Mechanisms of Endogenous HIV-1 Reactivation by Endocervical Epithelial Cells

Abstract

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia (“viral blips”) during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells’ HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption. IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped.

Buprenorphine Increases HIV-1 Infection In Vitro but Does Not Reactivate HIV-1 from Latency

“…There are two additional reasons why we chose to analyze potential reactivation effects on J-Lat 11.1. First, in our previous work [ 60 ], we showed it has less spontaneous reactivation than other J-Lat cells (~10% GFP + in media), but it is more easily reactivatable by latency-reversing agents, including TNF-α. Second, J-Lat 11.1 possesses an HIV structure more similar to the HIV provirus found in vivo, compared to some of the other J-Lats, such as A1 and A7, which contain only a mini-HIV cassette [ 56 ].…”

“…We also tested in vitro-differentiated Langerhans cells as a surrogate of mucosal Langerhans/dendritic cells and found they transcribed the NOP gene even more strongly than vaginal macrophages ( Figure 1 D), but were also negative for MOP , DOP , and KOP . Because epithelial cells can produce factors, such as TNF-α, which in turn influence HIV infection in leukocytes [ 60 ], we also assessed opioid receptor mRNA expression in primary epithelial cells generated from surgically excised vaginal, endocervical and ectocervical tissues. These cells also expressed NOP but not the classical opioid receptors ( Figure 1 D,E).…”

“…The deep submucosa was removed with surgical scissors and the remaining mucosa was cut into 5 × 5 mm pieces. Genital epithelial cell lines from vagina, endocervix and ectocervix were generated and expanded in the presence as feeder cells and the Rho kinase inhibitor Y-27632, as described before [ 60 ]. To isolate vaginal leukocytes from these tissue pieces, we followed a previously published method [ 61 ].…”

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir

“…Thus, it remains unknown whether mucosal sites, 51 especially the gut, [52][53][54][55] are clinically relevant reservoirs that cause intermittent plasma viremia during suppressive ART and/or viral rebound after ART cessation. 56,57 Therefore, optimizing our protocol for use with tissue specimens was a crucial step to expand future reservoir studies beyond peripheral blood. One important component of this optimization is the ability to normalize proviral copy counts to the number of HIV target cells.…”

Brief Report: Vaginal Viral Shedding With Undetectable Plasma HIV Viral Load in Pregnant Women Receiving 2 Different Antiretroviral Regimens: A Randomized Clinical Trial

“…This compartment has been hypothesized to be a source of low-level viremia in plasma with use of ARVs, associated with virologic failure, rarely with selection of drug-resistance mutations, and with clones of HIV-infected cells in the genital tract. 3 , 4 However, studies of genital shedding in sexual and perinatal HIV transmission are few. Sexual transmission in the largest randomized trial of ART on sexual transmission (HTPN 052) found no genetically linked transmissions from HIV-infected individuals when their virus replication was suppressed by antiretroviral therapy (ART) 5 when measured by PVL.…”