Single cultures of primary human cerebrocortical astrocytes (HA) (Sciencell, USA) and Human Umbilical Vein Endothelial Cells (HUVECs) (Lonza, Germany) were maintained at 37 °C in a humidified 5 % CO2 in high glucose DMEM (Life Technologies Corp., USA) supplemented with 10 % FCS (Biochrom, Germany) and 1 % N2 Supplement (Life Technologies Corp., USA) and complete EGM (Lonza, Germany) respectfully. HL2/3 cells (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HL2/3 from Dr. Barbara K. Felber and Dr. George N. Pavlakis), HeLa derived cells producing high levels of Gag, Env, Tat, Rev and Nef proteins, were maintained at the conditions mentioned above in high glucose DMEM (Life Technologies Corp., USA) supplemented with 10 % FCS (Biochrom, Germany). Cells were routinely sub-cultured before reaching confluence.
Cell numbers were determined using a haemocytometer following trypsinization and trypan blue staining. For 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays, HA and HUVECs were seeded in 6 well cell culture plates (500000 cells/well). For all other single culture experiments all cell types were seeded in 6 well cell culture dishes at the aforementioned cell density.
In order to simulate the blood–brain barrier, co-cultures of HA and HUVECs were established on opposite sides of fibronectin (BD Biosciences, USA) coated 3 μm pore size tissue culture inserts (BD Biosciences, USA) .
All cell culture experiments were done in triplicate and repeated a minimum of three times.
Preparation of S. frutescens aqueous extract
Commercially available plant material was kindly donated by Mr Ulrich Feiter (Parceval Pharmaceuticals Pty Ltd). Sutherlandia frutescens plants were cultivated from commercial stock seed which have been previously taxonomically verified as S. frutescens var. SU1 (registered product code 02P0058), harvested (well after flowering and seeding phase) and dry milled (leaves and stems only) by Parceval Pharmaceuticals Pty Ltd (Wellington, South Africa) using proprietary procedures. A warm water extract of dry-milled S. frutescens (moisture content 16.41 %) was prepared in boiling distilled water (25 mg/ml) using methods previously described for in vivo treatment [4, 7] and then sterile filtered using filter pore size 0.22 μm.
S. frutescens dose response cell viability assay
In order to determine the highest dose of S. frutescens tolerated with the least amount of cell death, human astrocytes, HUVECs and primary human monocytes were incubated with 50, 500 and 5000 μg/ml S. frutescens extract for 24 h.
Cell viability was assessed using a modified version of the MTT assay described by Gomez and colleagues . The assay is based upon the principle of reduction of MTT into blue formazan pigments by viable mitochondria in healthy cells. At the end of the experiment, the medium was removed from the 6 well plates and the cells washed twice with PBS. MTT (0.01 g/ml) was dissolved in PBS, and 500 μl was added to each well dish. Cells were subsequently incubated for 1 h at 37 °C in an atmosphere of 5 % CO2. After the incubation period, cells were washed twice with PBS, and one ml of HCl–isopropanol–Triton (1 % HCl in isopropanol; 0.1 % Triton X- 100; 50:1) was added to each well and gently agitated for 5 min. This lysed the cell membranes and liberated the formazan pigments. The suspension was then centrifuged at 131 × g for 2 min. The optical density (OD) was determined spectrophotometrically at a wavelength of 540 nm and the values expressed as percentages of control.
Full length HIV-1 subtype B & C Tat protein stimulation
Full length synthetic Tat proteins were kindly provided by Professor Ranga Udaykumar of the Jawaharlal Nehru Centre for Advanced Scientific Research (Bangalore, India) and were synthesized and purified as previously described . Tat proteins were reconstituted and subsequently diluted in Tris-Cl buffer (20 mM, pH8) supplemented with 1 mM DTT.
Human astrocytes, HUVECs, primary human monocytes as well as simulated BBB co-cultures were stimulated with either protein (10 ng/ml) for 2.5 h and 24 h, after which culture media was collected and stored at −80 °C for subsequent analyses. In order to test the efficacy of S. frutescens as a modulator of neuroinflammatory processes, cells were pre-treated for 4 h and 24 h respectively prior to HIV-1 Tat protein stimulation. Following stimulation culture supernatants were collected and stored at −80 °C until further analysis.
HL2/3 Cells – A more representative in vitro model of HIV-1 infection
As previously mentioned, HL2/3 cells produce and secrete high levels of most HIV-1 subtype B proteins into their culture media, and for this reason it was decided to co-culture these cells with the simulated BBB cultures in order to mimic the neuroinflammatory milieu at the interface between the infected central nervous system (represented by the HL2/3 cells seeded into the wells of a 24 well culture plate into which the tissue culture inserts, on which the simulated BBB has been constructed, are placed) and the neurovasculature (i.e., the BBB represented by the in vitro simulated BBB cultures). Firstly, to assess the effect of the HL2/3 derived HIV-1 proteins on the individual cell types used to construct the in vitro BBB, HL2/3 cells were seeded into 6 well plates at 200 000 cells per well and allowed to adhere to the culture surface. Once the HL2/3 cells had adhered, culture media was replaced. HL2/3 conditioned media was collected at 2.5 h and 24 h from separate cultures, and this media was then used to stimulate human astrocytes, HUVECs and primary human monocytes for either 2.5 h or 24 h. Also, as in the aforementioned section outlining the HIV-1 Tat experiments, cells were pre-treated with S. frutescens for either 4 h or 24 h prior to stimulation. Culture supernatants were collected post stimulation and stored at −80 °C until further analysis.
The aforementioned experiment was repeated in the co-culture system, with the omission of the 24 h time point. HL2/3 cells were seeded into 24 well plates at 50 000 cells/well and allowed to adhere. Culture media was refreshed after which the BBB co-cultures were transferred to the wells containing the HL2/3 cells. BBB co-cultures were exposed to the HL2/3 cells for a period of 2.5 h, after which culture supernatants were collected and stored at −80 °C for further downstream analyses. Additional BBB co-cultures were treated with S. frutescens for 4 h prior to stimulation.
Pro-inflammatory cytokine & chemokine analysis
Monocyte chemoattractant protein-1 (MCP-1), a key role player in HIV-1-associated neuroinflammation, was measured in all supernatants by a conventional ELISA kit (Biolegend, San Diego, CA), used according to the manufacturer’s instructions.
IL-1β was measured in all co-culture supernatants by AlphaLISA (PerkinElmer, Waltham, MA), according to the manufacturer’s instructions.
Transmigration of both infected and uninfected inflammatory cells, in particular those from the monocyte macrophage lineage, play a major role in the aetiology of HIV-1-associated neuroinflammation. For this reason monocyte transmigration was assessed in the BBB co-cultures by adding primary human monocytes to the top of the insert, allowing the cells to migrate in response to the various stimuli for 2.5 h, after which the BBB inserts and cells in the bottom of the well were fixed in 4 % paraformaldehyde and stained with a FITC-anti-human CD14 antibody (Biolegend, San Diego, CA). CD14 is a specific marker of cells from the monocyte/macrophage lineage. All CD14+ monocytes on top of the entire insert (unmigrated) and on the bottom of the culture well (migrated) were counted using a fluorescent microscope (Leica, Germany). Cells in suspension were not quantified, since we have previously shown that cell counts in these compartments are independent of interventions/treatments .
All statistical analyses were performed using Graphpad Prism Version 5 software (Graphpad Software, La Jolla, CA, USA). Results are expressed as mean ± SD. One- or two- way analysis of variance (ANOVA) as relevant, followed by a Bonferroni post hoc test, was used to assess differences between experimental groups and/or time points. Differences were considered to be of statistical significance when P value ≤ 0.05.