Material and Methods for Chapter 10 82

Isolation and culture of human monocyte-derived macrophages (MDM)

Human monocytes were isolated from healthy donors by Ficoll density gradient centrifugation as

previously described (42). Monocytes were plated at 10.5 × 103 cells/cm2 in Cell-Bind plates

(Corning) and cultured in DMEM supplemented with 10% FBS (Thermo Scientific), 10% horse serum (Invitrogen), 1% nonessential amino acids (Invitrogen), 2mM glutamine (Invitrogen), and

visually inspected for MDM differentiation before use in HIV-infection experiments. MDM were cultured for 7-10 DIV before use in noninfectious experiments.

HIV infection of MDM

Differentiated MDM were exposed to 0.2-50ng of p24 of HIV per 106 cells for 24 hours. All HIV-1

virus stocks were prepared by the University of Pennsylvania Center for AIDS Research Virology Core (Table 6.8) through transfection of 293T cells for molecularly cloned viruses or by amplification in primary human peripheral blood mononuclear cells. HIV-1 89.6 accessory gene mutants have been described previously (528). HIV-2 CBL-20 virus stock was obtained from Dr. Robin Weiss through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH. Supernatants from HIV-infected or noninfected (Mock) MDM were collected every 3 days through

12 days post-infection and stored at -80oC. Supernatants were monitored for HIV replication by

quantifying cell associated HIV-1 p24 content, as analyzed by Western blot, or by viral reverse transcriptase (RT) activity, as analyzed by the amount of radiolabeled deoxythymidine incorporation (529). Briefly, 10µl of MDM supernatant is incubated with 50µl of RT cocktail (50µM

Tris pH 7.8, 75µM KCl, 5µM MgCl2, 0.05% NP-40, 2µM DTT, 5µg/ml, 1.6 mU poly(rA)·p(dT)12-18,

and 10µCi/ml dTTP [α-32P]) at 37oC overnight. 30µl of sample is dotted onto DE81 Whatman ion

exchange cellular chromatography paper (Fisher) and air dried for 30 minutes, washed four times

with 2X UltraPure SCC (Invitrogen), washed once in absolute ethanol, and dried at 80-100oC for

30 minutes. Whatman paper is then placed in scintillation vial with 5ml of Scintiverse BD cocktail

84 Donor/ Sourcea Strain Isolate Type Co-receptor Usage Tissue Isolated From

Clinical Diagnoses Reference

A Jago Swarm CCR5 CSF AIDS Dementia

Complex Stage 0.5

(530)

B Doge Swarm CCR5 CSF AIDS Dementia

Complex Stage 1

(530)

C TYBE Swarm CXCR4 CSF CMV Encephalitis (530, 531)

D BR-2 Swarm CCR5 Brain Progressive

Dementia (532, 533) D CSF-2 Swarm CCR5 CSF Progressive Dementia (532, 533) E BL-3 Swarm CXCR4 PBMC (534) F SF162 Swarm CCR5 CSF Toxopplasmosis, Acute Meningitis (535, 536)

G JR-FL Swarm CCR5 Frontal Lobe AIDS Encephalopathy, Kaposi's Sarcoma

(537, 538)

G JR-CSF Swarm CCR5 CSF AIDS Encephalopathy,

Kaposi's Sarcoma

(537, 538)

H 89.6 Clone CCR5/ CXCR4 Blood AIDS with no

neurological disease

(538, 539)

I NL43 Clone CXCR4 Blood/

bone marrow

AIDS and non-AIDS (540)

J 3B Swarm CXCR4 Blood/

bone marrow

AIDS and non-AIDS (541, 542)

K YU2 Clone CCR5 Brain HIV-1 Associated

Encephalopathy

(543)

L Bal-1 Swarm CCR5 Lung Pediatric patient

with AIDS

(544)

M ADA Swarm CCR5 Blood Kaposi's Sarcoma (545)

Table 6.8 Characteristics of select HIV-1 clade B strains prepared by the Center for AIDS

Research Virology Core at the Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

Western blot analysis

MDM cultures were rinsed with ice-cold PBS, lysed in 75mM Tris-HCL (pH 6.8) containing 15% glycerol, 3.75mM EDTA, and 3% SDS, and supplemented with Complete Protease Inhibitor Cocktail (Roche) and PhosSTOP phosphatase inhibitor mixture (Roche). MDM protein lysates were subjected to SDS-PAGE as previously described (46) using primary and secondary antibodies listed in Tables 6.6 and 6.7 Protein expression as determined by Western blotting was normalized to GAPDH expression.

RNA quantification and qPCR analysis

Cell cultures were rinsed in ice-cold PBS and RNA was isolated using RNeasy Kit (Qiagen) per manufactures instructions. RNA concentration and quality was analyzed by NanoDrop 2000c (Thermo Scientific). Following cDNA generation using Reverse Transcriptase Kit (Applied Biosciences), quantitative real-time PCR (qPCR) was performed using RealMasterMix (5Prime)

on a MasterCycler RealPlex 2 (Eppendorf) with the primer sets listed in Table S4 in the

supplemental material.

MDM-mediated neurotoxicity and MDM extracellular glutamate

Rat cerebrocortical mixed neuroglial cultures (~80% neurons, 20% glia) were prepared from E17 embryos of Sprague-Dawley rats, as previously described (46). Cells were plated in tissue-culture plates pre-coated with poly-L-lysine (Peptides International) and maintained in neurobasal media

plus B27 supplement (Invitrogen) at 37oC and 5% CO2. After 7 DIV, approximately one-half

volume of fresh media was added to the cells to replace evaporation losses. All cultures were used between days 14 and 17 DIV. Cell-based MAP2 ELISAs were performed on primary rat

cerebrocortical cells plated at a density of 1x104 cells/well in 96-well plates as previously

86

replicates), neuronal cultures were fixed and fluorescently labeled using the following reagents:

mouse anti-MAP2 (Covance), goat anti-mouse β-lactamase TEM-1 conjugate (Invitrogen), and

Fluorocillin Green substrate (Invitrogen). Fluorescence intensity was measured using a flour metric plate reader with a 480/520-nm filter set. MDM supernatant was applied at 1:10-1:60 dilution; the dilution that gave values within the linear range of the assay is presented. For

uninfected MDM experiments pure neuronal cultures (~97-99% neurons, 5x104 cells/well) were

used in order to detect lower levels of neurotoxicity. To obtain pure neuronal cultures cells were treated with 10µM arabinosylcytosine 48 hours after plating and otherwise handled as described

above. Neuronal survival was expressed as a percentage of untreated (UT) cultures. In previous

publications we have demonstrated that this MAP-2 ELISA quantification robustly correlates with neuronal death as determined by hand counts of surviving MAP-2 stained neurons in this culture system (546, 547).

MDM extracellular glutamate

Glutamate concentration in MDM supernatant was assayed in triplicate using the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen) according to the manufacturer’s directions.

ART preparation

Stock solutions of Tenofovir disoproxil fumarate, Efavirenz, Atazanavir sulfate, Ritonavir, and

Raltegravir were prepared in DMSO and stock solution of Emtricitabine was prepared in H2O and

stored at -20oC until use. All ART drugs were provided by the NIH AIDS Research and Reference

Statistics

All quantifications are expressed as mean ± SEM. Two-tailed student’s t-test or one-way ANOVA followed by a Holm-Sidak post-test were performed on indicated comparisons. α = 0.05 unless otherwise noted in order to correct for multiple comparisons. Statistical input support was provided by the Biostatics and Data Management Core, Center for AIDS Research, Perelman School of Medicine, University of Pennsylvania.

Study approval

All animal studies and protocols were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and approved by the University of Pennsylvania IACUC. All human studies and protocols for monocyte isolation were reviewed and approved by the University of Pennsylvania IRB and all participants provided written informed consent.