Date of Award

2-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Pharmacometrics

Research Advisor

Santosh Kumar, Ph.D.

Committee

Theodore J. Cory Francesca-Fang Liao Wei Li Murali Yallapu

Keywords

Cytochrome P450, Cytokine, Extracellular Vesicles, HIV-1, Oxidative stress, Smoking

Abstract

Introduction. Smoking, which is highly prevalent in people living with HIV/AIDS, has been shown to exacerbate HIV-1 replication, in part via cytochrome P450 (CYP)-induced oxidative stress. CYP enzymes metabolize cigarette smoke condensate (CSC), causing oxidative stress and cytotoxicity. Our previous studies have demonstrated that CSC and specific CSC constituents, benzo(a)pyrene and nicotine, potentially induce CYPs, resulting in higher oxidative stress and subsequent exacerbation of HIV-1 replication in monocytes and macrophages. However, the exact mechanism behind tobacco-induced, oxidative stress-mediated enhancement of HIV-1 replication is still poorly understood. Extracellular vesicles (EVs) have recently gained attention for their unique nature as intercellular messengers which can package proteins, nucleic acids, lipids etc. EVs are known to alter HIV-1 pathogenesis through intercellular communication. Until now, the role of EVs in smoking-enhanced HIV-1 pathogenesis has been mostly unknown. In this study, we investigated the effect of CSC on the characteristics and differential packaging of monocyte- and macrophage-derived EVs, and their influence on HIV-1 replication. We hypothesized that CSC- and/or HIV-1-exposed monocyte and macrophage-derived EVs and their components, especially pro-oxidant factors, are key mediators of HIV-1 replication.

Methods. Two monocytic cell lines, U937 and HIV-1-infected U1 cells, and macrophages derived from these monocytes, as well as macrophages derived from primary human monocytes were used. Cells were treated with 10 μg/ml/day CSC. After treatment, the cells were harvested, and the supernatant was collected for isolating EVs by Total Exosome Isolation kit. The isolated EVs were characterized for their biophysical properties. Next, monocyte-derived macrophages were exposed to EVs, as well as subjected to downstream analysis (p24 ELISA, LDH cytotoxicity assay, DNA damage assay, rtPCR, western blot, cytokine analysis).

Results. Initially, we demonstrated that CSC reduced total protein and antioxidant capacity in EVs derived from HIV-1-infected and uninfected monocytes. The EVs from CSC-treated uninfected cells showed a protective effect against cytotoxicity and viral replication in HIV-1-infected macrophages. However, EVs derived from HIV-1-infected cells lost their protective capacity. The results suggested that the exosomal defense is likely to be more effective during the early phase of HIV-1 infection and diminishes at the latter phase.

Next, we investigated differential packaging of specific contents in EVs subjected to CSC and HIV-1 exposure. We observed CSC-induced upregulation of catalase in EVs from uninfected cells, with a decrease in the levels of catalase and PRDX6 in EVs from HIV-1-infected cells. We also observed higher expression of CYPs (1A1, 1B1, 3A4) and lower expression of antioxidant enzymes (SOD-1, catalase) in EVs from HIV-1-infected macrophages compared to those from uninfected macrophages. Together, they are expected to increase concentrations of oxidative stress factors in EVs derived from HIV-1-infected cells. Moreover, our results show that longer exposure to CSC increased the expression of cytokines in EVs from HIV-1-infected macrophages, when compared to the shorter exposure. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs from HIV-1-infected macrophages upon both long and short-term CSC exposures. Anti-inflammatory cytokines, particularly IL-10, had high packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-1-infected and uninfected macrophages.

Conclusion. Taken together, our results suggest a potential role of CSC-exposure in modulating HIV-1-infected and uninfected cell-derived EVs, thereby affecting HIV-1 replication in recipient cells. Our study also suggests the packaging of increased levels of oxidative stress-inducing and inflammatory elements in EVs upon exposure to tobacco constituents and/or HIV-1, which would ultimately enhance HIV-1 replication in macrophages via cell-cell interactions.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

http://orcid.org/0000-0003-1469-6232

DOI

10.21007/etd.cghs.2020.0506

Additional Files
2020-015-Haque-DOA.pdf (390 kB)
Declaration of Authorship
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