No recovery of replication-competent HIV-1 from human liver macrophages
Abraham J. Kandathil, … , Alan S. Perelson, Ashwin Balagopal
Abraham J. Kandathil, … , Alan S. Perelson, Ashwin Balagopal
Published September 10, 2018
Citation Information: J Clin Invest. 2018;128(10):4501-4509. https://doi.org/10.1172/JCI121678.
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Research Article AIDS/HIV Infectious disease

No recovery of replication-competent HIV-1 from human liver macrophages

Abstract

Long-lived HIV-1 reservoirs that persist despite antiretroviral therapy (ART) are a major impediment to a cure for HIV-1. We examined whether human liver macrophages (LMs), the largest tissue macrophage population, comprise an HIV-1 reservoir. We purified LMs from liver explants and included treatment with a T cell immunotoxin to reduce T cells to 1% or less. LMs were purified from 9 HIV-1–infected persons, 8 of whom were on ART (range 8–140 months). Purified LMs were stimulated ex vivo and supernatants from 6 of 8 LMs from persons on ART transmitted infection. However, HIV-1 propagation from LMs was not sustained except in LMs from 1 person taking ART for less than 1 year. Bulk liver sequences matched LM-derived HIV-1 in 5 individuals. Additional in vitro experiments undertaken to quantify the decay of HIV-1–infected LMs from 3 healthy controls showed evidence of infection and viral release for prolonged durations (>170 days). Released HIV-1 propagated robustly in target cells, demonstrating that viral outgrowth was observable using our methods. The t1/2 of HIV-1–infected LMs ranged from 3.8–55 days. These findings suggest that while HIV-1 persists in LMs during ART, it does so in forms that are inert, suggesting that they are defective or restricted with regard to propagation.

Authors

Abraham J. Kandathil, Sho Sugawara, Ashish Goyal, Christine M. Durand, Jeffrey Quinn, Jaiprasath Sachithanandham, Andrew M. Cameron, Justin R. Bailey, Alan S. Perelson, Ashwin Balagopal

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Figure 1

Purification of liver macrophages yields minimal T cell contamination.

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Purification of liver macrophages yields minimal T cell contamination. (...
(A) LMs were purified from fresh liver tissue using a multistep protocol that progressively depletes T cells: bulk liver tissue was disrupted, centrifuged on a Histodenz gradient (Sigma-Aldrich), and followed by depletion of T cells using CD3 microbeads. Mononuclear cell fractions were incubated on plastic to separate adherent LMs from nonadherent cells and then incubated with Resimmune, a high-affinity T cell toxin, to deplete remaining T cells. LMs were incubated for more than 30 days, deprived of T cell mitogens, and maintained in the presence of antiretrovirals (ARVs) to prevent ex vivo infection. (B) The purification protocol depletes T cells. In a sample isolation of LMs from an HIV-1 uninfected liver, a sensitive qPCR assay for CD3ε mRNA showed the absence of detectable T cells. LMs and hepatocytes were also detected by qPCR assays for CD68 and albumin mRNA, respectively. The fold increases were compared with GAPDH. However, when GAPDH was not detected, as shown by the open blue box (CD68), fold-change was calculated by assigning GAPDH the last cycle number of the qPCR. (C) LM purification resulted in minimal T cell contamination in tissues taken from HIV-1–infected people. LM purity and T cell contamination were measured in duplicate on LMs isolated from 9 HIV-1–infected people before Resimmune and compared with a dilution series of total T cells. The protocol resulted in no detectable T cell contamination of LMs from 8 of 9 participants. In LMs from LT02, 1% T cell contamination was detectable, which was removed by maintaining the LMs in culture devoid of T cell mitogens for more than 90 days and treatment with Resimmune. The T cell dilution series represents the standard curve derived by performing qPCR for CD3ε mRNA on total RNA extracted from 10-fold dilutions of unactivated CD3+ T cells isolated by MACS separation from PBMCs obtained from a healthy donor. Error bars indicate mean ± SD. *For N9, there appeared to be PCR inhibition in the sample as indicated by poor detection of a housekeeping gene by qPCR.