Background: Infection with human immunodeficiency virus type 1 (HIV-1) has transformed from a fatal disease into a chronic condition, as more than 70% of treated individuals achieve long-term viral suppression. Despite successful combination antiretroviral therapy (ART), comorbidities remain a significant clinical issue, causing a spectrum of cognitive, motor, and behavioral impairments known as HIV-associated neurocognitive disorders (HAND). One of the key factors in their pathogenesis is the presence of viral reservoirs in the central nervous system, particularly in microglia, where viral proteins such as Nef are expressed. Within the framework of this doctoral thesis, we aimed to define the role of the Nef protein in the release and properties of extracellular vesicles (EVs) on human neural progenitor cells (hNPCs), which also possess certain stem cell-like properties.
Hypotheses: We formulated the following hypotheses: (i) Nef expression in microglia affects the biophysical and molecular properties of released extracellular vesicles; (ii) Nef expression in T lymphocytes affects the biophysical and molecular properties of released extracellular vesicles; and (iii) extracellular vesicles containing Nef affect apoptosis, proliferation, and/or differentiation of neural stem cells.
Methods: In this study, we used human microglial cells (h-MG) and immortalized T lymphocytes (Jurkat) as sources of EVs, while hNPCs were used as a model of neural stem cells. Using lentiviral vectors with an inducible TET-ON system, we established stable h-MG and Jurkat cell lines expressing Nef.GFP prorein (or GFP as a control). Protein expression was verified by flow cytometry and fluorescence microscopy. EVs were isolated from cell culture supernatants by differential ultracentrifugation and additionally purified using an iodixanol density gradient. Their biophysical properties were analyzed using nanoparticle tracking analysis (NTA), nano-flow cytometry, and transmission electron microscopy (TEM), while their molecular composition was analyzed by Western blot (proteins) and nano-flow cytometry (lipids). The presence of Nef.GFP protein in EVs was additionally determined by TEM combined with immunolabeling, nano-Nef ELISA, and nano-flow cytometry. Functional effects of EVs were evaluated in hNPC by assessing metabolic activity, proliferation, apoptosis, and differentiation. For this purpose, fluorescence-based cell assays (metabolic activity, proliferation, apoptosis, necrosis) and luminescence-based assays (apoptosis) were employed, and the expression of specific markers was monitored using fluorescence microscopy and flow cytometry. All experiments included appropriate controls and replicates, and the results were analyzed using basic statistical methods.
Results: In our study, we established improved cellular models of translationally active HIV-1 reservoirs, enabling stable and controlled expression of the HIV-1 Nef.GFP protein in h-MG and Jurkat cells. We demonstrated that Nef.GFP selectively promotes the release of EVs from h-MG and Jurkat cells with distinct biophysical (higher abundance, small size, typical morphology, and density) and molecular (enriched with Nef, vesicular proteins, and lipids characteristic of lipid rafts) properties. Using single-vesicle analysis approaches, we showed that Nef.GFP is present in up to half of Nef-EVs, while immuno-TEM analysis revealed its localization on the luminal side of the vesicles. Within the framework of this dissertation, we investigated for the first time the functional role of Nef-EVs in hNPCs, with particular emphasis on their effects on differentiation into astrocytes. We indicated that Nef-EVs derived from h-MG and recombinant Nef (rNef) promote differentiation into astrocytes in one subset of the hNPC population, while increasing proliferation in another subset. In addition, we associated rNef with an increased level of apoptosis and necrosis in hNPCs directed toward astrocytic differentiation, whereas no such effect was observed for Nef packaged within EVs. Our study suggests a role for Nef-EVs in regulating the cellular state of hNPCs; however, additional independent biological experiments are required to more precisely define this role.
Conclusions: In this doctoral thesis, we demonstrated that Nef expression affects the biophysical and molecular properties of EVs released from microglia and T lymphocytes, thereby confirming the first and second hypotheses. The results also suggest that EVs isolated from microglia and T lymphocytes influence the apoptosis, proliferation, and differentiation of hNPCs, which were used as a model of neural stem cells, thereby partially confirming the third hypothesis.
Contribution to science: Our results contribute to a better understanding of the role of Nef and Nef-containing EVs in the central nervous system and suggest potential mechanisms associated with the development of HAND that should be further investigated in more complex experimental models. Furthermore, our findings open new possibilities for future research into intercellular communication and the potential development of targeted therapeutic approaches.
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