Inhibition of HIV-1 Replication in Human Monocyte-Derived Macrophages by Parasite Trypanosoma cruzi

and HIV-1 to date.0.01). inhibits HIV-1 replication at several replication stages in macrophages, a major cell target for both pathogens

HIV-1 inhibition by a different <i>T. cruzi</i> strain.

<p>(<b>A</b>) MDM were infected simultaneously with HIV-1_BaL and K98 clone blood trypomastigotes overnight and p24 antigen production was measured in supernatants. Results are expressed as mean ± SD of triplicates of infection and a representative experiment of 3 others is shown. (<b>B</b>) Cells were infected with BaL and VSV-G pseudotyped viruses and K98 strain blood trypomastigotes overnight. At day 4 p.i., cells were lysed and luciferase activity was measured from lysates. Results are expressed as relative light units per second (RLU/sec), presented as a percentage relative to the control (100%), where the histogram in white corresponds to the percentage of infection with the respective control virus and the histogram in black corresponds to the percentage of infection in the presence of the parasite. Results are representative of 4 independent experiments performed with cells from different donors.</p

Inhibition of HIV entry and reverse transcription by <i>T. cruzi</i>.

<p>MDM were infected with BaL pseudotyped virus in the presence of (<b>A</b>) trypomastigotes or (<b>B</b>) TcSn or VSV-G pseudotyped virus in the presence of (<b>C</b>) trypomastigotes or (<b>D</b>) TcSn, overnight. Single viral infections with both pseudotypes were performed as control. DNA was isolated and early (R-U5) and late (U5-<i>gag</i>) transcripts were quantified by real-time PCR 24 h p.i. Results are expressed as an n-fold difference with respect to the calibrator (x Cal) ± SD of infection duplicates. Results are representative of 3 independent experiments performed with cells from different donors. * <i>p<</i>0.05.</p

Implication of the major antigen cruzipain in the inhibition of HIV-1 replication cycle.

<p>(<b>A</b>) Cells were infected with either VSV-G or BaL pseudotyped viruses in the presence of 0.001; 0.1 and 10 µg/ml of cruzipain (Cz). Luciferase activity was measured in cell lysates 96 h p.i. Results are expressed as relative light units per second (RLU/sec), presented as a percentage relative to the control (100%), where the histogram in white corresponds to the percentage of infection with the respective control virus and the histogram in black corresponds to the percentage of infection in the presence of the parasite. A representative experiment of 3 is shown. (<b>B</b>) Production of p24 antigen at day 8 p.i. in cells treated with 0.1 and 10 µg/ml of cruzipain. Results are expressed as mean±SD of triplicates of infection and a representative experiment of 3 others is shown. (<b>C</b>) Influence of cruzipain on cell surface expression of CD4 and CCR5 after overnight infection in the presence of 0.1 µg/ml of cruzipain. Histograms in grey correspond to isotype control. Results are representative of at least 3 independent experiments. * <i>p</i><0.01. (<b>D</b>) Cells were infected with BaL isolate in the presence or absence of polymyxin, and p24 antigen production was measured at day 8 p.i. Results are expressed as mean±SD of triplicates of infection and a representative experiment of 3 is shown.</p

Inhibition of HIV-1 production by <i>T. cruzi</i> in MDM.

<p>MDM were infected with HIV in presence of <i>T. cruzi</i> blood trypomastigotes (<b>A</b>) or trypomastigotes free-supernatant (TcSn) (<b>B</b>) in three different schemes: HIV 24 h after <i>T. cruzi</i> (<i>T. cruzi</i>-HIV), HIV 24 h before <i>T. cruzi</i> (HIV-<i>T. cruzi)</i> or HIV at the same time as <i>T. cruzi</i> (HIV+<i>T. cruzi</i>), where <i>T. cruzi</i> indicates either trypomastigotes or TcSn. P24 antigen production was measured at days 4, 8 and 12 p.i. in culture supernatants. Results are expressed as mean ± SD of triplicates of infection and a representative experiment of at least 3 independent experiments performed with cells from different donors is shown.</p

Inhibition of pseudotyped viruses replication by <i>T. cruzi.</i>

<p>MDM were infected with both VSV-G or BaL pseudotyped viruses in the presence of <i>T. cruzi</i> blood trypomastigotes (<b>A</b>) or trypomastigotes free-supernatant (TcSn) (<b>B</b>) in three different schemes: HIV 24 h after <i>T. cruzi</i> (<i>T. cruzi</i>-HIV), HIV 24 h before <i>T. cruzi</i> (HIV-<i>T. cruzi</i>) or HIV at the same time as <i>T. cruzi</i> (HIV+<i>T. cruzi</i>), where <i>T. cruzi</i> indicates either trypomastigotes of TcSn. Luciferase activity was measured from cell lysates 4 days post-viral infection. Results are expressed as relative light units per second (RLU/sec), presented as a percentage relative to the control (100%), where the histogram in white corresponds to the percentage of infection with the respective control virus and the histogram in black corresponds to the percentage of infection in the presence of the parasite. (<b>C</b>) MDM were also infected with pseudotyped viruses and cell-derived trypomastigotes, and luciferase activity was evaluated. (<b>D</b>) Cell viability was evaluated by flow cytometry at day 4 p.i. in co-infected cells with blood trypomastigotes and controls stained with PI and Annexin-V-FITC. During the analysis 20000 events were acquired and the analysis includes all the ungated cells; percentages of PI and Annexin-V positive cells are indicated. Results are representative of 3 independent experiments performed with cells from different donors.</p

CCR5 expression impaired by <i>T. cruzi</i>.

<p>Expression of the cell surface of CCR5 and CD4 was evaluated on MDM infected or coinfected with (<b>A</b>) <i>T. cruzi</i> trypomastigotes or (<b>B</b>) TcSn. After overnight infection, cells were harvested, and CCR5 and CD4 expressions were assayed by flow cytometry. Histograms in grey correspond to isotype control. Results are representative of 4 independent experiments performed with cells from different donors.</p

Effect of <i>T. cruzi</i> on post-integration events.

<p>MDM were infected with VSV-G pseudotyped virus overnight and kept in culture for four days. Then, they were infected with trypomastigotes or treated with TcSn overnight. Cells were lysated 24 or 96 h after parasite infection and luciferase activity was measured in cell lysates. Results are expressed as mean ± SD of RLU/sec of triplicates of infection and a representative experiment of 3 is shown. * <i>p</i><0.01.</p

Inhibition of HIV-1 replication by <i>T. cruzi</i> in different cell types.

<p>Cells were infected with HIV-1 primary isolates with or without <i>T. cruzi</i> blood trypomastigotes and p24 production was measured at day 8 p.i. (<b>A</b>) PMBCs infected with R5 (BaL), X4 (Lai) and R5X4 (A204). (<b>B</b>) SupT-1 cell line infected with X4 (Lai) isolate and (<b>C</b>) MDM infected with R5 (BaL). Results are expressed as mean ± SD, and are representative of at least 2 independent experiments. * <i>p</i><0.001.</p