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Abstract
Background Microvesicles (MVs) expressing the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP) may play a pathogenic role in systemic lupus erythematosus (SLE). Co-expression of DNA on such MVs may render them immunogenic and target for anti-dsDNA antibodies. Little is known about the mechanisms underlying generation of this MV population. In this study, we investigated how Toll-like receptors, interferon- (IFN-) and T cells are related hereto in healthy subjects.
Methods Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy donors were stimulated in-vitro for 24 hours with a series of TLR-agonists or the T-cell activating antibody OKT3 or were subjected to apoptosis by incubation with staurosporine. MVs in the supernatants were subsequently isolated by differential centrifugation and were quantified and characterized with respect to expression of G3BP and DNA by flow cytometry.
Results Stimulation of PBMCs with the TLR9-agonist and strong IFN- inducer ODN2395 significantly increased the release of MVs expressing G3BP. A large proportion of these MVs expressed augmented levels of DNA on their surface. The production of MVs with this phenotype was markedly enhanced by co-stimulation of T cells. Furthermore, dependency on IFN- in the generation of G3BP-expressing MVs was indicated by a marked reduction following addition of the IFN- inhibitor IFN alpha-IFNAR-IN-1 hydrochloride.
PBMCs from healthy donors (n=6) were incubated for 24 hours with the TLR9-agonist ODN2395, alone (-) or in combination with the IFN- inhibitor IFN alpha-IFNAR-IN-1 hydrochloride (+). MVs released into the culture supernatants were subsequently isolated by differential centrifugation, quantified and characterized with respect to expression of G3BP and DNA by flow cytometry. (A) Concentration in the culture supernatant of G3BP-expressing MVs, DNA-expressing MVs, and G3BP/DNA double-positive MVs. (B) Corresponding concentration of MVs expressing these markers when co-stimulating T-cells with anti-CD3 antibody. (C) The G3BP-derived median fluorescence intensity (MFI) of the G3BP-expressing MV population and (D) the DNA-derived MFI of the DNA-expressing MV population released from the ODN2395-treated PBMCs in presence (black columns) or absence (gray columns) of anti-CD3 antibody (OKT3). Columns and error bars represent median values and interquartile range. (*) P<0.1. *P<0.05.
Conclusions The release of G3BP-expressing MVs from healthy donor PBMCs is induced by stimulation of TLR9 in an IFN--dependent manner. The co-expression of DNA accessible for anti-DNA antibodies on these MVs may render them relevant in lupus pathogenesis.
Funding Source(s): Lundbeck Foundation, Rigshospitalets Fund for Research, Gigtforeningen
Effect of IFN- inhibition on TLR9-induced release of MVs from mononuclear cells.