Author

Sabina Ranjit

Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

Santosh Kumar, Ph.D.

Committee

Sarka Beranova-Giorgianni, Ph.D. Subhash C. Chauhan, Ph.D. Theodore J. Cory, Ph.D. Anil Kumar, Ph.D. P. David Rogers, Ph.D.

Abstract

The advent of antiretroviral therapy (ART) has substantially increased the life span of people living with HIV-1 (PLWH). However, the presence of comorbid conditions such as high prevalence of drug abuse and coinfections have deteriorated the health outcomes in PLWH and progression of HIV-1 infection to AIDS. In this study, we will be focusing on the independent effect of two major comorbidities: cigarette smoking and human papilloma virus (HPV) coinfection on HIV-1 replication and pathogenesis in monocytic cells.

The prevalence of smoking is approximately 3-times higher in PLWH compared to the general population. Scientific evidences suggest that smoking aggravates HIV-1 pathogenesis and leads to decreased responses to ART. However, the exact mechanism by which smoking enhances HIV-1 replication is still not clear. The Kumar group has demonstrated the role of nicotine, at least in part, on HIV-1 replication in monocytic cells via cytochrome P450 (CYP)-mediated oxidative stress pathway. They have also shown the role of cigarette smoke condensate (CSC), which contains numerous organic compounds such as polyaryl hydrocarbons (PAHs), on cytotoxicity and HIV-1 replication in monocytic cells. Therefore, the goal in this study was to identify active ingredient of CSC, especially PAHs, which is responsible for cytotoxicity and HIV-1 replication in monocytic cells, and their underlying mechanism. PAHs such as benzo(a)pyrene (BaP), naphthalene (NPh), phenanthrene (Phe), benzo(a)antharacene (BeA), and benzo(b)fluoranthene (BeF) are known carcinogens present in cigarette smoke. As a first part of our study, we examined the relative effect of these constituents on the cytotoxicity of monocytic cells and the possible mechanism of PAH-mediated cytotoxicity. We examined the acute (6-24 hours) and chronic (7 days) effects of these PAHs on the expression of cytochromes P450 (CYPs), oxidative stress, and cytotoxicity. The treated cells were examined for mRNA and protein levels of CYPs (1A1 and 3A4) and antioxidants enzymes (AOEs) superoxide dismutase-1 (SOD1) and catalase. Further, we assessed the levels of reactive oxygen species (ROS), caspase-3 cleavage activity, and cell viability. We performed these experiments in U937 cell lines and/or primary monocytic cells. Of the five PAHs tested, after chronic treatment only BaP (100 nM) showed a significant increase in the expression of CYP1A1, AOEs (SOD1 and catalase), ROS generation, caspase-3 cleavage activity, and cytotoxicity. However, acute treatment with BaP showed only an increase in the mRNA expression of CYP1A1. These results suggested that of the five PAHs tested, BaP is the major contributor to the toxic effect of PAHs in monocytic cells, which is likely to occur through CYP and oxidative stress pathways.

Secondly, we investigated a molecular mechanism that would explain BaPinduced HIV-1 replication. We hypothesized that CYP-mediated BaP metabolism generates ROS, and the resultant oxidative stress aggravates HIV-1 replication. As expected, we observed ~4-fold increase in HIV-1 replication in differentiated U1 (HIV-1- infected U937) monocytic cell lines and human primary macrophages after chronic BaP exposure. We also observed ~30-fold increase in the expression of CYP1A1 at mRNA level, ~2-fold increase in its enzymatic activity, as well as, an elevated ROS level and cytotoxicity in U1 cells. The knock-down of the CYP1A1 gene using siRNA and treatment with selective CYP inhibitors and antioxidants significantly reduced HIV-1 replication. Further, we observed a nuclear translocation of NF-κB subunits (p50 and p65) after chronic BaP exposure, which was reduced by treatment with siRNA/inhibitors of CYP and antioxidants. Suppression of NF-κB pathway using specific NF-κB inhibitors also significantly reduced HIV-1 replication. Together, our results suggest that BaP enhances the HIV-1 replication in macrophages by CYP-mediated oxidative stress pathway via NF-κB signaling cascade.

Thirdly, we explored the underlying mechanism by which HPV increases HIV-1 pathogenesis. HPV infection is one of the major factors that contribute to a reduced suppression of the virus in HIV-1 patients. There is a high prevalence of comorbidity of HIV-1 and HPV (which leads to cervical cancer) among HIV-1-infected population. We proposed that exosomes secreted from HPV-infected cervical cancer cells exacerbate HIV-1 replication in HIV-1-infected macrophages. To test the hypothesis, we treated U1 cells (HIV-1-infected monocytic cell line) with the cell culture supernatant (CCS) obtained from caski cells (HPV-infected cervical cancer cells). We observed an ~2-fold increase in HIV-1 replication in the treated U1 cell. We also observed a significant increase in the expression of CYPs (CYP 1A1 and 2A6) at the mRNA level. However, we did not observe any significant change in the expression of CYPs as well as antioxidants enzymes (catalase, PRDX6) at the protein level. Furthermore, we isolated exosomes from the caski cell culture supernatant and observed the presence of CYPs (1A1, 2A6), SOD1 and HPV protein HPV16 E6 in caski exosomes. The exosomes derived from caski cells (CCS-Exo) significantly reduced cytotoxicity, while it increased HIV-1 replication in U1 cells. Treatment of antioxidant such as resveratrol, CYP1A1 inhibitor (ellipticine) and CYP2A6 inhibitor (Tryptamine) including chemodietary agents such as curcumin (20 μM) and curcubitacin-D (0.1 μM), significantly reduced the CCS and CCS-Exo mediated HIV-1 replication in U1 cells. These results suggest the role of specific CYP-induced oxidative stress pathway in HIV-1 replication. Altogether, we demonstrated that cervical cancer cells exacerbate HIV-1 replication in monocytic cells via transferring oxidative stress factors such as CYPs and HPV oncoproteins through exosomes. We also showed that the viral replication undergoes via a CYP-mediated oxidative stress pathway and it can be reduced by treatment of chemodietary agents like curcumin and Cucurbitacin-D. The present study therefore, illustrates that comorbidities such as smoking and HPV coinfection contribute to HIV-1 replication and pathogenesis in monocytic cells via a CYP-mediated oxidative stress pathway. The study also provides scientific rationale for the development of novel therapeutic treatment for PLWHs with these comorbidities by targeting CYP and specific oxidative stress pathway.

ORCID

http://orcid.org/0000-0001-5607-8403

DOI

10.21007/etd.cghs.2018.0467

Available for download on Friday, August 21, 2020

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