Cells and reagents
Purified human peripheral blood monocytes were obtained from Human Immunology Core at the University of Pennsylvania (Philadelphia, PA, USA). The Core has the Institutional Review Board approval for blood collection from healthy donors. Freshly isolated monocytes were cultured in 1640 RPMI medium supplemented with 10% fetal bovine serum, 1% nonessential amino acid, 1% L-glutamine and 1% penicillin-streptomycin solution. Rabbit antibodies against OAS2, GBP5, ISG56, Viperin, ISG15, IRF7, p-IRF7, STAT1, p-STAT1 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-HIV p24 antibody was obtained from the NIH AIDS Research and Reference Reagent Program (Bethesda, MD, USA). METH was purchased from Sigma Aldrich (St Louis, MO, USA). METH powder was dissolved in sterile endotoxin-free water (HyPureTM Cell culture grade water, GE Healthcare Life Science, Logan, UT, USA) and stored at 4℃.
HIV infection and METH Treatment
HIV Bal strain was obtained from AIDS Reagent Program (NIH, Bethesda, MD. Freshly isolated and purified monocytes in a 48-well plate were treated with METH at clinically relevant concentrations [51-54] (0, 100, 150, and 250 μM) for 24 h before being infected with HIV Bal strain (p24 60 ng/106 cells) overnight. The cells were then washed three times with plain RPMI to remove any unabsorbed virus and cultured in the presence of METH for 72 h.
The cytotoxic effect of METH on monocytes was evaluated by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, innersalt) assay. Freshly isolated human blood monocytes (2 ×104 cells/well) were placed in 96-well round bottom plates, and treated with different concentrations of METH (0,100, 150, 250, 400, 600, and 1000 μM) for 96h. The cells were then incubated with CellTiter 96® AQueous One Solution Reagent (Promega Corporation, Madison, WI) containing MTS and phenazine ethosulfate for 4 h at 37℃. Absorbance at 490 nm was measured by a plate reader (SpectraMax i3, Molecular Devices, Sunnyvale, CA, USA).
ELISA and western blot assays
HIV p24 protein levels in monocyte culture supernatant were determined by HIV p24 ELISA kit from Abnova (Taipei, Taiwan) as instructed by the manufacturer. HIV p24 protein levels in uninfected and infected monocytes were determined by Western blot assay. Briefly, the cell pellets were lysed with RIPA lysis buffer supplemented with protease/phosphatase inhibitors (Sigma Aldrich, St Louis, MO). The protein concentrations were determined by the bicinchoninic acid (BCA) assay (ChemCruz, Dallas, TX). The blots were incubated with primary antibodies in 5% nonfat milk in PBS overnight at 4℃, then washed with PBS containing 0.5% Tween. The blots were further incubated with horseradish peroxidase-conjugated second antibodies at room temperature for an hour, then washed with PBST and visulized by enhanced chemiluminescence (Amersham, Bucks, UK) in a Fuji Film LAS-4000 imaging analyzer (GE Life Sciences, NJ, USA).
RNA and microRNA extraction and quantification
Freshly isolated monocytes in 48-well plates were treated with or without METH (100, 150, and 250 μM) for different time points (0, 6, 12, and 24 h). Total RNAs were extracted with Tri-reagent (Molecular Research Center, OH, USA). RNA (1 μg) was subjected to reverse transcriptase PCR using reagents from Promega (Promega, WI, USA). The cDNA sample was then subjected to the real-time PCR using iQ SYBR Green Supermix (Bio-Rad Laboratories, CA, USA). All values were normalized to GAPDH mRNA. The sequences of oligonucleotide primers used in this study are listed in Table 1. Extracellular miRNAs were extracted from supernatant of monocyte cultures using the miRNeasy Mini Kit (Qiagen, CA, USA). The miRNAs from cells or supernatant were reversely transcribed with miScript Reverse Transcription Kit (Qiagen, CA, USA). The real-time PCR for the miRNAs quantification was carried out with miScript Primer Assays using miScript SYBR Green PCR Kit from Qiagen as previously described .
HIV GAG gene quantification
HIV GAG gene copy numbers in monocytes or monocytes culture supernatant were determined by the real-time PCR. RNAs from cells or the cell-free supernatant were extracted with Tri-reagent (for tissues, cells cultured in monolayer, or cell pellets) or Tri-reagent (for whole blood, serum/plasma or cell culture supernatant) according to the manufacturer’s instructions, respectively. HIV GAG standards with known copy numbers were used to quantify viral GAG gene expression in the culture supernatant.
Data were expressed as mean ± standard deviation (mean ± SD) of three experiments using monocytes from three different donors. Statistical signiﬁcance was measured by Student’s t-test using GraphPad Prism Statistical Software (GraphPad Software, La Jolla, USA). *P <0.05 and **P <0.01 indicate statistic difference between compared groups.