Anti-nerve growth factor Ab abrogates macrophage-mediated HIV-1 infection and depletion of CD4+ T lymphocytes in hu-SCID mice

Abstract

Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+ T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate the in vivo effect of HIV-1-infected macrophages on virus spread and CD4+ T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+ T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast, in vivo treatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustaining in vivo HIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.

Fig. 1.

HIV-infected macrophages induce infection and CD4⁺ depletion in hu-PBL-SCID mice. (A) Percentages of CD4⁺ T lymphocytes (open bars) and CD8⁺ T lymphocytes (filled bars) were measured in hu-PBL-SCID mice at day 28 after challenge with 5,000 HIV-infected macrophages (M/M+HIV), 5,000 HIV-infected CD4⁺ T lymphocytes (CD4-T-Ly+HIV), or mock-infected macrophages (M/M); *, P < 0.001 compared with CD4 of mice challenged with uninfected M/M. (B) Mice were killed 4 weeks after infection, then the spleen and the lymph nodes (L.N.) were analyzed for the presence of HIV-1 proviral copies by DNA PCR. HIV-1 p24 antigen was measured by ELISA in supernatants of peritoneal cells isolated from hu-PBL-SCID mice and cocultivated (peritoneal cells) with donor autologous PBLs. Controls for the DNA PCR experiments included the DNA from uninfected CEM cells [Ctr(-)] and DNA from 8E5 HIV-infected cells, which harbor one proviral HIV copy per cell (8E5). (C) HIV-1 RNA was measured in the plasma of hu-PBL-SCID mice challenged with HIV-infected M/M (▪), HIV-infected CD4⁺ T lymphocytes (▴), or mock-infected macrophages (•) at different time points after cell injection. The dashed line defines the threshold of HIV RNA detection. Four mice were used in each group.

Fig. 2.

Few M/M are sufficient to efficiently spread HIV infection in hu-PBL-SCID mice. (A) Percentage of CD4⁺ T lymphocytes (open bars) and CD8⁺ T lymphocytes (filled bars) were measured in hu-PBL-SCID mice at day 21 after challenge with 500 or 5,000 HIV-infected macrophages, or 5,000 mock-infected macrophages; *, P < 0.01 compared with CD4 of mice challenged with uninfected M/M. (B) HIV-1 proviral copies were evaluated in spleen and lymph nodes (L.N.) by DNA PCR; as a control, several dilutions of DNA from 8E5 HIV-infected cells are reported. Cocultivation of peritoneal cells with donor autologous PBLs was also performed. (C) HIV-1 RNA was measured in the plasma of hu-PBL-SCID mice at days 7 and 17 after challenge with 5,000 HIV-infected macrophages (5,000 M/M+HIV, stippled bars), 500 HIV-infected macrophages (500 M/M+HIV, filled bars), or 5,000 mock-infected macrophages (M/M, open bars); *, P < 0.001 compared with CD4 of mice challenged with uninfected M/M. The dashed line defines the threshold of HIV-RNA detection. Three mice were used in each group.

Fig. 3.

Macrophages produce and release NGF once infected by HIV. NGF production was measured in hu-PBL-SCID mice 3 and 7 days after challenge with HIV-infected macrophages (M/M+HIV, filled bars) or mock-infected microphages (M/M, stippled bars). Control mice were not injected with macrophages (control, open bars). Samples were run in pool for each different group of three mice. Results presented are from one representative experiment of two.

Fig. 4.

NGF starvation prevents HIV infection in hu-PBL-SCID mice challenged with infected macrophages. (A) Percentage of CD4⁺ T lymphocytes (white bars) and CD8⁺ T lymphocytes (black bars) were measured in hu-PBL-SCID mice at day 14 after challenge with HIV-infected macrophages and treatment with anti-NGF mAb (HIV-infected M/M+aNGF Ab). A group of mice was treated with an isotypic Ab (HIV-infected M/M+IgG); *, P < 0.05 compared with CD4 in infected but untreated hu-PBL-SCID mice. (B) HIV-1 RNA was measured in hu-PBL-SCID plasma at days 7 and 14 after HIV macrophage challenge and treatment with either anti-NGF Ab (▪) or an isotypic IgG Ab (▴), or no treatment (•); P < 0.05. The dashed line defines the threshold of HIV-RNA detection. (C) Mice were killed 2 weeks after infection, then the spleen, lymph nodes (L.N.), and peritoneal cells (P.C.) were analyzed for the presence of HIV-1 proviral copies and HLA-DQ expression by DNA PCR. (D) HIV-1 p24 antigen was measured by ELISA in supernatants of peritoneal cells isolated from hu-PBL-SCID mice challenged with HIV-infected M/M treated with aNGF Ab (black bars) or an IgG isotypic Ab (gray bars), or mock-treated (white bars). In this experiment, three mice for each group were used. Each bar corresponds to a single mouse.