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Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages.

Kang W, Marasco WA, Tong HI, Byron MM, Wu C, Shi Y, Sun S, Sun Y, Lu Y - J Neuroinflammation (2014)

Bottom Line: Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability.Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc.Our study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary hMDM and does not lead to any significant changes in hMDM immune-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Tangdu Hospital, The Fourth Military Medical University, 569 Xinsi Road, Xi'an, Shaanxi, 710038, China. kangwenkevin@gmail.com.

ABSTRACT

Background: HIV-1 Tat is essential for HIV replication and is also a well-known neurotoxic factor causing HIV-associated neurocognitive disorder (HAND). Currently, combined antiretroviral therapy targeting HIV reverse transcriptase or protease cannot prevent the production of early viral proteins, especially Tat, once HIV infection has been established. HIV-infected macrophages and glial cells in the brain still release Tat into the extracellular space where it can exert direct and indirect neurotoxicity. Therefore, stable production of anti-Tat antibodies in the brain would neutralize HIV-1 Tat and thus provide an effective approach to protect neurons.

Methods: We constructed a humanized anti-Tat Hutat2:Fc fusion protein with the goal of antagonizing HIV-1 Tat and delivered the gene into cell lines and primary human monocyte-derived macrophages (hMDM) by an HIV-based lentiviral vector. The function of the anti-Tat Hutat2:Fc fusion protein and the potential side effects of lentiviral vector-mediated gene transfer were evaluated in vitro.

Results: Our study demonstrated that HIV-1-based lentiviral vector-mediated gene transduction resulted in a high-level, stable expression of anti-HIV-1 Tat Hutat2:Fc in human neuronal and monocytic cell lines, as well as in primary hMDM. Hutat2:Fc was detectable in both cells and supernatants and continued to accumulate to high levels within the supernatant. Hutat2:Fc protected mouse cortical neurons against HIV-1 Tat86-induced neurotoxicity. In addition, both secreted Hutat2:Fc and transduced hMDM led to reducing HIV-1BaL viral replication in human macrophages. Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability. Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc.

Conclusions: Our study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary hMDM and does not lead to any significant changes in hMDM immune-activation. The neuroprotective and HIV-1 suppressive effects produced by Hutat2:Fc were comparable to that of a full-length anti-Tat antibody. This study provides the foundation and insights for future research on the potential use of Hutat2:Fc as a novel gene therapy approach for HAND through utilizing monocytes/macrophages, which naturally cross the blood-brain barrier, for gene delivery.

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Transduction of human cell lines HTB-11 and U937 as well as primary hMDM by lentiviral vectors HR-Hutat2 expressing anti-HIV-1 Hutat2:Fc and EGFP. HTB-11 cells (5 × 105) were transduced in a T25 flask in the presence of 8 μg/mL polybrene for 2 h (multiplicity of infection, MOI = 10). U937 cells (1 × 105) were transduced twice by spin-infection at 1,500 × g for 90 minutes (MOI = 100). Human MDM were infected with HR-Hutat2 vectors (MOI = 50 or MOI = 10) for 1.5 h on days 7 and 8 in vitro (DIV 7 and DIV 8), respectively. The transduction efficiencies were evaluated by calculating the percentage of GFP+ cells from five randomly selected microscopic fields under a fluorescence microscope on day 3 post-transduction for HTB-11, as well as on day 8 post-transduction for U937 and hMDM, respectively. HTB-11, Non-transduced HTB-11 cells; HTB-Hutat2, HR-Hutat2 transduced HTB-11 cells; U937, Non-transduced U937 cells; U937-Hutat2, HR-Hutat2 transduced U937 cells; EGFP, Enhanced green fluorescent protein; hMDM-Hutat2 MOI = 50, HR-Hutat2 transduced hMDM at the MOI of 50; hMDM-Hutat2 MOI = 10, HR-Hutat2 transduced hMDM at the MOI of 10. (A) Expression of EGFP in HR-Hutat2 transduced HTB-11 and U937 cells. (B) Co-location of the Hutat2:Fc and EGFP expression in HR-Hutat2 transduced HTB-11. Nuclei were counterstained with DAPI (blue). The Hutat2:Fc proteins (red) were expressed in the cytoplasm while EGFP proteins (green) were expressed both in the nuclei and cytoplasm. (C) Expression of EGFP in transduced hMDM. Fluorescently-labeled cells were visualized with an epi-microscope (Nikon Eclipse TE2000-U) using a numerical aperture lens (0.30 or 0.45) and a digital camera attachment. The pictures were overlaid using ImageJ software (Version 1.48, National Institutes of Health, USA). Data represent means ± s.e.m. of three independent experiments. Scale bar = 100 μm.
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Fig1: Transduction of human cell lines HTB-11 and U937 as well as primary hMDM by lentiviral vectors HR-Hutat2 expressing anti-HIV-1 Hutat2:Fc and EGFP. HTB-11 cells (5 × 105) were transduced in a T25 flask in the presence of 8 μg/mL polybrene for 2 h (multiplicity of infection, MOI = 10). U937 cells (1 × 105) were transduced twice by spin-infection at 1,500 × g for 90 minutes (MOI = 100). Human MDM were infected with HR-Hutat2 vectors (MOI = 50 or MOI = 10) for 1.5 h on days 7 and 8 in vitro (DIV 7 and DIV 8), respectively. The transduction efficiencies were evaluated by calculating the percentage of GFP+ cells from five randomly selected microscopic fields under a fluorescence microscope on day 3 post-transduction for HTB-11, as well as on day 8 post-transduction for U937 and hMDM, respectively. HTB-11, Non-transduced HTB-11 cells; HTB-Hutat2, HR-Hutat2 transduced HTB-11 cells; U937, Non-transduced U937 cells; U937-Hutat2, HR-Hutat2 transduced U937 cells; EGFP, Enhanced green fluorescent protein; hMDM-Hutat2 MOI = 50, HR-Hutat2 transduced hMDM at the MOI of 50; hMDM-Hutat2 MOI = 10, HR-Hutat2 transduced hMDM at the MOI of 10. (A) Expression of EGFP in HR-Hutat2 transduced HTB-11 and U937 cells. (B) Co-location of the Hutat2:Fc and EGFP expression in HR-Hutat2 transduced HTB-11. Nuclei were counterstained with DAPI (blue). The Hutat2:Fc proteins (red) were expressed in the cytoplasm while EGFP proteins (green) were expressed both in the nuclei and cytoplasm. (C) Expression of EGFP in transduced hMDM. Fluorescently-labeled cells were visualized with an epi-microscope (Nikon Eclipse TE2000-U) using a numerical aperture lens (0.30 or 0.45) and a digital camera attachment. The pictures were overlaid using ImageJ software (Version 1.48, National Institutes of Health, USA). Data represent means ± s.e.m. of three independent experiments. Scale bar = 100 μm.

Mentions: The efficiency of lentiviral vector-mediated gene transfer was evaluated initially in human neuronal and monocytic cell lines. Human neuroblastoma cell line HTB-11 and monocytic cell line U937 were transduced with lentiviral vectors HR-Hutat2 at a MOI of 10 and 100, respectively. Under the established experimental conditions, transduction efficiencies were calculated to be 98.5% ± 0.8% for HTB-11 cells and 95.4% ± 2.5% for U937 cells (Figure 1A). Furthermore, the expression of the integrated genes was confirmed by examining transduced HTB-11 for the Fc expression using immunofluorescent staining with an anti-human IgG Fc specific antibody. EGFP proteins were expressed in both the nuclei and cytoplasm, whereas Hutat2:Fc was predominately distributed in the cytoplasm (Figure 1B). HTB-11 cells were also transduced with control vectors HR-A3H5 containing a construct encoding the anti-Epstein-Barr virus latent membrane protein 1 scFv A3H5 fused to Fc. The transduction efficiency was as high as that obtained from HR-Hutat2 transduced HTB-11 cells (data not shown).Figure 1


Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages.

Kang W, Marasco WA, Tong HI, Byron MM, Wu C, Shi Y, Sun S, Sun Y, Lu Y - J Neuroinflammation (2014)

Transduction of human cell lines HTB-11 and U937 as well as primary hMDM by lentiviral vectors HR-Hutat2 expressing anti-HIV-1 Hutat2:Fc and EGFP. HTB-11 cells (5 × 105) were transduced in a T25 flask in the presence of 8 μg/mL polybrene for 2 h (multiplicity of infection, MOI = 10). U937 cells (1 × 105) were transduced twice by spin-infection at 1,500 × g for 90 minutes (MOI = 100). Human MDM were infected with HR-Hutat2 vectors (MOI = 50 or MOI = 10) for 1.5 h on days 7 and 8 in vitro (DIV 7 and DIV 8), respectively. The transduction efficiencies were evaluated by calculating the percentage of GFP+ cells from five randomly selected microscopic fields under a fluorescence microscope on day 3 post-transduction for HTB-11, as well as on day 8 post-transduction for U937 and hMDM, respectively. HTB-11, Non-transduced HTB-11 cells; HTB-Hutat2, HR-Hutat2 transduced HTB-11 cells; U937, Non-transduced U937 cells; U937-Hutat2, HR-Hutat2 transduced U937 cells; EGFP, Enhanced green fluorescent protein; hMDM-Hutat2 MOI = 50, HR-Hutat2 transduced hMDM at the MOI of 50; hMDM-Hutat2 MOI = 10, HR-Hutat2 transduced hMDM at the MOI of 10. (A) Expression of EGFP in HR-Hutat2 transduced HTB-11 and U937 cells. (B) Co-location of the Hutat2:Fc and EGFP expression in HR-Hutat2 transduced HTB-11. Nuclei were counterstained with DAPI (blue). The Hutat2:Fc proteins (red) were expressed in the cytoplasm while EGFP proteins (green) were expressed both in the nuclei and cytoplasm. (C) Expression of EGFP in transduced hMDM. Fluorescently-labeled cells were visualized with an epi-microscope (Nikon Eclipse TE2000-U) using a numerical aperture lens (0.30 or 0.45) and a digital camera attachment. The pictures were overlaid using ImageJ software (Version 1.48, National Institutes of Health, USA). Data represent means ± s.e.m. of three independent experiments. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256057&req=5

Fig1: Transduction of human cell lines HTB-11 and U937 as well as primary hMDM by lentiviral vectors HR-Hutat2 expressing anti-HIV-1 Hutat2:Fc and EGFP. HTB-11 cells (5 × 105) were transduced in a T25 flask in the presence of 8 μg/mL polybrene for 2 h (multiplicity of infection, MOI = 10). U937 cells (1 × 105) were transduced twice by spin-infection at 1,500 × g for 90 minutes (MOI = 100). Human MDM were infected with HR-Hutat2 vectors (MOI = 50 or MOI = 10) for 1.5 h on days 7 and 8 in vitro (DIV 7 and DIV 8), respectively. The transduction efficiencies were evaluated by calculating the percentage of GFP+ cells from five randomly selected microscopic fields under a fluorescence microscope on day 3 post-transduction for HTB-11, as well as on day 8 post-transduction for U937 and hMDM, respectively. HTB-11, Non-transduced HTB-11 cells; HTB-Hutat2, HR-Hutat2 transduced HTB-11 cells; U937, Non-transduced U937 cells; U937-Hutat2, HR-Hutat2 transduced U937 cells; EGFP, Enhanced green fluorescent protein; hMDM-Hutat2 MOI = 50, HR-Hutat2 transduced hMDM at the MOI of 50; hMDM-Hutat2 MOI = 10, HR-Hutat2 transduced hMDM at the MOI of 10. (A) Expression of EGFP in HR-Hutat2 transduced HTB-11 and U937 cells. (B) Co-location of the Hutat2:Fc and EGFP expression in HR-Hutat2 transduced HTB-11. Nuclei were counterstained with DAPI (blue). The Hutat2:Fc proteins (red) were expressed in the cytoplasm while EGFP proteins (green) were expressed both in the nuclei and cytoplasm. (C) Expression of EGFP in transduced hMDM. Fluorescently-labeled cells were visualized with an epi-microscope (Nikon Eclipse TE2000-U) using a numerical aperture lens (0.30 or 0.45) and a digital camera attachment. The pictures were overlaid using ImageJ software (Version 1.48, National Institutes of Health, USA). Data represent means ± s.e.m. of three independent experiments. Scale bar = 100 μm.
Mentions: The efficiency of lentiviral vector-mediated gene transfer was evaluated initially in human neuronal and monocytic cell lines. Human neuroblastoma cell line HTB-11 and monocytic cell line U937 were transduced with lentiviral vectors HR-Hutat2 at a MOI of 10 and 100, respectively. Under the established experimental conditions, transduction efficiencies were calculated to be 98.5% ± 0.8% for HTB-11 cells and 95.4% ± 2.5% for U937 cells (Figure 1A). Furthermore, the expression of the integrated genes was confirmed by examining transduced HTB-11 for the Fc expression using immunofluorescent staining with an anti-human IgG Fc specific antibody. EGFP proteins were expressed in both the nuclei and cytoplasm, whereas Hutat2:Fc was predominately distributed in the cytoplasm (Figure 1B). HTB-11 cells were also transduced with control vectors HR-A3H5 containing a construct encoding the anti-Epstein-Barr virus latent membrane protein 1 scFv A3H5 fused to Fc. The transduction efficiency was as high as that obtained from HR-Hutat2 transduced HTB-11 cells (data not shown).Figure 1

Bottom Line: Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability.Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc.Our study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary hMDM and does not lead to any significant changes in hMDM immune-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Tangdu Hospital, The Fourth Military Medical University, 569 Xinsi Road, Xi'an, Shaanxi, 710038, China. kangwenkevin@gmail.com.

ABSTRACT

Background: HIV-1 Tat is essential for HIV replication and is also a well-known neurotoxic factor causing HIV-associated neurocognitive disorder (HAND). Currently, combined antiretroviral therapy targeting HIV reverse transcriptase or protease cannot prevent the production of early viral proteins, especially Tat, once HIV infection has been established. HIV-infected macrophages and glial cells in the brain still release Tat into the extracellular space where it can exert direct and indirect neurotoxicity. Therefore, stable production of anti-Tat antibodies in the brain would neutralize HIV-1 Tat and thus provide an effective approach to protect neurons.

Methods: We constructed a humanized anti-Tat Hutat2:Fc fusion protein with the goal of antagonizing HIV-1 Tat and delivered the gene into cell lines and primary human monocyte-derived macrophages (hMDM) by an HIV-based lentiviral vector. The function of the anti-Tat Hutat2:Fc fusion protein and the potential side effects of lentiviral vector-mediated gene transfer were evaluated in vitro.

Results: Our study demonstrated that HIV-1-based lentiviral vector-mediated gene transduction resulted in a high-level, stable expression of anti-HIV-1 Tat Hutat2:Fc in human neuronal and monocytic cell lines, as well as in primary hMDM. Hutat2:Fc was detectable in both cells and supernatants and continued to accumulate to high levels within the supernatant. Hutat2:Fc protected mouse cortical neurons against HIV-1 Tat86-induced neurotoxicity. In addition, both secreted Hutat2:Fc and transduced hMDM led to reducing HIV-1BaL viral replication in human macrophages. Moreover, lentiviral vector-based gene introduction did not result in any significant changes in cytomorphology and cell viability. Although the expression of IL8, STAT1, and IDO1 genes was up-regulated in transduced hMDM, such alternation in gene expression did not affect the neuroprotective effect of Hutat2:Fc.

Conclusions: Our study demonstrated that lentivirus-mediated gene transfer could efficiently deliver the Hutat2:Fc gene into primary hMDM and does not lead to any significant changes in hMDM immune-activation. The neuroprotective and HIV-1 suppressive effects produced by Hutat2:Fc were comparable to that of a full-length anti-Tat antibody. This study provides the foundation and insights for future research on the potential use of Hutat2:Fc as a novel gene therapy approach for HAND through utilizing monocytes/macrophages, which naturally cross the blood-brain barrier, for gene delivery.

Show MeSH
Related in: MedlinePlus