Abstract
Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1β, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.
Highlights
Plasmacytoid dendritic cells are one of the two principal subsets of human dendritic cells (DCs) and represent a link between innate and adaptive immunity [1,2]. constituting only 0.2–0.8% of human blood cells, they have garnered interest because they are able to produce up to 1000-fold more type I interferon (IFN) (IFN-α) than any other cell types [3]
Peripheral Blood Mononuclear Cells (PBMCs) depleted of monocytes (PBLs), PBLs depleted of Plasmacytoid dendritic cells (pDCs) (PBLs-pDCs) and PBMCs depleted of pDCs (PBMCs-pDCs) were isolated by negative selection, and the cells were resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 Units/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated FBS
Representative images of two independent are shown. experiments are shown. Since both type I (α/β) and type III (λ) IFN can regulate the expression of mxA gene, and their expression depends on a similar transcription model that requires the previous activation and nuclear translocation of specific IFN regulatory factors (IRFs), such as IRF-7 [44], we evaluated whether Nef treatment induced the activation and nuclear translocation of this factor in pDCs
Summary
Plasmacytoid dendritic cells (pDCs) are one of the two principal subsets of human dendritic cells (DCs) and represent a link between innate and adaptive immunity [1,2]. Since the effects of the pathogenic accessory protein Nef on pDCs have not been fully characterized, in this study, we examined the alterations in intracellular signalling and in the release of EVs induced by the treatment of non-HIV infected pDCs with myrNef. In particular, we used the human pDC cell line GEN2.2 as an experimental model system, demonstrating that myrNef treatment of these cells induced the release of a set of cytokines/chemokines which, in turn, activated STAT-1/2 proteins and influenced the gene expression program by inducing STAT1, IRF-1 and ISG15 expression. We observed that myrNef treatment did not increase the EV release of GEN2.2 cells, and the protein was found to be associated with small (size < 200 nm) vesicles produced by the pDC cell line
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