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. 2020 Sep 1;34(11):1581-1591.
doi: 10.1097/QAD.0000000000002589.

"V体育官网" Proliferation of HIV-infected renal epithelial cells following virus acquisition from infected macrophages

Kelly Hughes  1 , Guray Akturk  2 , Sacha Gnjatic  2 , Benjamin Chen  2 , Mary Klotman  1 , Maria Blasi  1
Affiliations

Proliferation of HIV-infected renal epithelial cells following virus acquisition from infected macrophages

"VSports最新版本" Kelly Hughes et al. AIDS. .

Abstract

Objectives: HIV-1 can infect and persist in different organs and tissues, resulting in the generation of multiple viral compartments and reservoirs. Increasing evidence supports the kidney as such a reservoir. Previous work demonstrated that HIV-1 infected CD4 T-cells transfer virus to renal tubule epithelial (RTE) cells through cell-to-cell contact. In addition to CD4 T cells, macrophages represent the other major target of HIV-1. Renal macrophages induce and regulate inflammatory responses and are critical to homeostatic regulation of the kidney environment VSports手机版. Combined with their ability to harbour virus, macrophages may also play an important role in the spread of HIV-1 infection in the kidney. .

Design and methods: Multiparametric histochemistry analysis was performed on kidney biopsies from individuals with HIV-1 associated nephropathy (HIVAN). Primary monocyte-derived macrophages were infected with a GFP-expressing replication competent HIV-1. HIV-1 transfer from macrophages to RTE cells was carried out in a coculture system and evaluated by fluorescence-microscopy and flow-cytometry V体育安卓版. Live imaging was performed to assess the fate of HIV-1 infected RTE cells over time. .

Results: We show that macrophages are abundantly present in the renal inflammatory infiltrate of individuals with HIVAN. We observed contact-dependent HIV-1 transfer from infected macrophages to both primary and immortalized renal cells. Live imaging of HIV-1 infected RTE cells revealed four different fates: proliferation, hypertrophy, latency and cell death V体育ios版. .

Conclusion: Our study suggests that macrophages may play a role in the dissemination of HIV-1 in the kidney and that proliferation of infected renal cells may contribute to HIV-1 persistence in this compartment VSports最新版本. .

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VSports app下载 - Figures

Fig. 1.
Fig. 1.. Presence of macrophages in interstitial infiltrates observed in HIVAN.
Multiplexed immunohistochemical consecutive staining on single slide (MICSSS) analysis on a kidney biopsy from a HIV-1 positive individual with HIVAN. The mononuclear infiltrate was characterized by serially staining the tissue with markers for macrophages (CD68), T cells (CD3), cytotoxic T cells (CD8), B cells (CD20) and granulocytes (CD66b). Composite figure is produced by using each marker image with image registration, colour inversion and image overlay method. Composite figure shows CD3+ cells in red, CD8-positive cells in green, CD20 positive cells in cyan, CD66b+ cells in magenta and CD68-positive cells in yellow colour. The shown staining of interstitial inflammatory infiltrates is from one representative HIVAN renal biopsy from a formalin fixed paraffin embedded tissue sample.
Fig. 2.
Fig. 2.. HIV-1 infected macrophages mediate cell-to-cell infection when cocultured with renal tubule epithelial cells.
(a) Monocyte-derived macrophages were differentiated in presence of 20ng/ml of MCSF for 7 days and then infected with 2.5 MOI of each of the indicated GFP-expressing IMCs. Infection rates were evaluated by assessing the percentage of GFP+ cells by flow-cytometry 7 days post infection. (b) HPT-1b-mCherry cells were incubated with at least 15 MOI of cell-free virus or separated from infected macrophages by a transwell membrane. No GFP expression could be detected in those conditions. HIV-infected macrophages were cocultured with either the HPT-1b-mCherry renal epithelial cell line (c) or with urine-derived autologous renal cells previously stained with the CellTracker Deep Red dye (d). Transfer of virus from macrophages to HPT-1b-mCherry and primary renal cells was observed 3 days postcoculture as demonstrated by the presence of GFP/mCherry double positive cells. (e) Percentages of mCherry/GFP double positive Hpt-1b cells and GFP/Deep Red double positive primary RTE were assessed by flow cytometry at day 6 post coculture with infected macrophages. Data are shown as mean+standard deviation of three separate coculture experiments. (f-h) Primary macrophages were infected with NLGI/JRFL or the JRFL pseudotyped envelope mutant virus NLGIΔenv for 24 h prior to coculture with HPT-1b-mCherry renal cells. Infection of renal epithelial cells was evaluated by flow cytometry (f) or fluorescence microscopy (h) 6 days postcoculture with infected macrophages. (g) Percentage of mCherry/GFP double positive Hpt-1b cells were assessed by flow cytometry at day 6 post coculture with macrophages infected with NLGI/JRFL or NLGIΔenv. Data are shown as mean+standard deviation of three separate coculture experiments. White arrows in (h) indicate mCherry/GFP double positive renal cells.
Fig. 3.
Fig. 3.. HIV gene expression in renal epithelial cells post coculture with infected macrophages.
Renal epithelial cells were plated with macrophages 24 h prior to infection with 10 MOI of the HIV-ADA-GFP IMC, and cocultured for 6 additional days. mCherry/GFP double positive HPT-1b (a) or urine-derived primary renal cells (b) were flow-sorted 7 days postinfection and replated for further analysis. (c) Flow-sorted cells were analysed by fluorescence microscopy 24 h postsort to confirm the isolation of a pure population of mCherry/GFP double positive renal epithelial cells. (d) Time course microscopy analysis of HIV-GFP expression in flow-sorted mCherry/GFP double positive renal epithelial cells (days 1–7). (e) Number of cells, originally sorted as mCherry/GFP double positive, expressing GFP and/or mCherry at day 1, 4 and 7 postsort. Data are shown as mean+standard deviation of the number of cells positive for each marker in 3 different fields.
Fig. 4.
Fig. 4.. Infected renal cells produce infectious virus and transfer HIV-1 to target immune cells.
(a) Primary macrophages were infected with HIV-GFP for 24 h prior to coculture with HPT-1b-mCherry renal cells. HPT-1b-mCherry renal cells were treated with AZT (100μmol/l) or raltegravir (10μmol/l) for 1 h prior to coculture with infected macrophages and drugs were replenished every day throughout the remainder of the coculture experiments. Infection of renal epithelial cells (mCherry/GFP+ double positive cells) in the coculture was evaluate after 6 days by flow-cytometry. (b) Percentage of mCherry/GFP double positive Hpt-1b cells in AZT and raltegravir (RAL) treated or untreated cocultures shown as mean+standard deviation of three separate coculture experiments. (c) The GHOST(3) CXCR4+/CCR5+ indicator cell line was incubated with supernatants collected from flow-sorted mCherry/GFP double positive HPT-1b cells or primary urine-derived renal cells 4 days postsort; the presence of infectious virions was evaluated by assessing the percentage of GFP+ cells by flow-cytometry 48 h post infection. (d) CEM-SS T cells or THP-1 monocytes were incubated for 7 days with mCherry/GFP double positive HPT-1b cells that had been in culture for 5 days after being flow-sorted. Transfer of virus from renal cells to T cells or monocytes was evaluated by fluorescence microscopy (d) or flow-cytometry (e).
Fig. 5.
Fig. 5.. Proliferation of HIV-1 infected renal epithelial cells.
Macrophage HPT-1b-mCherry cocultures were analysed over time by fluorescence microscopy to assess viral transfer and the fate of infected renal cells. Clusters of mCherry/GFP double positive renal cells began to appear at day 4 postcoculture (white arrows in Merge images in panel a). Live imaging of cocultures between HIV infected macrophages and either HPT-1b-mCherry (b) or primary urine-derived renal epithelial cells (c) between days 5 and 7 postinfection. Imaging demonstrates three different cellular fates: Clustered proliferation of infected mCherry/GFP double positive renal cells (b–d); Hypertrophy, persistent GFP expression and no cellular division (white arrows in b); or cell death (white arrows in e). Red arrows in b indicate an HIV infected macrophage. Numbers indicate elapsed time since beginning of live imaging. White boxes in b and c delimit areas where cell proliferation was observed (see supplemental movies 1, http://links.lww.com/QAD/B760 and 2, http://links.lww.com/QAD/B761).
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