Figure 5. TNF-α and Pam3CSK4 enhance HIV-1 infection.

(A and B) Epidermal sheets were stimulated with TNF-α or Pam3CSK4. After 6 hours, the sheets were inoculated with HIV-1–eGFP. After 3 days, the epidermal sheets were removed, the migrated cells were harvested, stained for CD86, and subsequently analyzed for GFP expression by flow cytometry. (A) Infection is depicted in dot plots. The percentage of GFP⁺ cells ± SD is depicted in the upper-right corner. (B) HIV-1 infection is depicted as percentage of cells positive for GFP expression. Error bars represent the mean ± SD of duplicates. The 3 depicted donors have been measured in 2 independent experiments. (C–E) Epidermal single-cells suspensions were stimulated with TNF-α or Pam3CSK4 for 30 minutes and inoculated with NL4.3-BaL. After 6 hours, the cells were washed and analyzed by quantitative real-time PCR analysis for HIV-1 replication by Tat/Rev transcripts and viral uptake by full-length viral RNAs (LTR). The Ct values were normalized for cellular GAPDH, and relative mRNA expression of HIV-1–inoculated medium control samples was set at 1. (C) Transcription per viral copy in cell was determined by the ratio of Tat/Rev mRNA and full-length viral RNA. A representative experiment out of 4 donors is depicted. Error bars represent the mean ± SD of duplicates.