FIG. 2.
C5a treatment or C5aR1 blockade decreases HIV infection of THP-1 and MDM but does not decrease infection with vesicular stomatitis virus (VSV)-pseudotyped HIV. (a) Differentiated THP-1 cells were cultured at 37°C with C5aRA (87.4 μM), anti-C5aR1 mAb S5/1, isotype control mAb (both at 1 μg/ml), or TAK779 (20 μM). The cells were infected with YK-JRLF at an MOI = 1 for THP-1 or MOI = 3 for MDM. After 6 h, cells were washed, cultured for another 24 h, and then lysed. Integrated HIV-1 DNA was measured in cell lysates by Alu-RT-PCR. ΔCt was used to calculate the relative expression of HIV, normalized to CD3 expression. Eventually, results were normalized to infection in untreated cells. The graphs show the mean normalized infection levels ± SEM from four independent experiments. (b) C5aRA and C5aR1-specific antibody treatment decreases HIV infection of THP-1 cells in a dose-dependent manner. (c) As in (a), except that MDM were used instead of THP-1 cells. The graph shows the mean normalized infection levels ± SEM of MDM from five individual donors. (d) As in (a), except that THP-1 cells were incubated with hC5a (100 μM) for 1 h. (e) C5aR1 and CCR5 blocking reagents do not affect the number of cells. Differentiated THP-1 cells or MDM were cultured at 37°C with C5aRA, anti-C5aR1 mAb S5/1, or TAK779 and infected with YK-JRLF as described above. CD3 DNA levels were measured in cell lysates by Alu-RT-PCR. The graph shows the mean ± SEM of the cycle threshold (Ct) values from ≥4 independent experiments. (f) Infection of differentiated THP-1 cells with VSV-pseudotyped YU2 in the presence or absence of C5aR1 blockade with C5aR1 mAb (1 mg/ml). Data are the mean values ± SEM from three independent experiments.