pathogenesis. Further research is warranted to elucidate the molecular mechanisms underlying these effects and to explore the therapeutic potential of targeting EV-mediated signaling in SSc.

Keywords: Jurkat; U937; extracellular vesicles; fibroblasts; fibrosis; systemic sclerosis.

Figure 1

Evaluation of the effects of three different concentrations of extracellular vesicles on fibroblast proliferation. HDFs reduce proliferation when incubated with 25,000/50,000 EVs derived from (A) Jurkat or (B) U937 cells treated with PMA + IONO, as well as with 50,000 EVs obtained from U937 cells stimulated with TNFα (* p < 0.05). HDFs: human dermal fibroblasts; EVs: extracellular vesicles; PMA: phorbol 12-myristate 13-acetate; IONO: ionomycin; TNFα: tumor necrosis factor-alpha.

Figure 2

Evaluation of the effects of three different concentrations of extracellular vesicles on scleroderma fibroblast proliferation. SScHDFs reduce proliferation when incubated with (A) 12,500/25,000/50,000 EVs derived from Jurkat cells treated with PMA + IONO or 25,000/50,000 EVs treated with TNFα. (B) Incubation with 25,000/50,000 EVs from U937 cells reduces proliferation, and incubation with 50,000 EVs obtained from U937 cells stimulated both when incubated with PMA + IONO rather than TNFα (* p < 0.05, ** p < 0.005). SScHDFs: scleroderma human dermal fibroblasts; EVs: extracellular vesicles; PMA: phorbol 12-myristate 13-acetate; IONO: ionomycin; TNFα: tumor necrosis factor-alpha.

Figure 3

Evaluation of collagen production after stimulation with extracellular vesicles on fibroblasts. At baseline, the collagen production induced by HDFs and SScHDFs demonstrated a statistically significant difference. HDFs exhibited a notable difference from the control group when incubated with EVs derived from Jurkat cells stimulated with either TNFα or PMA + IONO. Similarly, EVs from U937 cells stimulated with PMA + IONO caused a significant difference. SScHDFs showed a significant reduction in collagen production only when incubated with EVs stimulated with PMA + IONO (* p < 0.05, ** p < 0.005). HDFs: human dermal fibroblasts; SScHDFs: scleroderma human dermal fibroblasts; EVs: extracellular vesicles; PMA: phorbol 12-myristate 13-acetate; IONO: ionomycin; TNFα: tumor necrosis factor-alpha.