Background We have shown recently that microparticles (MP) from monocytes stimulated with Poly(I:C) (PIC), a model for dsRNA, decrease the sensitivity of RA synovial fibroblasts (RASF) to TRAIL-induced apoptosis in an NF-kB-dependent manner. Extracellular nucleic acids can be incorporated in cell-derived MPand PIC is known to activate NF-kB signalling in different target cells.
Objectives To investigate whether PIC can associate with monocyte-derived MP and, thus, contribute to the observed anti-apoptotic effects of PIC-induced MP in RASF.
Methods MP were isolated by differential centrifugation from supernatants of the monocytic cell line U937, untreated or stimulated with PIC (16h), and then were analyzed by flow cytometry, Nanosight and BCA Protein Assay. The association of PIC with MP and the transfer of PIC to RASF were studied by flow cytometry using FITC-labelled PIC. Agarose gel electrophoresis and flow cytometry were used to define the susceptibility of soluble and MP-associated PIC to RNase III degradation. TRAIL-induced apoptosis of RASF was determined by Western blot for activated caspase 3 and by flow cytometry using Annexin V/PI staining. Additionally, MP from untreated U937 (control MP) were pre-incubated with PIC (1.5h) and their effect on the apoptosis of RASF was assessed by Annexin V/PI staining.
Results Number (control MP: 3.0*1010/mL vs PIC-induced MP: 3.1*1010/mL; n=2), size (median diameter 207 vs 199nm, n=2), Annexin V binding (mean ± SD: 66±10% vs 63±9%, n=3), the presence of CD4 on MP (20.9±1.4% vs 21.7±3.4%) and total protein content (330±50 vs 325±83 ng/mL) did not differ significantly between control MP and PIC-induced MP. FITC PIC was detected in 14.5±1.3% of U937-derived MP (n=3). In contrast to soluble PIC, MP-associated FITC-PIC was resistant to degradation by RNAse III (FITC-PIC MP + RNaseIII: 33.3±0.7; FITC-PIC MP: 29.0±2.6, n=2-3) and was stably present in cell culture supernatants from RASF treated with MP for 24h (FITC-PIC MP: 12.9 ± 0.2, n=3). PIC was transferred to RASF by MP as confirmed by a 27±15% increase in fluorescence intensity of RASF (n=7, p=0.002) treated with FITC PIC-induced vs PIC-induced MP. The sensitivity of RASF to TRAIL-induced apoptosis decreased after treatment of RASF with PIC-induced MP but not control MP, as shown by inhibition of caspase 3 cleavage (PIC-induced MP: 72.0±23.5% vs TRAIL: 100%, p=0.03, n=6; control MP: 102.1±9.8%, n=5) and a decrease in Annexin V staining (PIC MP: 21.5±15.7% vs TRAIL: 25.5±18.0%, p=0.006, control MP 26.4±17.2%, n=16) in RASF treated with PIC-induced MP. Furthermore, MP from untreated U937, preincubated with PIC, decreased TRAIL-induced apoptosis of RASF to a simlar extent as PIC-induced MP (PIC-preincubated MP: 11.9%±4.8%, PIC MP: 11.8±5.5%, TRAIL: 14.0±4.9%), confirming the anti-apoptotic effect of MP-associated PIC.
Conclusions Extracellular dsRNA PIC can associate with monocyte-derived MP and exist in a form protected from RNAse degradation. PIC, associated with MP, can be transferred to RA synovial fibroblasts and protect RASF from TRAIL-induced apoptosis. These findings suggest a mechanism by which MP can incorporate nucleic acid to modulate their immunological activity.
Acknowledgements SUPPORTS Articulum, IMI-BT Cure, Masterswitch, IAR.
Disclosure of Interest None Declared
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