The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Abstract

Background: Monocytes/Macrophages (M/M) play a pivotal role as a source of virus during the whole course of HIV-1 infection. Enhanced oxidative stress is involved in the pathogenesis of HIV-1 infection. HIV-1 regulatory proteins induce a reduction of the expression and the activity of MnSOD, the mitochondrial isoform leading to a sustained generation of superoxide anions and peroxynitrite that represent important mediators of HIV-1 replication in M/M. MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphrin chloride), a synthetic peroxynitrite decomposition catalyst, reduced oxidative stress subsequent to peroxynitrite generation.

Results: Virus production was assessed by p24 ELISA, western blot, and electron microscopy during treatment with MnTBAP. MnTBAP treatment showed a reduction of HIV-1 replication in both acutely and chronically infected M/M: 99% and 90% inhibition of p24 released in supernatants compared to controls, respectively. Maturation of p55 and p24 was strongly inhibited by MnTBAP in both acutely and chronically infected M/M. EC50 and EC90 are 3.7 (+/- 0.05) microM and 19.5 (+/- 0.5) microM, in acutely infected M/M; 6.3 (+/- 0.003) microM and 30 (+/- 0.6) microM, in chronically infected M/M. In acutely infected peripheral blood limphocytes (PBL), EC50 and EC90 are 7.4 (+/- 0.06) microM and of 21.3 (+/- 0.6) microM, respectively. Treatment of acutely-infected M/M with MnTBAP inhibited the elevated levels of malonildialdehyde (MDA) together with the nitrotyrosine staining observed during HIV-1 replication. MnTBAP strongly reduced HIV-1 particles in infected M/M, as shown by electron microscopy. Moreover, in presence of MnTBAP, HIV-1 infectivity was reduced of about 1 log compared to control.

Conclusion: Results support the role of superoxide anions in HIV-1 replication in M/M and suggest that MnTBAP may counteract HIV-1 replication in combination with other antiretroviral treatments.

Figure 1

Antiviral activity of MnTBAP in acutely HIV-1 infected macrophages. Monocytes were cultured for 5 days to generate monocyte-derived macrophages which where infected with 300 TCID50/ml HIV-1 BaL and treated acutely (i.e. treated with drugs prior to virus challenge). Supernatants were collected day 14 after infection and tested for virus production by analysis of HIV-1 p24 gag Ag production with a commercially available kit ELISA.

Figure 2

Antiviral activity of MnTBAP in chronically HIV-1 infected macrophages. Monocytes were cultured for 5 days to generate monocyte-derived macrophages which where infected with 300 TCID50/ml HIV-1 BaL and treated chronically (i.e. treated with drugs 10 days after infection) with MnTBAP at indicated doses, and Amprenavir (4 uM). Supernatants were collected at day 8, 10, 15, 20 after infection and tested for virus production by analysis of HIV-1 p24 gag Ag production with a commercially available kit ELISA.

Figure 3

MnTBAP reduces p24 and p55 expression in HIV-infected macrophages. Western blots of lysates of acutely and chronically infected M/M. Line 1: Mock-infected macrophages. Line 2: macrophages HIV-1 BaL infected and treated with MnTBAP (30 μM). Line 3: macrophages HIV-1 BaL infected.

Figure 4

MnTBAP inhibits MDA in HIV-infected macrophages in a dose dependent fashion. MDA increased within HIV-1-infected macrophages. Treatment with MnTBAP (0,24–30 μM) antagonized MDA overproduction dose-dependently while AZT (0.05 μM) was not able to inhibit macrophages HIV-related MDA formation. † P < 0.001 when compared to control; * P < 0.05 and ** P < 0.001 when compared to HIV-infected cells.

Figure 5

MnTBAP inhibits nitrotyrosine formation in HIV-infected macrophages. Photomicrographs (optical microscopy) of nitrotyrosine staining in HIV-1-infected macrophages. HIV-1 infection enhance the immunocytochemical expression of nitrotyrosine (Panel A) in compared to mock-infected macrophages (Panel B), indicating an increased production of peroxynitrite. Acute treatment with MnTBAP (30 μM) (Panel D), but not with AZT (0.05 μM) (Panel C) is able to inhibit in macrophages HIV-related peroxynitrite formation.

Figure 6

Removal of free radicals by MnTBAP is involved in macrophages HIV replication. Infectivity of virus particles produced by HIV-1-infected macrophages was evaluated on macrophages obtained from a different seronegative donor exposed to serial dilution of supernatants from MnTBAP treated or not-treated HIV-1-infected macrophages. The TCID50/ml was calculated according to Reed and Muench method. MnTBAP reduces TCID50 about a log both in acutelly (Panel A) as in chronically (Panel B) HIV-1-infected macrophages compared to infected and non treated macrophages.

Figure 7

Electron microscopy of untreated or MnTBAP-treated HIV-1 infected macrophages. Untreated macrophages show accumulation of many mature particles, at different stages of maturation, in cytoplasmatic vacuoles and in the extracellular space. By contrast in MnTBAP (30 μM) treated macrophages no viral particles are found. This observation support the hypothesis that MnTBAP treatment is able to prevent enveloped and unenveloped virions production.