Purification of extracellular vesicles from the plasma of HIV + patients and preparation for omics assays

Abstract

Cells release extracellular vesicles (EVs) by different mechanisms that involve cell membrane turnover in response to stimuli or during apoptosis. These are important for intercellular communication as they carry membrane, cytoplasmic, and nuclear components, which contain proteins, lipids, messenger RNAs, and metabolically active microRNAs. The involvement of EVs in the immune system modulation has been demonstrated, including during HIV infection, which is characterized by an inflammatory response with exacerbated production of cytokines and induction of early senescence of the innate immune system. In this context, our group has shown that immunological dysfunctions involving cytokine production and effector function of monocytes from HIV-infected individuals are correlated with epigenetic alterations in histones and DNA, which can be induced by proteins and microRNAs present in EVs, thus regulating gene expression. The production of EVs has also been associated with the development of neurocognitive diseases in HIV+ patients, as it has been reported that HIV-infected macrophages in vitro produce EVs containing glutaminase, the enzyme responsible for the synthesis of the neurotransmitter glutamate. In addition, HIV -1-infected dendritic cells in vitro produced EVs, which are involved in the activation and trans-infection of the virus to T cells during immunological synapse. Despite the importance of EVs, little is known about the content of the vesicles of HIV-infected patients. Therefore, our research group has sought to identify EV molecules related to disease progression, as we believe that the vesicles released into the circulation carry components capable of modulating the gene expression of immune cells, regulating the inflammatory response, and inducing early immunosenescence in HIV-infected individuals. We also sought to assess how the disease alters the content of extracellular vesicles circulating in the blood of chronic HIV+ patients, especially regarding the presence of components that play a role in epigenetic alterations. These components have the potential for clinical use, as they are targets for prospecting biomarkers in various pathological processes. (AU)