Cytokine and chemokine levels (including IL-12, IL-8, IL-6, RANTES, and IL-17A) [31], and reorganize lipid rafts, potentiating an inflammatory response in bystander cells [77]. We hypothesize that in vivo soluble Nef released by necrosis of infected cells may well be internalized straight or by way of FcRs following forming immune complexes with anti-Nef antibodies produced in infected men and women. Then, the acidic environment with the endosomal compartments could favour the release of the viral protein in totally free kind, which may interact with the endosome membrane and, eventually, be translocated through a flip-flop mechanism by inducing signal transduction pathways involved within the regulation from the cellular secretome. This speculation may also clarify the results we previously obtained in THP-1 differentiated cells, applying the silencing procedure and co-immunoprecipitation approaches, indicating that (i) Nef is capable to kind a Trypanosoma Inhibitor MedChemExpress complex with TRAF2 through its conserved 4E acidic domain and (ii) extracellular Nef-induced production of inflammatory cytokines and IFN- in THP-1 demands the precise intracellular adaptors TRAF2 and six and the 4E acidic domain [19]. Finally, Nef, free inside the extracellular space, could possibly represent a danger signal, inducing cellular response distinct from that of Nef transferred through nanotubes, EVs or cell-to-cell contact. This should be taken into consideration inside the development of an HIV vaccine, primarily based also around the expression/presence of this viral protein or its conserved domain. In conclusion, the outcomes presented right here lay the foundation for extending the study to primaryViruses 2022, 14,30 ofpDCs, to determine the destiny from the internalized protein and analyse the content plus the biological activity from the exosomes released by treated cells.Supplementary Materials: The following are offered on the web at https://www.mdpi.com/article/ ten.3390/v14010074/s1, Table S1: Antibodies’ reagents for FC analysis. Figure S1: Purity of isolated cells; Figure S2: Morphology and phenotype of GEN2.two cell line. SSTR3 Agonist Biological Activity Author Contributions: All the experiments were conceived by A.A., F.G, Z.A.P. and E.A. methodology and formal evaluation of experiments had been performed by A.A., F.G., C.A. and Z.A.P.; A.A. analysed experimental data and wrote the original manuscript draft; A.A., F.G. and K.F. setup the Bodipy C16 methodology; S.L. and M.C. performed flow cytometry analyses; R.G. and E.D. performed the Bioplex secretome analysis; L.C. supplied the cell line GEN2.two and reviewed the manuscript; M.S. offered Bodipy reagent and methodology, E.A. and Z.A.P. reviewed and edited the manuscript, E.A. administered the project. All authors have read and agreed towards the published version in the manuscript. Funding: This research was funded by the grant “Excellence Departments, MIUR-Italy (ARTICOLO 1, COMMI 314–337 LEGGE 232/2016)” and “BE-VIN (protocollo GeCoWEB n. A0375-2020-36641, CUP F85F21003690009)”. Institutional Overview Board Statement: Not applicable. Informed Consent Statement: Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats obtained from human healthy donors at Centro Trasfusionale-Cattedra di Ematologia, Universitdegli Studi “La Sapienza” Rome. No ethical approval from University La Sapienza or Roma Tre ethics committees nor formal or verbal informed consent from blood donors had been necessary to use buffy coats as sources of cells. Information Availability Statement: Data sharing isn’t applicable to this short article. Acknowledgments: We thank P.