Mip 1β

Standard ELISA methods were used to measure concentrations of Gal-9 (R&D Systems), cytokines and chemokines such as IL-12p40, IL-12p70, MIP-1β (macrophage inflammatory protein [MIP]), monocyte chemoattractant protein 2 (MCP-2), tumour necrosis factor-alpha (TNF-α), IL-6, transforming growth factor-beta1 (TGF-β1), IL-18, IL-23, IL-27, and IL-1β (R&D Systems) and IFN-α (Verikine™ Human IFNα ELISA Kit, PBL Assay Science, Piscataway, NJ, USA).

Based on the differential usage of co-receptors (CCR5 and CXCR4), HIV isolates have been referred to as R5-, X4-, or dual-tropic strains [32 (link)]. The HIV R5-tropic strains (Bal, Jago, and YU2) were obtained from the AIDS Research and Reference Reagent Program of the National Institute of Health (NIH, Bethesda, Rockville, MD, USA). Imiquimod, a synthetic TLR7 ligand, was purchased from InvivoGen (San Diego, CA, USA). MIP-1α, MIP-1β, and RANTES antibodies were purchased from R&D System (R&D system Inc., Minneapolis, MN, USA). Mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-Mouse IgG (horseradish peroxidase (HRP)-linked) antibody, and anti-rabbit IgG (HRP-linked) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse antibody against HIV-1 p24 was purchased from Abcam (Abcam, Cambridge, UK). All antibodies and reagents for flow cytometry assay were purchased from BD Bioscience (BD Bioscience, San Jose, CA, USA). Trichloroacetic acid (TCA) and acetone were purchased from Sigma-Aldrich.

To measure CCR5 receptor occupancy, PBMC were incubated in the presence or absence of the chemokine MIP-1β (R&D Systems; 271-BME) which binds to CCR5 and inhibits antibody binding and detection of cell surface CCR5 otherwise measured without MIP-1β incubation, as previously described [44] (link). CCR5 values calculated by using the receptor occupancy assay were determined as the frequency of CCR5-expressing CD4 T cells subtracted from those values measured with MIP-1β incubation.

Standard ELISA methods were used to measure concentrations of IFN-λ1 (R&D Systems), and other cytokines and chemokines, including IL-12p40, MIP-1α, MIP-1β, MCP-2, IL-6, IL-8, IL-10, RANTES and IL-1β (R&D Systems).

Mouse MIP-1α, MIP-1β and MIP-2 ELISAs (R & D Systems, Minneapolis, MN, USA) were performed on the samples and the LPS-stimulated culture supernatants. The kits contained all the reagents required for the experiment and experiments were performed as per the manufacturer’s instructions. The samples were all diluted in reagent diluent, 1% human serum albumin (HSA) (w/v). The MIP-1α in unstimulated samples were assayed at 1/270 v/v and the LPS stimulated supernatant at 1/2000 v/v in diluent. For the MIP-1β ELISA, the unstimulated culture supernatants were assayed at 1/100 v/v while the LPS stimulated supernatants were assayed at 1/5000 v/v in diluent. The MIP-2 ELISA unstimulated supernatants were assayed at 1/20 v/v and the mitogen stimulated supernatant was at 1/500 v/v in assay diluent.

LAD2 and LUVA cells were stimulated for 6 hours in the presence of L-cysteine (3 mmol/L) with LTD₄ and LTE₄ (both 100 nmol/L) and vehicle control. CCL4 and CSF2 concentrations were measured in supernatants using human CCL4 (MIP-1β) and CSF2 (GM-CSF) duo set kits (R&D Systems, UK) following manufacturer’s protocol.